REVIEW OF INGREDIENTS ADDED TO CIGARETTES
PHASE ONE: THE FEASIBILITY OF TESTING INGREDIENTS
ADDED TO CIGARETTES
LSRO
Life Sciences Research Office
9650 Rockville Pike
Bethesda, Maryland
Daniel M. Byrd III, Ph.D., D.A.B.T.
Editor
For internal use of Philip Morris personnel only.
Copyright © 2004 Life Sciences Research Office
No part of this document may be reproduced by any mechanical, photographic, or
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the U.S. Government.
Copies of the publication may be obtained from the Life Sciences Research
Office. Orders and inquiries may be directed to: LSRO, 9650 Rockville Pike,
Bethesda, MD 20814-3998. Tel: 301-634-7030; Fax 301-634-7876; web site:
www.LSRO.org
ISBN: 0-9753167-0-2
Library of Congress Catalog Number: 2004102833
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FOREWORD
The Life Sciences Research Office, Inc. (LSRO) provides scientific assessments
of topics in the biomedical sciences. Reports are based on comprehensive literature
reviews and the scientific opinions of knowledgeable investigators engaged in work
in relevant areas of science and medicine. This LSRO report was developed for
and supported by Philip Morris, USA, Inc., P.O. Box 26583, Richmond, VA 23261
(Philip Morris) in accordance with a contract between Philip Morris and LSRO.
An Expert Panel provided scientific oversight and direction for all aspects of this
project. The LSRO independently appointed members of the Panel based on their
qualifications, experience, judgment, and freedom from conflict of interest, with due
considerations for balance and breadth in the appropriate professional disciplines.
The Panel was selected with the concurrence of the LSRO Board of Directors.
The Expert Panel convened eight times (seven full meetings and one conference
call) to assess the available data. LSRO invited submission of data, information, and
views bearing on the topic under study, held a widely advertised Open Meeting on
August 26, 2002, and accepted written submissions. Information about the process,
including the critical literature and presentations upon which the Expert Panel based
their deliberations, was made publicly available by posting on the LSRO web site.
The LSRO staff and special consultants considering all available information and
the deliberations of the Expert Panel drafted the report. The LSRO report was
edited by Daniel M. Byrd III, Deputy Director, LSRO who, with the assistance of
LSRO staff, submitted the report for review by an independent reviewer, incorporated the reviewer’s comments, and provided additional documentation and viewpoints for incorporation into the final report. The Expert Panel and the LSRO
Board of Directors reviewed and approved the final report. On completion of
these reviews, the report was transmitted to the Sponsor for technical comments
by the Executive Director, LSRO.
The listing of members of the Expert Panel, others who assisted in preparation of
this report, and the LSRO Board of Directors, does not imply their endorsement of
all statements in the report. The report was developed independently of Philip
Morris and conclusions drawn therein do not necessarily represent the view of
Philip Morris or any of its employees. The LSRO accepts full responsibility for the
study conclusions and accuracy of the report.
Michael Falk, Ph.D.
Executive Director
Life Sciences Research Office, Inc.
January 9, 2004
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For internal use of Philip Morris personnel only.
Table of Contents
TABLE OF CONTENTS
Forward .............................................................................................................. iv
1 Executive Summary .....................................................................................1
2 Introduction ..................................................................................................5
2.1 Intent & Purpose .....................................................................................6
2.2 Public Health Considerations ...................................................................7
2.3 Feasibility .................................................................................................9
2.4 The Scientific Review Process .............................................................. 10
2.5 Summary ................................................................................................ 14
3 Cigarettes and Cigarette Smoke ............................................................. 16
3.1 Intent & Purpose ................................................................................... 18
3.2 Cigarettes ............................................................................................... 18
3.3 Cigarette Smoke .................................................................................... 25
3.4 Components of Cigarette Smoke ........................................................... 36
3.5 Summary ................................................................................................ 59
4 Cigarette Smoke: Exposure and Dosimetry ........................................ 61
4.1 Intent & Purpose ................................................................................... 62
4.2 Human Smoking ..................................................................................... 63
4.3 Dosimetry of Cigarette Smoke .............................................................. 73
4.4 Biological Monitoring of Smoking Exposure .......................................... 87
4.5 Experimental Animal Exposure to Cigarette Smoke .............................. 94
4.6 Summary ................................................................................................ 98
5 Adverse Human Health Effects ............................................................. 100
5.1 Intent & Purpose ................................................................................. 101
5.2 Premature Mortality from Cigarette Smoking ..................................... 102
5.3 Cigarette-Related Causes of Premature Mortality .............................. 114
5.4 Summary .............................................................................................. 121
6 Testing: Issues and Methods ............................................................... 123
6.1 Intent & Purpose ................................................................................. 125
6.2 Testing Issues ...................................................................................... 126
6.3 Testing Methods ................................................................................... 139
6.4 Some Test Data: Menthol as an Example .......................................... 150
6.5 Summary .............................................................................................. 161
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Evaluation of Cigarette Ingredients: Feasibility
7 Research Needs ....................................................................................... 163
7.1 Intent & Purpose ................................................................................. 165
7.2 The Chemistry and Physics of Cigarette Smoke ................................. 165
7.3 Smoke Exposure .................................................................................. 170
7.4 Adverse Effects of Smoke .................................................................. 175
7.5 Testing Issues and Methods ................................................................. 183
7.6 Summary .............................................................................................. 184
8 Conclusions ............................................................................................... 185
8.1 Primary Conclusions ............................................................................ 186
8.2 Scientific Rationale .............................................................................. 187
8.3 Details & Exceptions ........................................................................... 193
8.4 Priority Setting ..................................................................................... 195
9 Literature Citations ................................................................................. 197
10 Study Participants ..................................................................................... 269
ad hoc Expert Panel ................................................................................... 269
Life Sciences Research Office Staff .......................................................... 270
Tables
Table 3-1 Component levels of mainstream smoke from Kentucky
research cigarettes (85 mm) as measured under standard
laboratory conditions. ................................................................................... 34
Table 3-2 Numbers of substances in different chemical classes identified
in tobacco leaf and/or cigarette smoke. ....................................................... 38
Table 3-3 Chemical substances and chemical classes in the vapor phase
of mainstream smoke of cigarettes without filters. ...................................... 40
Table 3-4 Chemical substances and chemical classes in the particulate
matter of mainstream smoke of cigarettes without filters. ........................... 41
Table 3-5 Percent distribution of radioactivity in mainstream and
sidestream cigarette smokes from 14C-labeled nicotine or
n-dotriacontane (based on tobacco consumed). ........................................... 45
Table 3-6 Carcinogens in cigarette smoke. ......................................................... 56
Table 4-1 Regional deposition of tar 24 weeks after switching to
a lower tar cigarette (mean ± SE). .............................................................. 72
Table 4-2 Some estimates of inhaled tar depositon using different
exhaled particle collection methods. ............................................................. 75
Table 4-3 Human deposition of cigarette smoke components after a
two-second, 35 mL puff and inhalation or mouth-only exposure. ................. 79
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vii
Table 4-4 Percentage of inhaled particles (diameter 0.2 µm) predicted
to deposit in human lung regions by three models. ....................................... 86
Table 4-5 Comparison of exposure biomarkers in nonsmokers a
nd active smokers. ........................................................................................ 87
Table 4-6 Breath concentrations (µg/m3) pf selected smoke constituents
by smokers (S) and nonsmokers (NS) [Data given as unweighted
geometric averages]. .................................................................................... 93
Table 5-1 Summary of smoking-related deaths. ............................................... 106
Figures
Figure 3.1 The components of a typical filtered cigarette. ................................. 19
Figure 3.2 Diagram of the major processes generating cigarette
smoke during a puff. ..................................................................................... 26
Figure 3.3 Variation in temperatures (°C) inside the combustion zone
of a cigarette following one second of a 2-second puff. .............................. 27
Figure 3.4 Cumulative number of chemical substances identified in
tobacco and cigarette smoke by year. .......................................................... 37
Figure 3.5 Sales-weighted tar and nicotine values for U.S. cigarettes. .............. 47
Figure 4.1 Typical puff profiles. .......................................................................... 67
Figure 4.2 Average puff volume changes with puff number. .............................. 67
Figure 4.3 Schematic representation of the nicotine-acetylcholine
receptor. ....................................................................................................... 69
Figure 4.4 Relative deposition of particles in a lung model. ................................ 76
Figure 4.5 Pankows’s depiction of four processes for molecular
absorption. .................................................................................................... 78
Figure 4.6 Schematic diagram of cigarette smoke deposition in a
cast model of the human tracheobronchial tree. .......................................... 79
Figure 4.7a Ciliated epithelium at a tracheal bifurcation in an
unexposed rat. .............................................................................................. 84
Figure 4.7b Deciliation at a tracheal bifurcation in a rat exposed
to cigarette smoke. ...................................................................................... 84
Figure 4.8 Average benzene concentrations in breath as function of
number of cigarettes smoked. ...................................................................... 94
Figure 4.9 Schematic representation of whole body exposure of rats
to cigarette smoke. ....................................................................................... 95
Figure 6.1 Biomarker categories along the continuum from
exposure to disease. ................................................................................... 142
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viii
Evaluation of Cigarette Ingredients: Feasibility
Appendices
A. Added Ingredient Review Ad Hoc Expert Panel ....................................... 274
Added Ingredient Review Meeting Speakers ............................................ 278
LSRO Board of Directors .......................................................................... 283
LSRO Staff ................................................................................................ 284
LSRO Staff Writers ................................................................................... 288
B. Glossary ...................................................................................................... 289
C. Public and Invited Comments ..................................................................... 302
D. List of Ingredients ...................................................................................... 306
E. Definition of a Cigarette ............................................................................. 355
F. Standard Methods for Tobacco Smoke Analyses ...................................... 356
G. Added Ingredient Review Committee Closure Letter ................................ 361
H. Power Calculations .................................................................................... 362
Index ................................................................................................................ 366
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1
EXECUTIVE SUMMARY
REVIEW OF INGREDIENTS ADDED
TO CIGARETTES
Governments throughout the world do not regulate ingredients added to cigarettes.
A U.S. National Academy of Sciences (NAS) report, Clearing the Smoke, recommended testing of ingredients added to cigarettes as a public health and scientific research objective (Institute of Medicine, 2001). Clearing the Smoke also
posed this testing as a manufacturer’s responsibility. Philip Morris U.S.A., a manufacturer of cigarettes, engaged the Life Sciences Research Office (LSRO) to evaluate independently the additional health risks associated with the nontobacco ingredients of cigarettes. LSRO gave the overall project the following organization:
Phase One. Determination of the feasibility of testing
Phase Two. Establishing scientific criteria for the review
Phase Three. Reviews of specific ingredients
By added ingredients, LSRO means chemical substances directly added during
manufacturing, not indirect additives like pesticides applied to tobacco and thus,
present in cigarettes indirectly. In this report, LSRO also has focused attention on
the existing, not new, ingredients typified by the substances listed in Appendix D.
LSRO defines “feasibility” in the sense of practicality, not in the sense of measuring cost-benefit. This report of the first phase examines two questions about the
feasibility of testing ingredients added to cigarettes: (A) What objectives would
tests of ingredients added to cigarettes achieve? (B) If testing is feasible, is it also
worthwhile?
Like the NAS panel in Clearing the Smoke, LSRO believes that the primary, if not
the only objective of testing ingredients added to cigarettes should be to assure, to
the extent practical, that ingredients do not increase the premature mortality and
morbidity known to be associated with cigarette smoking. Prospective epidemiological studies established this association with cigarette smoking. However, epidemiological associations alone do not prove causation. Understanding causation
requires diverse kinds of data. For LSRO, the descriptive standard for adverse
health effects of cigarettes remains the long-term observation of human smokers.
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2
Evaluation of Cigarette Ingredients: Feasibility
Thus, the best comparison would be between smokers of cigarettes containing an
added ingredient with smokers of nearly identical cigarettes lacking this ingredient.
However, observational studies such as these present many challenges. For the
foreseeable future, the optimal public health goal will remain prevention of smoking
initiation and encouragement of smoking cessation.
Manufacturers make cigarettes from tobacco, a natural product. Burning a cigarette produces smoke. Normally a combustion aerosol, like cigarette smoke, consists of both particles and gases and also contains a mixture of many thousands of
chemical substances, some short-lived and reactive. Cigarette smoke is dynamic
in nature, changing rapidly with time, airflow, puff frequency, and type of cigarette.
Aerosol particles exchange substances with the gas phase, and their chemical
composition(s) vary over time. When humans inhale cigarette smoke, their mouths
and respiratory tract surfaces exposed to the smoke react with, and absorb, many
of the chemical substances, sometimes producing effects at the site of deposition.
Once deposited, these chemicals and/or their reactive products may be transported
to systemic tissues by circulation. Biological effects depend in part on complex
interactions between the substances and/or their components and the target tissues.
Epidemiological studies have shown that certain major causes of death, cancer,
cardiovascular diseases, and chronic obstructive pulmonary disease (COPD), contribute more than half of the premature mortality associated with cigarette smoking. All of these diseases increase in incidence as unexposed nonsmokers age, as
smokers age, as smokers consume more cigarettes, and as smokers consume cigarettes for longer periods of time. Among smokers, latency and reversibility vary by
disease. For example, the induction of lung cancer has a long period of latency, and
lung cancer incidence eventually decreases to nearly background rate after cessation of smoking. In contrast, COPD does not revert to background rate after
cessation.
The testing of the adverse effects of ingredients added to cigarettes should evaluate adverse effects associated with (A) inhalation of the ingredient or its pyrolysis
products within the hot smoke matrix, (B) inhalation of cigarette smoke constituents altered by the ingredient, and/or (C) changes in smoking behavior, so that
smoke inhalation pathways change. Tests to predict these effects must have both
internal validity (reliability, reproducibility, data quality, and positive signal-to-noise
characteristics) and external validity. An invalid test cannot eliminate a false hypothesis. Useful tests should generate data that also have external validity consistent with observed human outcomes.
In addition to observational studies of human smokers, scientists artificially generate cigarette smoke using machines. Test data reveal many interesting characteristics of the cigarette smoke matrix and the chemical substances in it, but the results of these tests do not yield satisfactory explanations of the diseases seen in
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Review of Ingredients Added to Cigarettes
3
human smokers. In addition, few test data have addressed the effects of ingredients added to cigarettes. Thus, testing these ingredients will reveal significant
research needs.
Data about the pyrolytic fate of an ingredient under cigarette smoking conditions
would be uniformly desirable for all ingredients, whether the ingredient transfers to
smoke or, pyrolyzes in whole or in part. Research strategies will be necessary to
support the testing of potential ingredient pyrolysis in burning cigarettes. In determining the feasibility of testing ingredients, the key is preparation of comparative
test cigarettes, nearly identical to a commercial product but lacking or containing
lesser, defined amounts of an ingredient. Within the precision of standard methods,
test data about the effects of combustion within the cigarette also will reveal whether
the ingredient shifts the chemical composition of other substances in the smoke
matrix.
In evaluating the feasibility of testing ingredients added to cigarettes, LSRO has
concluded that the application of every conceivable test is neither necessary nor
desirable. While research proceeds into methods of pyrolysis and transfer during
human smoking and while a scientific consensus arises about new methods, LSRO
recommends that investigators continue to include reference cigarettes, such as
those developed at the University of Kentucky for comparison, and to use the
reference smoke generation conditions specified by the Federal Trade Commission
or International Standards Organization, unless a compelling scientific rationale dictates otherwise (Davies & Vaught, 1990; Federal Trade Commission, 1967a; International Organization for Standardization, 2000). Extensive comparative data are
available for these reference cigarettes, providing internal standards.
Research needs exist on the pyrolysis and transfer of ingredients under common
cigarette smoking conditions. There is a need to know, for example, whether transfer in smoke is concentration dependent. Consensus methods for data acquisition
about pyrolysis and transfer do not exist. Research needs also exist about observation studies of changes in relative mortality and morbidity, particularly for studies to
achieve pre-specified limits of statistical power. Consensus methods for data acquisition are not available when conducting studies of comparative mortality, although smoking is an everyday activity. In addition, LSRO has not yet specified
tests that would constitute a balanced (or optimal) set. More research about general testing approaches would aid this task.
Because manufacturers already use many ingredients, setting priorities on the evaluations of these ingredients makes good sense. The need to set priorities explains
why LSRO regards an incremental approach as beneficial. A checklist of specific
tests to apply to all added ingredients is neither feasible, nor desirable. A program
of testing ingredients added to cigarettes will reduce the possibility of an additiverelated adverse human health effect, beyond the premature mortality and morbidity
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4
Evaluation of Cigarette Ingredients: Feasibility
already seen with cigarette smoking. In this regard, manufacturers should retest
their proprietary mixtures of added ingredients, in the proportions used in their brands
of cigarettes, to self-assure that their mixtures lack effects which were not seen
with individual ingredients, generated through some interaction between ingredients.
Scientists can evaluate data from balanced, diverse tests of an ingredient and identify a maximum level of the ingredient per cigarette, based on these data, or specify
what additional testing would be required to identify the level.
LSRO approaches the testing by estimating relative risk, defined as the risk of
exposure to a cigarette with the ingredient, relative to the risk of exposure to a
cigarette without the ingredient. An “unchanged relative risk” does not imply that
the underlying activity, cigarette smoking, is safer or that addition of the ingredient
has changed the risk of cigarette smoking. LSRO is not attempting to test ingredients in an effort to achieve safer cigarettes. Relative risk depends on testing paired
cigarettes that differ only in the presence or absence of an ingredient or mixture of
ingredients. The endpoint here is no change in risk.
Although the addition of ingredients to tobacco is unlikely to change significantly
the adverse health effects of cigarettes based on the magnitude of the health effects of cigarettes and the incremental mass of pyrolyzed materials contributed by
the added ingredients; guidelines for cigarette labeling and specification of excursion limits for ingredients added to cigarettes developed by an independent, outside
group of scientists will insure an objective and uniform interpretation of data and
further minimize the possibility of a contribution to adverse health effects. Overall,
LSRO concludes that testing of ingredients added to cigarettes with this objective
is both feasible and worthwhile.
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Introduction
5
2
INTRODUCTION
OUTLINE
2.1
INTENT & PURPOSE
2.2
PUBLIC HEALTH CONSIDERATIONS
2.3
FEASIBILITY
2.4
THE SCIENTIFIC REVIEW PROCESS
2.4.1 The Committee to Assess the Science Base for Tobacco
Harm Reduction of the Institute of Medicine (IOM)
2.4.2 The Added Ingredients Review Ad Hoc Expert Panel of the
Life Sciences Research Office (LSRO)
2.5
SUMMARY
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2
INTRODUCTION
2.1 INTENT & PURPOSE
The goal of this report is to assess whether non-tobacco ingredients added to
cigarettes can feasibly be tested for their toxicological effects within the appropriate
context of their intended delivery. If such testing can be accomplished, the human
health risks associated with smoking cigarettes containing specific added ingredients
can be compared to the risks associated with smoking cigarettes that lack those
ingredients. The ability to perform such a comparison could lead to significant
advances in the current scientific knowledge regarding the ability of non-tobacco
ingredients to alter the risks of human cigarette smoking. Therefore, a process
has been initiated to answer unresolved questions about the risks of added ingredients
in cigarettes and, if possible, remove doubts surrounding the safety of these
substances. Specifically, this report seeks to address two important issues:
(1) If the testing of ingredients added to cigarettes is scientifically possible,
what specific objectives do these tests achieve?
(2) If testing is feasible, is it worthwhile?
The Life Sciences Research Office (LSRO), its staff and their advisors, the Added
Ingredients Review Ad Hoc Expert Panel (referred to collectively as “LSRO” in
the remainder of this report), first addressed the feasibility of testing non-tobacco
ingredients added to cigarettes in October of 2002. Given its positive determination
of feasibility, the purpose of this Phase One report is to explain the scientific rationale
for LSRO’s conclusion. LSRO will ultimately continue this process by undertaking
two subsequent steps. In Phase Two, the scientific criteria for specific tests as
well as the optimal types of tests to employ will be presented. State-of-the-art
information about test data for each commonly used additive will also be reviewed.
In Phase Three, preferred additive testing strategies along with their scientific
rationale will be described. (See Appendix H)
This LSRO review will not address the safety of the use of tobacco products in
general, nor will it weigh the risk of human exposure to any specific commercial
tobacco product, since the aim is not to conduct a review of existing tobacco
testing programs or products. In addition, this report will not address directly
which substances in cigarette smoke cause the most harm to human health.
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Introduction
7
A potential method to address the health effects of added ingredients is to determine their relative risk. To calculate relative risk, the absolute risk of one substance
is divided by the absolute risk of another. Absolute risk is defined as an increased
probability of an adverse outcome due to exposure to a defined substance. Therefore, if no difference exists between two substances, the relative risk must equal
1.0. For example, if two groups of smokers consume identical cigarettes, the relative risk will be 1.0, since the risk of one cigarette relative to the risk of the exact
same cigarette is equivalent. Alternatively, if the absolute risk to smokers of cigarettes containing a specific ingredient (the experimental group) is divided by the
absolute risk to smokers of cigarettes without that ingredient (the control group),
with the assumption that the cigarettes are otherwise identical, the relative risk will
be 1.0, unless the ingredient induces a change in the absolute risk of smokers in the
experimental group. Therefore, risk common to both the control and experimental
cigarette are removed from consideration. Only altered risk contributed by the
added ingredient is assessed.
During its deliberations of feasibility, LSRO adopted an approach based on relative
risk. LSRO argues that a relative risk approach most effectively addresses the
health consequences of added ingredients and can be supported experimentally,
based on the ability of manufacturers to make paired experimental cigarettes nearly
identical in every aspect except for the presence and absence of a particular
ingredient. If the health effects from inhaling the smoke of a cigarette containing
a specific ingredient compared to the same cigarette without the ingredient are
adequately tested, any direct and indirect effects of the ingredient on the inhaled
smoke toxicity should be evident and scientifically defensible.
The development of reliable testing methods able to detect both direct and indirect
adverse health effects attributable to added cigarette ingredients is critically
important. Direct health effects result from exposure to an ingredient, or its pyrolysis
products as a result of cigarette smoking. Indirect health effects differ from direct
effects in that they do not result from an individual’s cigarette use, but instead from
exposure to environmental tobacco smoke. However, the general applicability of
scientific results obtained from a particular brand of cigarettes will need to be
considered carefully, since changes in the kind of tobacco, smoker behavior,
cigarette structure, or composition also may alter the health effects of the added
ingredient under investigation.
2.2 PUBLIC HEALTH CONSIDERATIONS
Cigarette smoking is associated with adverse human health effects. Whether
most non-tobacco ingredients added to cigarettes augment, diminish, or do not
change these health effects is unknown (Institute of Medicine, 2001). Scientific
advances associating cigarette smoking with increased morbidity and mortality
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8
Evaluation of Cigarette Ingredients: Feasibility
have been attributed predominantly to early research conducted within the
Framingham Heart Study sponsored by the National Heart Institute, now known
as the National Heart, Lung and Blood Institute. In 1948, this study began
investigations into the major risk factors contributing to near epidemic rates of
cardiovascular disease associated mortality. Notably, the investigators identified a
significant relationship between cigarette smoking and the average annual death
rate of a cohort of 5,209 individuals (Freund et al., 1993). Subsequent epidemiologic
studies supported an association between smoking and premature death from
cardiovascular disease, pulmonary disease, cancer, and other disorders (National
Institutes of Health, 1997).
Available data appears to underestimate global smoking-related mortality (Forey
et al., 2002). Death rates are calculated from death certificates – the raw material
for tabulation of lists of causes of death (Kircher & Anderson, 1987). To be
useful, these certificates need to be accurate, complete, and consistent. However,
different countries, and even different regions within those countries, have different
systems of assigning causes of death. In addition, autopsies are not always
performed, and in some areas of the world they are extremely rare. Although
primary care practitioners, specialists, urban and rural doctors, and other physicians,
fill out death certificates, their technical levels of expertise vary widely. Studies of
the accuracy of death certificates reported significant discrepancies between
autopsy diagnoses and entries on death certificates (Nielsen et al., 1991).
Adjustments for factors such as age make the data more useful, as do formulas
that include so-called “risk factors.” These formulas attempt to control for
confounding because many diseases have multi-factorial causes. The use of these
formulas, however, is controversial (Lee, 1996; Malarcher et al., 2000). One
complicating factor in assessing worldwide disease incidence with respect to added
ingredients in cigarettes is that certain added ingredients are used in some parts of
the world that are not used elsewhere. Therefore, global comparisons may prove
difficult.
Animal models have been developed to analyze the effects of added cigarette
ingredients. Measurements of general toxicity, expressed as mortality, inhibition of
weight gain, or other biological indicators, following chronic use or inhalation, appear
to translate to human exposure levels, as recalculated by investigators (Carmines,
2002; Heck et al., 2002; Vanscheeuwijck et al., 2002). However, the results of
animal bioassays cannot be extrapolated to human health endpoints with confidence,
thus necessitating comparison to human studies (Elcombe et al., 2002; Goodman
& Wilson, 1991).
Despite the declining prevalence of cigarette smoking over the last fifty years in
the developed world, smoking still causes substantial morbidity and contributes to a
variety of diseases. Smoking increases the risk of all leading causes of disease
among Americans and also takes an indirect toll on society in the form of subsidized
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Introduction
9
and unsubsidized health care costs, lost productivity in the workplace, and reduction
of personal wealth, as well as other human costs, which are not easily calculated.
The principle of harm reduction assumes continuation of a behavior (i.e., cigarette
smoking) and aims to lower the total adverse consequences associated with that
behavior (Institute of Medicine, 2001). Therefore, in the context of cigarette
smoking, harm reduction would seek to achieve a low enough level of a specific
substance so that no ill health effects result from the addition of the substance to
cigarettes. In this context, an ingredient-by-ingredient review will contribute to
determining the ability of added substances to change the relative risk of cigarette
smoking.
2.3 FEASIBILITY
Smoking cessation is the most feasible approach to the problem of cigarette-related
premature mortality and morbidity. Despite the well-publicized health risks caused
by tobacco, a large portion of the U.S. public continues to smoke cigarettes. Many
efforts have been made to persuade smokers to quit with varying degrees of success
(Institute of Medicine, 2001). The smoking public, however, has the right to know
whether steps have been taken to minimize their health risks, given their predilection,
and whether these steps have any scientific validity.
Cigarette smoking poses chronic health risks, but some scientific hypotheses
oversimplify the association between cigarette smoke and its adverse health effects.
Thousands of chemical substances have been identified in smoke and many systems
have been developed to measure them. However, certain experimental methods,
such as the fractionation of cigarette smoke to identify potential carcinogens, have
yielded unconvincing results, and no single component can explain the carcinogenicity
of cigarette smoke (Institute of Medicine, 2001). Adding further complication is
the fact that many measured biological activities do not equal the total activity of
the original mixture nor do they account directly for human disease, since many
biomarkers are not themselves primarily responsible for the observed increases in
morbidity and premature mortality.
Many scientific studies have investigated the association between cigarette smoking
and cancer, particularly the association between cigarette smoking and lung cancer.
The relationship between human lung cancer risk and cigarette smoking is, therefore,
well defined. However, substantial disagreement still exists over whether certain
categories of substances, such as polyaromatic hydrocarbons or nitrosamines, are
responsible for the carcinogenic effects of smoking. In order to consider additives
scientifically, LSRO determined that it must examine the impact of an additive or
some measured effect of it. This effect may arise from the amount of a specific
chemical substance in the smoke or from a biological activity of the smoke in total.
Some ingredients are expected to produce no effect on human health. A “no
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10
Evaluation of Cigarette Ingredients: Feasibility
detectable change” standard, therefore, was defined to accommodate these
situations.
This review distinguishes between premature mortality, causes-of-death, morbidity,
and other indicators of product safety. As outlined above, premature mortality and
causes-of-death are available from death certificate data. From this information
epidemiologists should be able to reconcile estimates of risk between different
studies. Such an approach makes the evaluation of scientific test data
straightforward, if difficult. Based on this information, scientists may evaluate the
ability of a test to predict overall premature mortality, or a cause-of-death, observed
as a specific increased mortality in the population. Without this evaluation, test
data are not useful for purposes of risk estimation. One caveat to the use of
epidemiological data needs to be addressed. If a single cause-of-death is exclusively
tabulated, it is impossible to determine whether all other causes-of-death change in
proportion to it. As an illustration, suppose that LSRO discovers that the addition
of a particular ingredient to cigarettes causes a small decrease in lung cancer
incidence. The result, no doubt, would be worldwide addition of this ingredient to
cigarettes. A later discovery that the same ingredient doubles cardiovascular deaths
would promptly reverse such attitudes.
LSRO is disinclined to recommend alterations in the levels of ingredients in a
product, in an effort to provide assurances, when the effects of these substances,
if any, are poorly understood. Therefore, in preparing this review, LSRO has
evaluated industrial cigarette production methods, identified the ingredients added
in the manufacture of cigarettes, evaluated biological endpoints and current
methodologies for the assessment of cigarette smoke and its risks, and considered
potential future endpoints and methodologies. LSRO has also addressed methods
to evaluate total body burden, subpopulations at risk (i.e., gender or ethnic groups),
and interactions between ingredients.
2.4 THE SCIENTIFIC REVIEW PROCESS
2.4.1 The Committee to Assess the Science Base for Tobacco
Harm Reduction of the Institute of Medicine (IOM)
In 2001, the Institute of Medicine’s (IOM’s) Committee to Assess the Science
Base for Tobacco Harm Reduction published Clearing the Smoke: The Science
Base for Tobacco Harm Reduction (Institute of Medicine, 2001). The IOM
report emphasized policy and regulatory considerations. It established a
comprehensive framework, including a set of eleven principles for the regulation
of tobacco products. Some principles in Clearing the Smoke underscore the
value of reviewing added ingredients. In referring to “potential reduced-exposure
products” (PREPs), the IOM panel wrote:
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Introduction
11
“Regulatory Principle 3. Manufacturers of PREPs should be required to conduct
appropriate toxicological testing in preclinical laboratory and animal models as
well as appropriate clinical testing in humans to support the health-related claims
associated with each product and to disclose the results of such testing to the
regulatory agency.”
“Regulatory Principle 8. All added ingredients in tobacco products, including
those already on the market, should be reported to the agency and subject to a
comprehensive toxicological review.”
The IOM report explains these two Principles in greater detail in Chapter 7 (Institute
of Medicine, 2001). The two Principles also interrelate in that they both would: (1)
require that government list ingredients or additives in tobacco products and (2)
establish the need for a structured process to assess the safety of such additives.
The Principles extend naturally to ingredients intended not to change exposure or
cigarette-related health effects. Results of the safety assessment would be reported
to the public in an organized framework. In this way the public would have a way
to verify claims about the risk of additives in tobacco products. Thus, the process
would be valuable, even if no regulatory agency exists.
The precedent for this kind of regulatory requirement would be the regulation of
food additives by the U.S. Food and Drug Administration (FDA), which began
addressing the safety of food additives with the passage of the Pure Food and
Drugs Act of 1906 (U.S. Congress, 1906). However, testing an added ingredient
in the matrix produced by the products that consumers use, is not a usual procedure
in product safety. Instead, food additives, pesticides and industrial chemicals undergo
regulatory review in relative independence from the matrix of their intended use.
For example, FDA seldom requires testing of a food additive in the matrix of a
food, although consumers may primarily experience exposure in this way. In 1938,
the Federal Food, Drug and Cosmetic Act required drug manufacturers to provide
scientific proof that new products could be safely used before putting them on the
market (U.S. Congress, 2000). As a result of 1949 hearings chaired by Rep.
James Delaney, three amendments passed, the Pesticide Amendment (1954), the
Food Additives Amendment (1958), and the Color Additive Amendments (1960)
(Janssen, 1981; National Research Council, 1987). The Delaney clause in the
Food Additives Amendment provided that FDA could not approve an additive, if it
caused cancer in man or experimental animals (Janssen, 1981). The Delaney
clause led to the establishment of compliance protocols as new regulations ensued
(Janssen, 1981).
The Center for Food Safety and Applied Nutrition (CFSAN) in FDA enforces
food additive regulations. CFSAN derives its authority from the 1958 Food Additives
Amendment. CFSAN provided guidance for regulatory compliance in 1982 by
publishing “Redbook I,” which contained guidelines for testing direct and indirect
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Evaluation of Cigarette Ingredients: Feasibility
food and color additives (U.S. Food and Drug Administration et al., 1982). As
changes occurred in safety assessment procedures, and as the body of literature
on metabolism and pharmacokinetics of food and color additives grew, CFSAN
made a revised edition of these guidelines available for public comment in 1993 in
the Draft Redbook II (U.S. Food and Drug Administration et al., 1993). Since its
publication, FDA has refined its thinking about test end points and methods. The
latest version of Redbook is now available in an electronic form as Redbook 2000:
Toxicological Principles for the Safety of Food Ingredients (U.S. Food and Drug
Administration et al., 2001), which provides guidance for a broad range of substances
added to food. It covers information for safety assessment of food ingredients
including direct food additives, color additives, Generally Recognized as Safe
(GRAS) substances, food contact substances, and constituents or impurities of
any of these (Collins et al., 1999). These guidelines describe a safety assessment
paradigm that emphasizes the identification of adverse effects and no observed
adverse effect levels (NOAEL) that are used to establish acceptable exposure
levels (Gaylor et al., 1998).
Most of the ingredients added to cigarettes have “Generally Regarded As Safe”
(GRAS) status. This means that a reasonable certainty exists that the substance
is not harmful under the intended conditions of use. GRAS status does not mean
that a substance is safe when used for any purpose in any amount. It applies to the
safety of substances directly or indirectly added to food. LSRO previously published
a report evaluating the safety assessment procedure for food ingredients (Raiten,
1999). Assurances that ingredients added to cigarettes have GRAS status are
irrelevant to the purpose of this report, as the conditions of use involve burning in a
cigarette and inhaling combustion products.
Literally interpreted, IOM’s Regulatory Principle 3 envisions that manufacturers
of tobacco product additives would conduct the same kinds of tests required of
manufacturers of food additives (Institute of Medicine, 2001). Redbook 2000
provides requirements and recommendations for safety testing. Historically,
manufacturers performed these tests on a wide variety of food additives, including
sweeteners and flavorings (Goldsmith, 2000; Munro & Kennepohl, 2001; Soni et
al., 2001; Takayama et al., 2000). Revolutions in molecular biology and transgenic
science have generated even newer methods of testing (Elespuru, 1996; MacGregor
et al., 1995; Pauluhn & Mohr, 2000; Sills et al., 2001).
LSRO views clearly differ from a literal interpretation of the two IOM passages
(above). Testing each ingredient in a standard set of Redbook tests would make
no sense, when most of the ingredients pyrolyze. Even if the pyrolysis products of
ingredients underwent testing, the data would have no use when an ingredient
undergoes complete combustion. Yet, such testing might leave crucial questions
unanswered. The key to the IOM’s advice in Regulatory Principle 3 is the term
“appropriate.” Guidelines are ideal approaches for ideal circumstances. No
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Introduction
13
regulatory agency ever intended a guideline to override case-by-case needs or
scientific judgment. Instead, an approach tailored to the circumstances of cigarette
smoking is in order.
The burning of a cigarette usually changes the nature of the added ingredient, and
the route of exposure will differ. The collection of smoke for testing purposes may
actually interfere with the chemical species relevant to the human health effects, if
it is evanescent. FDA acknowledges the possibility of this effect in Guidance for
Industry - Preparation of Premarket Notifications for Food Contact
Substances: Toxicology Recommendations (U.S. Food and Drug Administration
et al., 2002).
Section 309 of the FDA Modernization Act of 1997 amended Section 409 of the
Federal Food, Drug, and Cosmetic Act (21 U.S.C. 348), and established a procedure
for the FDA to regulate food additives that are classed as food contact substances
- substances used in the manufacturing, packaging, transporting or holding of food.
FDA states: “Some food contact substances decompose to other substances that
exert technical effects during the manufacture of the food contact substance. There
are other food contact substances that are known to decompose during processing,
in storage, and in food or food-simulating solvents. In such cases, decomposition
products of the food contact substances may be appropriate test substances for
toxicity studies” (U.S. Food and Drug Administration et al., 2002).
LSRO’s report presents a scientific rationale for a strategy to test ingredients
added to cigarettes that implements Principles 3 and 8 of the IOM report. In part,
the IOM Committee anticipated the establishment of a regulatory body within the
U.S. government that has not, subsequently, come to pass. However, many
statements in Clearing the Smoke make equally good sense without a regulatory
agency. To achieve this interpretation of the text, the reader has to parse the
document, while omitting phrases such as, “to the regulatory agency,” or “to the
agency.” Similarly, the IOM report concerned claims of harm reduction, but many
of its conclusions extend to claims of no change in effect from the addition of
ingredients.
Cigarette smoking induces premature mortality and morbidity. The IOM Committee
appeared willing to accept data other than direct observation of mortality and
morbidity, but only to the extent that other data, such as bioassays, predicted changes
in human mortality and morbidity. LSRO agrees with this interpretation. In the
context of this report, test data, other than direct observations of human smokers,
are useful to the extent that such data are prognostic of premature human mortality
and morbidity, or allow a better understanding of related disease mechanisms.
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Evaluation of Cigarette Ingredients: Feasibility
2.4.2 The Added Ingredients Review Ad Hoc Expert Panel of
the Life Sciences Research Office (LSRO)
This Phase One report, which is a scientific review, follows on, and closely agrees,
with the primary objective of the IOM report, Clearing the Smoke (Institute of
Medicine, 2001). To avoid potential misunderstandings, LSRO reiterates several
key statements from that document.
An ingredient added to a cigarette does not change the risk of adverse health
effects of smoking, if cigarette-related mortality and morbidity do not change.
Manufacturers of cigarettes with added ingredients should conduct appropriate
testing to determine whether expected cigarette-related mortality and morbidity
changes after addition of the ingredient.
Manufacturers should report all ingredients added to cigarettes, including those
already on the market, as a published list. Manufacturers should report these
data to an appropriate agency, if one exists.
All ingredients added to cigarettes, including those already on the market, should
be subject to comprehensive review of relative risk.
A structured process needs to be established to assess the relative risks of
cigarettes containing added ingredients.
To conduct an independent, third party review, LSRO selected an expert panel of
qualified scientists and developed specific review standards, criteria, and procedures.
It conducted the review in such a manner so as to prevent the influence of factors
other than scientific principles and scientific data. LSRO invited public comments.
By publishing the current and future reports of its activities, LSRO will make the
data, decisions, and deliberations of the panel available to the public.
2.5 SUMMARY
The plan of this report is first to review cigarette smoke, including its physics and
chemistry in Chapter 3, because testing the smoke matrix for changes will be
particularly feasible. Physical, chemical, and biological analysis of the smoke matrix
is intrinsically more accurate and more precise than tests of smoke exposed
recipients. If the smoke does not change, and if the ingredient does not transfer,
understanding how an ingredient could cause an effect, other than a change in
smoking behavior, will be difficult.
Chapter 4 reviews human exposure to cigarette smoke, to enable dosimetric analysis
of ingredients added to cigarettes or their pyrolysis products that transfer in smoke.
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Introduction
15
Only in this way can investigators understand whether the exposure to the substance
or its pyrolysis products would merit additional testing. If the ingredient, or a
pyrolysis product of the ingredient, transfers in smoke, estimation of exposure and
dose will become crucial to considerations of potential human health effects. If
neither the ingredient, nor any pyrolysis product of the ingredient, transfers in smoke,
but the ingredient does induce a change in the composition of the smoke matrix,
estimation of exposure and dose from the changed components may similarly become
crucial to considerations of potential human health effects, if only to reinforce that
an excursion limit can be estimated below which change in the matrix cannot be
detected.
Chapter 5 briefly reviews the adverse human health effects of cigarette smoking.
For changes induced in the smoke matrix, these outcomes are the gold standard
that other tests attempt to predict. With testing in mind, the report moves on to
Chapter 6, which reviews some principles of testing. Compliance with these
principles is a key element in making a decision about the feasibility of testing.
Chapter 6 also reviews the possible linkage of test methods and test data in part to
provide some insight into how LSRO might proceed under different circumstances
and briefly reviews existing testing procedures and includes a case study of menthol,
which transfers in smoke.
Since LSRO noted some areas where additional scientific data would remove
uncertainty from the question of feasibility, Chapter 7 reviews research needs
associated with the feasibility of testing ingredients added to cigarettes, as distinct
from testing the ingredients themselves.
The penultimate chapter, Chapter 8, summarizes LSRO’s conclusions for the
convenience of the reader. The Appendices, A-H provide supporting information
to assist readers’ in obtaining more detailed and referenced information quickly.
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3
CIGARETTES AND CIGARETTE SMOKE
OUTLINE
3.1
INTENT & PURPOSE
3.2
CIGARETTES
3.2.1 Cigarette Definition
3.2.2 Cigarette Tobaccos
3.2.3 Blends of Different Tobaccos
3.2.4 Cigarette Papers
3.2.5 Cigarette Filters
3.2.6 Ingredients
3.3
CIGARETTE SMOKE
3.3.1 Smoking
3.3.2 Types of Smoke
3.3.3 Processes in the Formation of Smoke
3.3.3.1 Combustion
3.3.3.2 Change of state
3.3.3.3 Distillation and chromatography
3.3.3.4 Pyrolysis
3.3.3.5 Pyrosynthesis
3.3.3.6 Transfer
3.3.3.7 Filtration, agglomeration, and dilution
3.3.4 Smoke Characteristics
3.3.5 Aging of Smoke
3.3.6 Sensory Evaluation of Smoke
3.3.7 Reference Cigarettes
3.3.8 Mechanical Methods of Smoke Generation
3.4
COMPONENTS OF CIGARETTE SMOKE
3.4.1 Intent of Analysis of Cigarette Smoke
3.4.2 Measurement of Some Substances in Cigarette Smoke
3.4.2.1 Tar
3.4.2.2 Nicotine
3.4.2.3 Carbon monoxide
3.4.2.4 Water
3.4.2.5 Free radicals
3.4.2.6 Metals
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17
3.4.3 Separation of Smoke
3.4.3.1 Filtration (particulate and vapor phases)
3.4.3.2 Chromatographic separation
3.4.4 Manufacturers’ Data
3.4.5 Classes of Chemical Substances
3.4.5.1 Hydrogen ion
3.4.5.2 Phenols
3.4.5.3 Pyridines
3.4.5.4 Aldehydes
3.4.6 Specific Carcinogens
3.4.6.1 Mutagenic activity
3.4.6.2 Polycyclic aromatic hydrocarbons
3.4.6.3 Nitrosamines
3.4.6.4 Interpretation of carcinogenicity and mutagenicity data
3.4.7 Other Biological Properties
3.5
SUMMARY
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CIGARETTES AND CIGARETTE SMOKE
3.1 INTENT & PURPOSE
Historically, an initial pharmacological approach to complex mixtures with important
biological properties has been the isolation of a substance or a family of related
substances responsible for the biological effect. Identification of these
pharmacologically active substances represents significant research achievements.
Often overlooked by non-scientists however, but just as valuable to the research
community, have been demonstrations of the absence of explanatory substances;
that is, demonstrations that neither a single substance nor a family of related
substances confers the important biological properties of a complex mixture. To
scientists, ruling out hypotheses is as important as substantiating them and is
necessary for scientific progress. Non-scientists, however, tend to view these
explorations as failures and undervalue them.
The presence of a single chemical substance, nicotine, explains many of the attractive
properties of cigarette smoking. However, chemical and biological analyses of
cigarette smoke have not shown that any single chemical substance or family of
chemical substances explains the variety and extent of adverse human health effects
of cigarette smoking, as observed in epidemiological studies. Chemical analyses
of cigarette smoke have documented a variable, complex mixture of many thousands
of substances. LSRO emphasizes that the investigators who established these
facts should be viewed as succeeding in their research enterprise. Besides overall
properties, such as the physical properties outlined in this chapter, quantitative
measurements of the chemical fractions or individual chemical substances
characterize cigarette smoke. These physical and chemical properties will prove
useful in testing ingredients added to cigarettes.
3.2 CIGARETTES
3.2.1 Cigarette Definition
By definition, a cigarette consists of tobacco and a paper wrapper. (See Appendix
E and Fig. 3.1, which illustrates the components of a cigarette). Although manufacturers attempt to control the qualitative and quantitative composition of cigarettes, even within a specific brand, cigarettes vary inherently, because both major
components of cigarettes arise from biological sources (CORESTA, 1991c).
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19
3.1 The components of a typical filtered cigarette.
A typical U.S. cigarette is approximately 84 mm long with a cellulose acetate filter
approximately 21 mm long. It has a tobacco rod of approximately 63 mm and a
circumference of approximately 25 mm (diameter of 8 mm). It contains tobacco
weighing approximately 0.8 grams (Norman, 1999). Despite high variation in smoke
yields from smoker behavior and cigarette properties, an average cigarette yields
ten puffs of smoke during ten minutes of smoking, approximately one, fifty milliliter
puff each minute (Djordjevic et al., 2000).
At least four physical characteristics of the tobacco rod influence smoke yields:
length, circumference, fineness of the tobacco cut, and packing density (Federal
Trade Commission, 2000b; Hoffmann & Hoffmann, 1997). Changing the length
and/or circumference (keeping the packing density of tobacco constant) will alter
the amount of tobacco burned and the amounts of smoke constituents generated
(Hoffmann & Hoffmann, 1997; Terrell & Schmeltz, 1970). Changing the resistanceto-draw will make drawing a puff of mainstream smoke more difficult. Resistanceto-draw is the amount of negative pressure applied through suction by the mouth
and lungs to the butt end of a cigarette, causing mainstream smoke to flow from
the butt end (International Organization for Standardization, 2000).
Engineering changes in cigarette structure to control airflow, such as changes in
paper permeability (higher porosity) and alterations in the number of air holes in
the filter, can modify the chemical composition of cigarette smoke.
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Evaluation of Cigarette Ingredients: Feasibility
Engineering changes modify the dilution and the concentration of the substrate for
pyrosynthesis (Baker, 1999). Engineering changes modify delivered smoke
constituents to a greater degree than changes in the composition of the tobacco
rod and its added ingredients (Norman, 1999).
3.2.2 Cigarette Tobaccos
Tobacco is the principal ingredient of cigarettes. The tobacco plant belongs to the
family Solanaceae, genus Nicotiana. Tobacco grows indigenously in the Americas
as 64 different species (Tso, 1999). The properties of any single kind of tobacco
can vary with the quality of the seed, soil, fertilizer, geographical area, weather
conditions, growing season, harvesting time, and other variables (Fisher, 1999).
U.S. cigarettes use the leaves of N. tabacum, which are classified according to
curing method and/or by geographic region where grown. Tobacco leaves are
complex biological substances that differ in content of nicotine, sugar and nitrogen,
depending not only on the growing conditions mentioned above, but also on the
type of tobacco plant, the position of the leaf on the stalk [e.g., higher leaves
produce less acidic smoke (Brunnemann & Hoffmann, 1974)], the portion of the
leaf sampled (Jenkins & McRae, 1996) and the method of curing. Differences in
curing conditions, even with the same tobacco lot, contribute to variation in the
tobacco constituents found within the cigarette. Stems, reconstituted sheet and
other manufactured products of tobacco plants also may contribute to the nicotine
content of cigarettes.
Three traditional curing methods, flue, air, and sun, yield different tobaccos (Institute
of Medicine, 2001). The curing process preserves the inherent qualities of tobacco
leaves and imparts flavor. Flue curing, which uses tightly constructed barns to
apply artificial heat to freshly harvested tobacco, best controls temperature and
humidity (Peedin, 1999). Barn temperatures begin at approximately 35oC and end
at approximately 75oC, during a five- to seven-day curing period. The main chemical
change in the first part of the curing process involves degradation of complex
carbohydrate (starch) to sugar. High dry heat applied in the second part of the
drying process stops cellular respiration and retains simple sugar content. Such
chemical changes increase aromas and decrease bitter tastes in flue cured tobacco,
relative to air cured tobacco. The nicotine content of flue cured tobacco (Virginia
tobacco) ranges from 2% to 4.5% (Fisher, 1999).
Air curing places tobacco in well-ventilated barns with little or no artificial heat
and uses ambient environmental conditions for a duration of four to eight weeks
(Jenkins, Jr. et al., 1971). During slow air-drying, cellular respiration in the leaf
continues, resulting in decreased natural starch and sugar. The main air cured
tobaccos are Burley and Maryland. Burley tobacco was originated in southern
and northern Kentucky in 1864. Today, Burley is still grown in Kentucky and
surrounding states, setting a standard for full flavor (Palmer & Pearce, 1999).
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21
Burley is less likely to extinguish while smoldering between puffs (Institute of
Medicine, 2001). The range of nicotine content of Burley is from 2.5% to 5.0%
(Fisher, 1999).
Water loss and shrinkage occur during the curing process. The moisture content
of tobacco affects the yield of nicotine and phenols in its smoke. Both yields
decrease as the moisture content increases (DeBardeleben et al., 1978).
Manufacturers control the moisture content of the tobacco leaves during processing,
primarily to control leaf pliability prior to the final cut.
In addition to the field grown tobacco leaf, manufacturers of U.S. cigarettes use
two processed tobaccos: reconstituted and expanded tobaccos. Processed tobaccos
serve as fillers to reduce tar yields (Fisher, 1999; Norman, 1999). The Federal
Trade Commission (1967a) refers to tar as the weight in grams of total particulate
matter collected on a Cambridge filter minus the weight of alkaloids, as nicotine,
and water. Reconstituted tobacco also delivers lower yields of phenols, carbon
monoxide, and benzo[a]pyrene than standard tobacco, because the methods of
manufacturing reconstituted tobacco result in a loss of a portion of the tobacco
components which apparently generate more of these substances (Fisher, 1999;
National Institutes of Health, 1996). Processed filler tobaccos (reconstituted and
expanded) add bulk to a cigarette while maintaining desirable characteristics (e.g.,
ease of draw). Their effects on the smoke depend on the type of reconstituted
tobacco and the relative amount in the blend in a cigarette.
Expanding tobacco increases filling capacity (bulk). Today, the most widely used
method of manufacturing expanded (“puffed”) tobacco uses carbon dioxide. Each
type of tobacco in a blend is separately expanded, rather than expanding the whole
blend at one time. More than 150 patents concern the tobacco expansion process.
Expanded tobacco has a lower density than other types of tobacco, allowing more
aeration, enhancing combustion (Hoffmann & Hoffmann, 2001). Norman (1999)
gives specific details of different methods of expanding tobacco.
3.2.3 Blends of Different Tobaccos
Manufacturers blend tobaccos to achieve specific tastes (Fisher, 1999; Hoffmann
& Hoffmann, 2001). Changes in the proportions of different tobaccos in the blend
contribute to variations from season to season in the final product. According to
Hoffmann and Hoffmann (2001) and Fisher (1999), the U.S. blend of tobacco is
widely recognized as the preferred cigarette by Americans and many world
markets.
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Evaluation of Cigarette Ingredients: Feasibility
3.2.4 Cigarette Papers
Cigarette papers are constructed of an inorganic filler and cellulose fiber from
either wood pulp or flax fiber (Norman, 1999). Cigarette papers also typically
contain added calcium carbonate. Among 26 American brands, permeability to air
varied (Taylor, Jr. et al., 2000). The type of paper wrapping, its weight, chemical
composition, bulk density, and porosity (air permeability of the paper) will influence
the burn rate of a cigarette and will contribute to the concentration of cigarette
smoke by modulating the inward transport of air and the outward diffusion of the
smoke during and between puffs (Hoffmann & Hoffmann, 2001). By altering
airflow, cigarette papers affect burn temperature and concentrations of mainstream
and sidestream smoke constituents (See Appendix B and Section 3.2.5, below).
Thus, the composition and engineering of cigarette paper strongly contribute to
cigarette smoke.
Highly porous papers reduce nitrogen oxides in cigarette smoke and reduce volatile
and tobacco specific N-nitrosamines in mainstream smoke (Brunnemann et al.,
1994; Brunnemann et al., 1996; Hoffmann & Hoffmann, 2001). Highly porous
cigarette paper only minimally changes the yields of tar, nicotine, and
benz(a)anthracene in the particulate phase (Hoffmann & Hoffmann, 2001).
Phosphate and citrate impregnated into paper reduce the burn rate and can reduce
smoke yields of tar and carbon monoxide (Norman, 1999; Owens, 1978).
3.2.5 Cigarette Filters
In 1995, cigarettes with filters comprised 97% of the U.S. domestic market (Federal
Trade Commission, 1997). Cellulose acetate filters presently account for nearly
the entire market in the U.S. (Norman, 1999). Mainstream smoke is the smoke
drawn through the butt end of the cigarette into the mouth as a smoker puffs on a
cigarette, while sidestream smoke is the smoke emitted directly into the air from
the burning end of the cigarette, largely during the smolder interval between puffs.
In general, filtered cigarettes have reduced mainstream smoke yields relative to
unfiltered counterparts (wt/wt) but have similar sidestream yields (Baker, 1999;
Browne et al., 1980; Chortyk & Schlotzhauer, 1989; Hoegg, 1972). According to
Hoffmann and Hoffmann (2001), cellulose acetate filters remove up to 80 percent
of the phenols in cigarette smoke and up to 75 percent of the volatile nitrosamines.
The presence of phenols (section 3.4.5.2) and nitrosamines (section 3.4.6.3) in
tobacco smoke is discussed later in this chapter.
Filter ventilation (air dilution of the mainstream draw) represents the major
engineering innovation associated with the low-tar cigarettes (Kozlowski et al.,
2001b). The vents usually consist of one or more rings of small holes along the
circumference of the filter (Burns & Benowitz, 2001; Hoffmann & Hoffmann,
2001). These perforations allow for a variable amount of air dilution of the smoke
during a puff. This dilution slows the velocity of smoke through the burning cone
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Cigarettes And Cigarette Smoke
23
of the cigarette as well as slows the flow of smoke through the tobacco rod.
Ventilation holes can become blocked with smokers’ lips, fingers or tape which
may increase the delivered levels of tar and nicotine (Kozlowski & O’Connor,
2002). When not occluded, the result of filter ventilation can be a more complete
combustion and higher retention of particulate matter in the cigarette filter. Presently,
more than 50 percent of all cigarettes marketed in the U.S. have perforated filter
tips (Benowitz, 2001; Burns et al., 2001).
3.2.6 Ingredients
Literally, a cigarette ingredient is any substance used in making a cigarette.
Cigarette ingredients vary by type and brand of cigarette but typically include
tobacco, paper wrapping, adhesives, monogram ink and filter, as well as ingredients
inherent to tobacco processing and manufacturing, and substances within or added
to each of these components. For example, filters are treated with glycerol
triacetate, to remove some of the volatile and semi-volatile compounds in mainstream
smoke (Hoffmann et al., 1995).
Legal and common definitions of cigarettes encompass tobacco and paper. With
this report LSRO is reviewing added ingredients that are any chemical substance,
other than tobacco or paper, used to make a cigarette. Paper, inks and filters as
well as chemicals used in their manufacture have been excluded from this review
and these will be addressed at a later date. Although design and engineering
features such as filter ventilation can alter the chemistry of cigarette smoke, this
report focuses on the effect of added ingredients that are chemical substances.
The term often used to describe such a chemical substance is an “additive.”
For purposes of this report, LSRO restricted considerations of added ingredients
to chemical substances currently used as ingredients in cigarettes, excluding
engineering controls, such as paper porosity. For testing purposes, an added
ingredient can be a single added chemical compound or substance, or a specific
recipe of compounds or substances, e.g., a proprietary flavor formula, or a complex
mixture, e.g., cocoa. In theory, testing of engineering changes in cigarette design,
such as paper porosity, should be amenable to the same strategy as the one LSRO
outlines in this report. However, changes in paper porosity are not familiar to
LSRO and might not change cigarette smoke in increments as expected in response
to changes in amounts of an ingredient. LSRO will defer application of the strategy
outlined here until experience accumulates with direct, existing chemical substances
added to cigarettes.
No agency of the federal government directly regulates chemical substances added
to cigarettes. In 1994, six cigarette manufacturers published a list of 599 substances
(now commonly referred to as the “599 list”) that might be added to some cigarettes
(Appendix D) (Doull et al., 1994; Select Committee on Health, 2000; Tobacco
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Evaluation of Cigarette Ingredients: Feasibility
Institute, 1994). In addition, cigarette manufacturers disclose a list of current
tobacco additives to the Department of Health and Human Services, as indicated
by the industry’s different websites (Philip Morris USA, 2002), (R.J.Reynolds
Tobacco Company, 2000), (Brown & Williamson Tobacco Corporation, 2000).
LSRO distinguishes these substances as “existing additives,” distinct from newly
arising chemical substances that a manufacturer might want to add to a particular
brand of cigarette. Most product safety laws and regulations similarly differentiate
new from existing substances (Viscusi et al., 2000). The World Health Organization,
the Province of British Columbia in Canada and the United Kingdom have issued
inquires about ingredients added to cigarettes (Government of British Columbia,
1998; United Kingdom Department of Health, 2003; World Health Organization,
2003).
Connolly and coworkers (2000) summarized the added ingredients that are used to
reduce the odor, irritation, visibility and emission of sidestream smoke. For example,
vanillin may reduce odors, aluminum sulfate may reduce irritation, calcium carbonate
may reduce the visibility of smoke, and magnesium oxide may reduce the quantity
of sidestream emissions (Connolly et al., 2000). Three major groups of ingredients
added to tobacco include: casings, humectants and flavorings.
A casing refers to a solution that is sprayed onto the whole tobacco leaf or into
which the tobacco leaf is dipped. Some examples of casings are water, sugars,
licorice, cocoa, and fruit extracts (Fisher, 1999). Manufacturers add casings to
improve “processability” of the tobacco in that the tobacco leaf is more pliable
than if the casings had not been applied. This provides more consistency in the
size and shape of the cut tobacco (Fisher, 1999). Casings also improve smoke
quality by retarding the burn rate (Fowles, 2001) and by moderating or masking the
harshness and irritation of the smoke, particularly for Burley tobacco (Fisher, 1999).
For these reasons, Burley tobaccos receive the greatest application of casings
compared to the other tobacco types in the blend of U.S. cigarettes.
Humectants retain moisture and plasticity of tobacco (Hoffmann & Hoffmann,
2001). Presently, the principal humectants added in cigarette manufacturing are
glycerol and propylene glycol. Less frequently, sorbitol and diethylene glycol are
added (Hoffmann & Hoffmann, 2001). Humectants preserve flavor compounds
and produce a less harsh tasting cigarette smoke.
Flavorings add an aroma to packaged cigarettes and improve cigarette quality by
enhancing the taste of tobacco without masking it (Fisher, 1999). Flavorings are
added near the end of cigarette processing and applied to cut tobacco in relatively
small amounts (concentrations in parts per million by weight) (Fisher, 1999).
Therefore, flavorings are not ordinarily measured with quantitative analytic methods
because the concentrations in smoke fall below detection levels and because the
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Cigarettes And Cigarette Smoke
25
complex matrices add to the difficulty of obtaining precise and accurate data (Clinton
et al., 2000; International Organization for Standardization, 2000).
Flavoring substances number in the hundreds (Fisher, 1999). Ingredients added to
cigarettes are essentially unregulated by governments in the U.S. and throughout
the world, although the World Health Organization, the Province of British Columbia
in Canada, and the United Kingdom have published inquires about the health effects
of ingredients added to cigarettes (Government of British Columbia, 1998; United
Kingdom Department of Health, 2003; World Health Organization, 2003). See
Appendix D for examples of flavorings added to tobacco in the U.S. Manufacturers
usually dissolve flavorings in ethanol, which evaporates near the end of the
manufacturing process and leaves the flavorings adhered to the tobacco (Fisher,
1999). Menthol, a common flavoring agent can be added up to approximately 2%
by weight in some mentholated cigarettes (Select Committee on Health, 2000;
Tobacco Institute, 1994). Other flavoring substances include sucrose up to 4%
(by weight), glycerol (7%) propylene glycol (2.5%), and cocoa and cocoa products
(1.5%). It is possible that added ingredients could increase the level of exposure
to combustion products compared to cigarettes without these additives.
3.3 CIGARETTE SMOKE
3.3.1 Smoking
LSRO is primarily interested in the health effects of smoking cigarettes and the
potential influence of nontobacco ingredients added to cigarettes on these health
effects. The behavior of individual smokers is highly variable, and the smoke they
inhale is relatively inaccessible to investigators (Adams, 1976; Creighton et al.,
1978; Creighton & Lewis, 1978; Höfer et al., 1992). Thus, the study of the toxic
material to which smokers are exposed is a relatively difficult scientific subject.
3.3.2 Types of Smoke
Most of a cigarette will burn to smoke and ash (Baker, 1999). Cigarette smoke is
a complex mixture of heterogeneous inorganic and organic compounds consisting
of vaporized chemicals and particulate matter suspended as an aerosol formed by
the combustion of tobacco (Sethi & Rochester, 2000).
Cigarette smoke occurs in three relatively independent forms: mainstream smoke,
sidestream smoke, and exhaled smoke. A smoker draws mainstream smoke through
the non-burning, butt end of the cigarette into the mouth while puffing on a cigarette
(Institute of Medicine, 2001). Frequent reference also is made to environmental
tobacco smoke (ETS), which is found in the air around smokers or air carried
away from smokers. ETS is composed of sidestream smoke and exhaled smoke
For internal use of Philip Morris personnel only.
26
Evaluation of Cigarette Ingredients: Feasibility
in varying proportions, greatly diluted by ambient air. The terms “passive” and
“second hand” smoke are often used interchangeably for ETS.
Smoke emitted during the interval between puffs is sidestream smoke, including
the smolder stream emitted from the butt end. The amount of sidestream smoke
produced will fluctuate during the puff interval, and the total amount of sidestream
smoke produced during the puff interval correlates with the weight of tobacco
burned during this interval (Baker, 1984). Smokers inhale all types of smoke including
mainstream smoke while puffing, sidestream smoke from the immediate breathing
zone, air mixed with some previously exhaled mainstream smoke, and even some
environmental tobacco smoke (Institute of Medicine, 2001; Sethi & Rochester,
2000).
Figure 3.2 Diagram of the major processes generating cigarette smoke during a puff. *Flameless
ignition specified by the International Organization for Standardization (2000) and the Centre de
Coopération Pour Les Recherches Scientifiques Relative au Tobac (CORESTA) (1991a).
3.3.3 Processes in the Formation of Cigarette Smoke
The formation of cigarette smoke is a dynamic process. Many processes contribute
to the generation of smoke, including combustion, pyrolysis, pyrosynthesis, direct
transfer, distillation, filtration and dilution (Fig. 3.2). Jenkins and McRae (1996)
reviewed the last half-century of research on the formation of smoke.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
27
3.3.3.1 Combustion
After ignition of the end of the tobacco rod, a process of combustion ensues,
actively driven by heat and the oxygen from the ambient air the smoker draws
through the cigarette. A burning cone forms within the cylinder of a lit cigarette.
Atmospheric conditions and suction applied by the smoker influence the burning.
Cigarette structure and ingredients, such as sodium citrate added to the cigarette
paper, also affect the burn rate.
The burn region lacks oxygen, temperatures range up to 950°C, and heating rates
go as high as 500 degrees per second (Baker, 1975; Baker, 1999). Maximum heat
production occurs at the tip (ash) side of the paper burn line while the smoker
puffs (Fig. 3.3). Using a thermocouple inserted to a depth of approximately 4 mm
into the cigarette at 1.5 cm from the tip end before lighting, Langer and coworkers
(1989) measured burn temperatures until the combustion zone passed the
thermocouple. The peak temperature of burn ranged from 520°C to 620°C and
averaged 575 ± 30°C for 17 different U.S. cigarettes.
Figure 3.3 Variation in temperatures (°C) inside the combustion zone of a cigarette following one
second of a 2-second puff. The estimated direction of vapor flow pattern is shown. Reprinted
with permission from (Baker, 1975). Reproduced with permission of Humana Press.
The region of combustion continually advances toward its source of fuel, unburned
tobacco. Complete combustion generates carbon dioxide, carbon monoxide, nitric
oxide, moisture (water), and ash. Ash is primarily an inorganic mineral residue and
amounts to an average of 116 ± 16 mg/cigarette (average of 17 U.S. brands) or
For internal use of Philip Morris personnel only.
28
Evaluation of Cigarette Ingredients: Feasibility
approximately 14-15% of the original weight of the tobacco, when burned at 550°C
(Langer et al., 1989). An invisible plume of vapor rises from the combustion zone,
4 to 8 mm from the tip side of the paper burn line (Baker, 1999), which can mix
with the visible aerosol plume arising from the pyrolysis-distillation zone. (See
below.) The rest of the vapor generated in the combustion zone is drawn deeper
into the tobacco rod.
3.3.3.2 Change of state
As a cigarette burns, many chemical substances present in the solid state in tobacco
liquefy and/or turn to gas, as the temperature climbs. As these gases pass down
the column of tobacco and cool, they re-liquefy and re-solidify on the surface of
the tobacco. Changes of state advance ahead of the heat generating combustion
zone, moving down the burning tobacco rod. Thus, the materials undergoing
combustion change, as the region of combustion advances.
3.3.3.3 Distillation and chromatography
Distillation is a physical process. Application, then removal, of heat separates a
liquid mixture of two or more chemical compounds. The application of heat
selectively vaporizes the compounds according to their boiling points. Subsequent
removal of heat condenses the vapor back into a liquid.
Distillation in a smoked cigarette occurs as the heated smoke moves down the
tobacco column. When highly volatile, low-boiling point substances enter the vapor
phase, they move ahead of the burning zone. Ambient air enters the cigarette
adjacent to the butt side of the paper burn line, cooling the internal temperature of
the cigarette. The gas containing suspension of fine liquid and solid particles cools,
condensing into an aerosol. Some vaporized compounds become trapped in aerosol
droplets. Examples of substances distilled out of tobacco while cigarettes undergo
smoking include saturated and unsaturated aliphatic and terpenoid hydrocarbons,
lactones, esters, carbonyls, alcohols, sterols, alkaloids, amino acids and aliphatic
amines (Green, 1977; Hecht et al., 1977; Stedman, 1968).
Chromatography is a related physical process through which one chemical
compound passes down the tobacco rod with a different velocity than a second,
different chemical compound. In either gas or liquid state, the two compounds
absorb to tobacco differentially and the hot gas passing along the column acts as a
carrier, moving the two compounds along. Progression of the pyrolysis-distillation
zone and paper burn line will eventually destroy the two substances by combustion
or pyrolysis, or displace them through volatilization.
Distillation and chromatography play roles in changing the chemical composition
of cigarette smoke as rod length decreases during the smoking of a cigarette.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
29
3.3.3.4 Pyrolysis
A pyrolysis-distillation zone forms just behind the combustion area, on the butt side
of the paper burn line (Baker, 1975; Baker, 1981; Baker, 1999). The majority of
organic smoke products form in the pyrolysis-distillation zone (Dube & Green,
1982; Jenkins & McRae, 1996). High temperatures in the pyrolysis-distillation
zone thermally decompose tobacco. However, pyrolysis is not a synonym for
combustion, as it includes nonoxidative processes. Nonvolatile substances in tobacco
which decompose through pyrolytic degradation include sugars, polysaccharides,
polyphenols, and proteins (Baker, 1999).
Pyrolysis and combustion will completely eliminate some substances found in
cigarettes, whereas other highly volatile and thermally stable substances (e.g.,
menthol or n-dotriacontane) can transfer from the tobacco into smoke (Baker,
1999; Stedman, 1968; Wakeman, 1972). Small amounts of substances of poor
volatility and/or high molecular weight in hot, fine particles of tobacco and ash,
may propel into the smoke stream without pyrolysis (Baker, 1999). Such entrainment
of solids can include inorganic salts, metals, sterols, carbohydrates, and/or pigments
(Jenkins & McRae, 1996; Stedman, 1968).
Dube and Green (1982) estimated that analytical chemists have identified 1135
compounds which could transfer unchanged from tobacco to smoke. That is,
approximately one-third of the chemically identified substances in mechanically
generated cigarette smoke might escape pyrolysis and transfer directly from tobacco
into smoke (Green, 1977). A recent pyrolysis study of 291 added ingredients
predicted that almost two thirds of these ingredients would transfer into the
mainstream smoke 95% intact (Baker & Bishop, 2004)).
3.3.3.5 Pyrosynthesis
Pyrosynthesis involves the formation of new chemical substances in smoke through
recombination of pyrolysis fragments. Pyrolysis and pyrosynthesis occur
simultaneously. Chemically reactive fragments, including free radicals, subsequently
recombine, particularly in the hot gas phase within the burning cigarette. These
reactions create new chemical substances, which include, pyridines, indoles, nitriles,
aromatic amines, furans, phenols and carbonyls (Baker, 1987; Brunnemann &
Hoffmann, 1982; Chortyk & Scholtzhauer, 1973; Hecht et al., 1977; Jenkins, Jr. et
al., 1975; Jenkins, Jr., 1990; Scholtzhauer & Chortyk, 1987). If these new
substances have sufficiently high boiling points, they may liquefy or even solidify
onto tobacco between the pyrolysis-distillation region and the butt end. Few
secondary oxidation products form in the migrating pyrolysis-distillation region,
because concentrations of oxygen are low.
The chemical reactions that occur during pyrolysis and pyrosynthesis do not produce
random chemical fragmentation. Instead, the patterns of bond breaking and bond
For internal use of Philip Morris personnel only.
30
Evaluation of Cigarette Ingredients: Feasibility
formation involve distinctive patterns dictated by thermodynamic considerations,
analogous to the uniform patterns obtained when chemical substances undergo
fragmentation during mass spectrometry. Some of these reactions and the
associated, overall considerations are theoretically predictable. The RiceRamsperger-Kassel-Marcus (RRKM) theory describes chemical energy transfer
and activation under the steady state and non-steady state conditions important to
the unimolecular bond breaking and formation events, such as those involved in
pyrolysis and pyrosynthesis (Davis et al., 1999; Kiefer et al., 1997; Schranz &
Sewell, 1996).
3.3.3.6 Transfer
The physical process of transfer of a chemical substance in smoke remains poorly
characterized. In principle, a volatile substance that resists pyrolysis will have the
optimal thermodynamic properties to transfer in cigarette smoke. Thus,
transferability seems highly predictable. As the heat generating combustion zone
moves down the burning tobacco rod, a substance that resists pyrolysis will undergo
changes of state, become a gas, and move with the plume of smoke. Eventually,
such substances will enrich mainstream smoke in higher concentrations as it exits
the end of the cigarette.
3.3.3.7 Filtration, agglomeration, and dilution
Liquid smoke particles (~20 per cent water) vary in size from < 0.1 to 1.0 µm.
Particles less than 0.1 µm in diameter attach either to larger particles in the aerosol
or to the surface of tobacco (Institute of Medicine, 2001). Tobacco also filters out
particles greater than approximately 1 µm in diameter from the smoke stream
(Institute of Medicine, 2001). Thus, the length of unburned cigarette will change
smoke characteristics. Fibers in the filter at the butt end of the cigarette also
remove aerosol particles from the smoke stream. Impaction removes particles,
when a particle is to large to follow the air flow, as it bends around the fibers in the
filter, by diffusion. The filter fibers capture smaller particles, by interception as the
particles in the smoke stream rub against the filter fibers (Jenkins & McRae, 1996).
Gaseous components of smoke may exchange into the particulate phase as it
migrates through the rod. Air diffusing into the filter, before the smoker inhales it,
dilutes the smoke.
Smoke yields vary with the length of the remaining, unburned cigarette. As the
tobacco burns, and as cigarette length decreases, fewer smoke constituents escape
through the paper wrapper, and fewer constituents filter from the smoke into the
tobacco rod. Thus, a cigarette delivers relatively more of its components, such as
nicotine, in mainstream smoke per cm, near the butt end, as the burning tobacco
rod shortens (Baker, 1999).
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
31
3.3.4 Smoke Characteristics
Most of the knowledge of cigarette smoke comes from studies of mechanically
generated cigarette smoke that reflect the composition and properties of the smoke
as it is exposed to human tissues during smoking. Thus, scientists interpret the
database about cigarette smoke with caution. Whole cigarette smoke is an aerosol,
which scientists usually partition into two phases, a vapor (gas) phase and a
particulate phase (Pankow, 2001). Molecules in the smoke distribute between the
vapor and particulate phases. Each substance seeks a state of vapor/particulate
equilibrium (Pankow, 2001).
A portion of the aerosol rises out of the tobacco rod, cooling from ~ 600°C to
nearly ambient temperature in a few milliseconds (Baker, 1999; Institute of Medicine,
2001). A visible plume forms from the butt side of the burn line in areas where the
temperature has cooled to less than 150°C (Baker & Robinson, 1990). Less volatile
components quickly condense into airborne droplets or particles. Another fraction
of the aerosol is drawn through the tobacco rod. Some particles in this portion of
the aerosol condense on the surface of tobacco. These particles re-volatilize,
pyrolyse or combust during the next puff, as the burning zone reaches the zone of
condensation. A smoker’s inhalation draws the remaining aerosol through the
cigarette, away from the burning zone.
The conditions that generate sidestream smoke differ from those producing
mainstream smoke in at least two ways: (1) natural convection drives the air flow
that results in sidestream smoke, and (2) the burning zone temperature is nearly
800°C as the cigarette smolders during the puff interval, compared with the ~
950°C of the burning zone during puffs (Baker, 1999).
The total volume of mainstream smoke generated from approximately 10 puffs
(50 mL each) of a typical cigarette, is approximately 500 mL; of which about 350
mL (~70%) is vapor (Djordjevic et al., 2000). The vapor phase consists mostly of
common atmospheric gases (e.g., oxygen, nitrogen, carbon dioxide) that constitute
90 to 96% of its total weight (Pankow, 2001).
The particulate phase of smoke consists largely of non-aqueous (~10% water)
liquid droplets containing a heterogenous mixture of condensed organic compounds
(e.g., nicotine or benzofurans). These particulate droplets suspend in the vapor
phase. Together the two phases form an aerosol (Pankow, 2001). The molecular
weights of the chemical compounds in the particulate phase usually exceed 200
(Institute of Medicine, 2001). The concentrations of particles in a smoker’s puff
range from 0.3 to 3.0 x 109 per cm3 (Martonen, 1992; Sethi & Rochester, 2000).
The estimated densities of particles in mainstream and sidestream smokes are
approximately 1.0 g/cm3 (McRae, 1990). The amount of total particulate matter in
smoke varies. Rustemeier and coworkers (2002) reported that added ingredients
For internal use of Philip Morris personnel only.
32
Evaluation of Cigarette Ingredients: Feasibility
commonly used in cigarette manufacturing increase total particulate matter by 13
to 28 percent.
Ogg (1964) reported data from an early, collaborative study of twelve laboratories
concerning the determination of particulate matter collected from smoke of
commercial cigarettes with and without filters. Smoke generated under conditions
similar to those recommended by the Federal Trade Commission (FTC) generated
an average of 32 ± 1.1 mg of particulate matter for non-filtered cigarettes and 24.6
± 0.8 mg for filtered cigarettes.
3.3.5 Aging of Smoke
The aerosol of cigarette smoke changes both chemically and physically over time
as it migrates from the combustion zone, through the tobacco rod, and eventually
into the ambient air. Baker (1999) has reviewed changes in the composition of
smoke during aging. Cigarette smoke contains many highly reactive chemical
fragments, such as beta-carbolines and free radicals (Totsuka et al., 1999). Synthesis
of substances with decreased chemical reactivity continues, as time and distance
from tobacco combustion increases. Thus, the age of cigarette smoke is a crucial
parameter in experimental studies.
In an early effort to measure the chemical changes of mainstream smoke, Vilcins
and Lephardt (1975) scanned undiluted mainstream smoke immediately after it
exited a mechanically smoked cigarette with infrared spectroscopy. They scanned
every 3.5 seconds for three minutes. An initial and rapid decrease in the
concentration of nitric oxide (NO) occurred, accompanied by a rapid build up in
nitrogen dioxide (NO2), which reached a maximum after approximately one minute
and slowly decreased. Oxygen in ambient air mixed with the smoke and converted
nitric oxide to nitrogen dioxide. Similar kinetic changes in smoke chemistry were
reported by Cueto and Pryor (1994).
As smoke forms, components of the smoke randomly collide, at all ranges of physical
observation, from molecular to macroscopic. Larger components coalesce into
droplets ranging in size from <0.1 to 1.0 µm in diameter, with ~45% of the droplet
particles in the range of 0.15 to 0.20 µm (Baker, 1999). These droplets further
coalesce. Aerosols, including cigarette smoke, generally do not attain a stable
equilibrium of particle size and mass concentration. Volatile and semi-volatile
substances evaporate out of smoke particles as cigarette smoke ages (Baker, 1999).
Further decay and elimination of the smoke aerosol is caused by eddy, Brownian
diffusion and sedimentation. With additional aging (catabolic chemical
transformations) and continuing dilution, the smoke constituents disperse and
eventually dissipate into (or precipitate out of) the atmosphere (Hollander & Stober,
1986).
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
33
3.3.6 Sensory Evaluation of Smoke
Judging from the published literature, the perception of smoke by human smokers
remains poorly understood. If smokers decline cigarettes, the effects of changes
induced by alterations in ingredients or structure become irrelevant. Cigarettes
made only with Virginia tobacco (flue cured; high natural sugar content of 15.5%
by weight) may be more acidic than those made with blended tobaccos. Smokers
of American blended cigarettes perceived them as having an “unbalanced” flavor
(Leffingwell, 1999). Schievelbein (1982) demonstrated that cigarettes made with
Virginia tobacco yielded higher amounts of nicotine and produced higher blood
nicotine concentrations in intubated dogs than cigarettes made with black type
tobacco or a German blend of tobacco, the latter thought more alkaline.
Based on available evidence, nicotine yield influences the sensation of impact
perceived by the smoker. Kochhar and Warburton (1990) determined that cigarettes
with higher nicotine yields (1.7 mg) associated with greater mouth, throat and
chest sensations than lower yielding cigarettes (0.8 mg). For each type of cigarette,
impact increased puff-by-puff over the duration of smoking. Bevan (1991)
determined that the addition of 5 mg of naturally occurring S-isomer of nicotine to
denicotinized cigarettes resulted in a sensation of “impact” for two of three smokers
compared to cigarettes spiked with other alkaloids. Leffingwell (1999) and Pankow
(2001) suggested that the unprotonated (freebase) form of nicotine associated
with sensory and physiological perceptions of harshness and intensity. However,
under experimental conditions, Burch and coworkers (1993) found no statistical
difference in symptoms of cough, throat burning or salivation among ten volunteers
who inhaled aerosols varying in pH from 5.6 to 11 and containing 0.484 mg of
nicotine per puff, delivered every 30 seconds over a five minute period (10 puffs
total). The particle size of the aerosol in this study exceeded that in cigarette
smoke.
3.3.7 Reference Cigarettes
In 1968, the Scientific Advisory Board of the Council for Tobacco Research asked
the Industry Technical Committee to arrange for the development of a reference
cigarette (Davies & Vaught, 1990). In response, scientists at the Tobacco and
Health Research Institute (THRI) of the University of Kentucky developed several
reference cigarettes, beginning with 1R1 in 1969 (Table 3-1) (Coggins, 2000; Davies
& Vaught, 1990). The latest reference cigarette that THRI designed, the 2R4F,
yields tar and nicotine near the sales-weighted average of cigarettes currently sold
in the U.S. (Tobacco and Health Research Institute, 2002). Table 3-1 provides the
yields of tar, nicotine and carbon monoxide of various reference cigarettes. Other
reference cigarettes have been developed (e.g., low alkaloid yield) (Davies &
Vaught, 1990; World Health Organization, 1986).
For internal use of Philip Morris personnel only.
For internal use of Philip Morris personnel only.
d
c
b
a
Year
introduced
1969
1974
1974
1974
1975
1969
1969
1969
1983
1989
2002
23 mm
34.3
36.8
36.4
31.1
34.
29.0
35.5
-
30 mm
30.1
32.9
23.4
31.8
26.8
29.5
25.8
29.8
9.2d
1.67d
9.9d
Butt length
23 mm
2.16
2.45
0.48
0.27
2.08
1.28
2.61
30 mm
1.98
2.19
1.74
0.42
0.24
1.75
1.14
2.20
0.80d
0.16d
0.81d
23 mm
20.8
25.1
25.2
19.4
21.4
21.9
19.6
-
30 mm
18.1
22.2
22.0
22.8
16.9
17.1
18.2
17.4
11.6d
2.95d
12.3d
Carbon monoxide
(mg/cigarette)
_________________
Data from Davies and Vaught (1990), International Agency for Research in Cancer, Table 9 (World Health
Organization, 1986) and Tobacco and Health Research Institute (2002).
No longer available; codes designate the run, series, blend and whether or not the cigarette had a filter (F)
(e.g., 1R1 indicates first run, R series, blend 1, without a filter).
Equivalent to the original 1A1, produced in 1969 and no longer available; 4A1 was the fourth run in this low
alkaloid series, produced in 1990.
35-mm butt length.
Kentucky
series
1R1b
2R1b
2R1F b
2A1c
3A1
1A2 b
1A3 b
1A4 b
1R4F
1R5F
2R4F
Nicotine
(mg/cigarette)
__________________
Tar
(mg/cigarette)
________________
Table 3-1. Component levels of mainstream smoke from Kentucky research cigarettes
(85 mm) as measured under standard laboratory conditions.a
34
Evaluation of Cigarette Ingredients: Feasibility
Cigarettes And Cigarette Smoke
35
While the sales-weighted tar was 12 mg in 1998, the range for 1294 domestic
brands analyzed was less than 0.5 to 27 mg per cigarette and 1 to 26 mg per
cigarette for 26 brands of U.S. cigarettes analyzed one year later (Federal Trade
Commission, 2000a) (Gray & Boyle, 2000) (Taylor, Jr. et al., 2000). Tar yields
from cigarettes vary widely, and exposures should vary commensurately.
The scientific community uses standardized cigarettes to generate smoke for
research, to obtain standardized materials for inter-laboratory comparisons, and to
validate analytical methods. Analysis of the smoke from a reference cigarette in
parallel with an experimental cigarette, which is the object of study, serves as an
internal standard to the study. The FTC monitors cigarettes of known yields for
tar, nicotine and carbon monoxide in four or more of twenty ports of their automated
smoking machine to verify its proper function (National Institutes of Health, 1996).
Inclusion of reference cigarette data would shed light on the precision and accuracy
of the methods used, when chemical composition of cigarette smoke is tested.
Descriptions of smoke composition in the literature typically refer to “plain, unfiltered
cigarettes of various types” or “high tar” or “low tar” cigarettes, without
accompanying indications that the investigators used reference cigarettes (Baker,
1999; World Health Organization, 1986). Quantitative data exist for the tar, nicotine
and carbon monoxide contents of mainstream smokes generated with reference
cigarettes under standard U.S. Federal Trade Commission (FTC) procedures
(1967a). However, U.S. consumers smoke many brands of cigarettes (e.g., 1294
in 1998)(Federal Trade Commission, 2000a). Care should be taken to select an
appropriate reference cigarette. Reference cigarettes made decades ago do not
necessarily represent cigarettes manufactured today.
3.3.8 Mechanical Methods of Smoke Generation
Automated smoking machines usefully improve consistency in generating smokes
from cigarettes and make the smokes accessible to study. One early standard,
promulgated by the FTC, acquired regulatory status. LSRO recommends a single
smoke generation protocol be used throughout a study for consistency between
experiments. The protocol should deliver smoke components in sufficient quantity,
not deliver smoke so diluted as to be below the limits of detection of current assays.
One crucial matter of interpretation is that machines generate FTC protocol cigarette
smoke that differs in composition from the smoke inhaled by a typical smoker.
Many experimental studies use mainstream smoke obtained by drawing air through
cigarettes, either in a pulsatile or a continuous manner, although human smokers
inhale a mixture of mostly mainstream smoke and variable amounts of sidestream
smoke (Benowitz et al., 1983; Benowitz, 2001; Guerin, 1996). Special chambers
to generate sidestream smoke were developed by Brunnemann and Hoffmann
(1974; World Health Organization, 1986). Klus (1990) also described devices to
For internal use of Philip Morris personnel only.
36
Evaluation of Cigarette Ingredients: Feasibility
collect sidestream cigarette smoke. Witschi and coworkers (1997b) used an experimental mixture of sidestream and mainstream smoke in a mouse lung adenoma model.
Private standard setting organizations also have developed specifications for cigarette
smoking machines (CORESTA, 1991a; International Organization for
Standardization, 2000). Baumgartner and Coggins (1980) described older models
of smoking machines. Jaeger (2002) reviewed the functioning of a modern smoking
machine. Computers now control the functions of smoking machines. A puff
profile graphically represents the rate of mainstream smoke flow, plotted as a
function of time during the puff (International Organization for Standardization,
2000). Puff recorders have been designed to measure the smoking patterns of
human smokers (Bentrovato et al., 1995; Djordjevic et al., 1995; Puustinen et al.,
1987). This approach allows the drawing of each puff and smoking of each cigarette
in a manner similar to human smoking (Bentrovato et al., 1995; Creighton et al.,
1978; Puustinen et al., 1987). (See Chapter 4.)
3.4 COMPONENTS OF CIGARETTE SMOKE
3.4.1 Intent of Analysis of Cigarette Smoke
Throughout this report, LSRO refers to the “chemical matrix,” or the “smoke matrix,”
as a shorthand way of communicating the concept of cigarette smoke as a complex
mixture of many chemical substances, which changes with time. Scientists collect
smoke from cigarettes (mainstream smokes and/or various smoke fractions) to
assure consistency in manufacturing, to compare brands, especially for tar, nicotine
and carbon monoxide, and to understand the effects of the cigarettes (Dube &
Green, 1982; Kozlowski et al., 2001b). Many research studies test the effects of
smoke and smoke constituents on parameters of health outcomes. Standardized
methods of testing cigarette smoke are also important for the accuracy and
reproducibility of industrial and research studies.
3.4.2 Measurement of Substances in Cigarette Smoke
Various factors alter the chemical composition of mainstream smoke. Bright tobacco
(flue-cured) produces less nicotine and more benzo[a]pyrene in smoke than Burley
(air-cured) tobacco (Gori, 1980). Cigarettes with filters have lower mainstream
smoke yields than unfiltered cigarettes but may have similar sidestream yields
(Baker, 1999; Browne et al., 1980; Chortyk & Schlotzhauer, 1989; Hoegg, 1972).
Therefore, most authors include ranges, instead of median or mean values, in surveys
of smoke constituents (Baker, 1999; Davies & Vaught, 1990; Hoffmann &
Hoffmann, 2001). Concentrations of various substances in cigarette smoke can
vary widely (reportedly threefold to twenty-fold) between brands (Gray & Boyle,
2002). In addition, the yields of constituents will vary with the method of smoking
the cigarette.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
37
Reviewers frequently refer to the smoke composition data as “approximate” or
“typical” (Institute of Medicine, 2001; World Health Organization, 1986). Most of
the data in this section came from model studies, not direct measurements of human
smoking, which adds uncertainty to interpretations that attempt to relate the
composition of cigarette smoke to the delivery of chemical constituents to the
human respiratory tract.
The chemical characteristics of smoke differ from the chemical characteristics of
unburned tobacco. Roberts (1988) identified more than 25 classes of chemical
substances in unburned tobacco and cigarette smoke, which he categorized by
functional groups. Hoffmann and coworkers (2001) added N-nitrosamines to
Robert’s list (1988). Table 3-2 identifies classes of compounds detected in tobacco
leaf, cigarette smoke, or both. Gudzinowicz and Gudzinowicz (1980) also listed
chemical substances detected in tobacco leaf and cigarette smoke.
The number of chemical substances identified in tobacco and cigarette smoke has
increased rapidly over the last 30 years (Fig 3.4) (Green & Rodgman, 1996). Dube
and Green (1982) found 1414 substances in the tobacco leaf pyrolysed during
smoking and not transferred to smoke. They also found that cigarette smoke
contained 3875 substances. Of these substances, an estimated 1135 transferred
directly with the smoke. These substances did not pyrolyse during smoking. They
estimated that 2740 substances occurred uniquely in smoke that they did not detect
in tobacco. These components apparently formed through pyrolytic degradation
of tobacco and/or subsequent pyrosynthesis. They did not report the types of
cigarettes smoked and the methods used to generate and analyze the smoke. Later,
Green and Rodgman (1996) estimated approximately 4800 chemical substances in
cigarette smoke. (Fig. 3.4)
Figure 3.4 Cumulative number of chemical substances identified in tobacco and cigarette smoke by
year. Reprinted with permission and modified from (Green & Rodgman, 1996).
For internal use of Philip Morris personnel only.
38
Evaluation of Cigarette Ingredients: Feasibility
Table 3-2. Numbers of substances in different chemical classes identified
in tobacco leaf and /or cigarette smoke.a
Chemical category
•Functional Groups
Carboxylic acids
Amino acids
Lactones
Esters
Amides and imides
Anhydrides
Aldehydes
Carbohydrates
Nitriles
Ketones
Alcohols
Phenols
Amines
N-nitrosaminesb
Sulfur compounds
N-Heterocyclics
Pyridines
Pyrroles and indoles
Pyrazines
Non-aromatics
Polycyclic aromatics
Other heterocyclicss
Ethers
Hydrocarbons
Saturated aliphatics
Unsaturated aliphatics
Monocyclic aromatics
Polycyclic aromatics
•Pesticidesb
•Miscellaneous
•Inorganic and metallic
a
b
# in both
leaf and
smoke
Additional # in
leaf
Additional #
in smoke
Total #
detected
140
16
39
314
32
4
48
12
4
122
69
40
37
19
2
450
95
129
529
205
10
111
138
4
348
334
58
65
23
3
69
18
135
456
227
10
106
30
101
461
157
188
150
18
37
659
129
303
1299
464
24
265
174
109
931
560
286
252
60
42
46
3
18
7
0
2
15
63
9
21
13
1
4
53
324
88
55
43
36
50
88
433
100
94
63
37
56
156
44
10
25
35
25
19
69
58
38
33
55
28
112
105
113
178
138
317
25
110
111
215
226
196
407
78
241
285
Modified from Roberts (1988).
Two additional groups, N-nitrosamines and pesticides, added by Hoffmann et al. (2001).
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
39
Hoffmann and coworkers (2001) identified 48 major groups of chemical compounds
in the vapor phase of mainstream smoke from cigarettes without filters. The data
in Table 3-3 provide ranges that illustrate the variation in chemical composition of
the vapor phase, assuming accurate analytical methods and absolute separation of
the two phases by filtration. Table 3-4 lists the major constituents in the particulate
matter phase of mainstream smoke of unfiltered cigarettes, as originally reported
by Hoffmann and coworkers (2001). Undiluted sidestream smoke contains 30-to90% more particulate matter, 2-to-4 times more nicotine, 20-to-30 fold more aromatic
amines, and more trace metals than mainstream smoke (Klus, 1990; US Consumer
Product Safety Commission, 1993).
3.4.2.1 Tar
The FTC (1967a) refers to tar as the weight in grams of the total particulate
matter collected on a Cambridge filter minus the weight of alkaloids, as nicotine,
and water. Cigarette smoke aerosols typically contain particulate matter which is
approximately 7% nicotine, 10% water, and 83% tar (Pankow, 2001). Tar is a
heterogenous condensate of myriad substances filtered from smoke. Under FTC
definitions, a cigarette will generate 10 puffs of 35 ml, which in turn typically yields
40,000 to 60,000 mg/m3 of tar.
3.4.2.2 Nicotine
Analysts estimate nicotine in the particulate phase from the measured amount of
extracted particulate matter injected into a gas chromatograph. The resulting nicotine
peak is compared against a standard curve. The gas chromatographic method for
nicotine was modified from the method of Wagner and Thaggard (Federal Trade
Commission, 1979; Federal Trade Commission, 1980). Earlier data, such as the
widely quoted review by Dube and Green (1982), reflected the composition of
smoke from typical cigarettes used in the early 1980’s, which had higher levels of
nicotine (Institute of Medicine, 2001). Baker (1999) cited the yields of mainstream
smoke from 17 reports published between 1966 and 1990. His compilation of data
reflected the chemical compositions of unfiltered cigarettes. Baker (1999) found
a threefold range of values for nicotine. Nicotine accounts for 90% or more of the
alkaloid fractions of cigarette smoke (Gudzinowicz & Gudzinowicz, 1980).
Manufacturers control the tobacco blend and cigarette design (porosity of papers
and filters to yield target concentrations of tar and nicotine (Fisher, 1999; Fowles,
2001). Cigarette filters alter the amount of nicotine in smoke. Ogg (1964) reported
data from an early collaborative study of 12 laboratories concerning the
determination of nicotine collected from smoke of commercial cigarettes with and
without filters. Smoke, generated under conditions similar to those of the FTC,
produced an average of 1.57 ± 0.06 mg nicotine for unfiltered cigarettes and 1.26
± 0.54 mg for filtered cigarettes. Perforation of the filter with ventilation holes
further alters nicotine yields. Browne and coworkers (1980) determined that nicotine
For internal use of Philip Morris personnel only.
40
Evaluation of Cigarette Ingredients: Feasibility
Table 3-3. Chemical substances and chemical classes in the vapor phase of
mainstream smoke of cigarettes without filters.a
Constituent
Yield/cigarette smoked
(% weight of mainstream smoke)b
Nitrogen
280-320 mg (56 - 64%)
Oxygen
50-70 mg (11-14%)
Carbon Dioxide
45-65 mg (9-13%)
Carbon Monoxide
14-23 mg (2.8 – 4.6%)
Water
7-12 mg (1.4-2.4%)
Argon
5 mg (1.0%)
Hydrogen
0.5-1.0 mg
Ammonia
10-130 µg
Nitrogen oxides (NOx)
100-600 µg
Hydrogen cyanide
400-500 µg
Hydrogen sulfide
20-90 µg
Methane
1.0-2.0 mg
Other volatile alkanes (20)
1.0-1.6 mgc
Volatile alkenes (16)
0.4-0.5 mg
Isoprene
0.2-0.4 mg
Butadiene
25-40 µg
Acetylene
20-35 µg
Benzene
12-50 µg
Toluene
20-60 µg
Styrene
10 µg
Other volatile aromatic hydrocarbons (29)
15-30 µg
Formic acid
200-600 µg
Acetic acid
300-1700 µg
Propionic acid
100-300 µg
Methyl formate
20-30 µg
Other volatile acids (6)
5-10 µg c
Formaldehyde
20-100 µg
Acetaldehyde
400-1400 µg
Acrolein
60-140 µg
Other volatile aldehydes (6)
80-140 µg
Acetone
100-650 µg
Other volatile ketones (3)
50-100 µg
Methanol
80-180 µg
Other volatile alcohols (7)
10-30 µg
Acetonitrile
100-150 µg
Other volatile nitriles (10)
50-80 µgc
Furan
20-40 µg
Other volatile furans (4)
45-125 µgc
Pyridine
20-200 µg
Picolines (3)
15-80 µg
3-vinylpyridine
10-30 µg
Other volatile pyridines (25)
20-50 µg c
Pyrrole
0.1-10 µg
Pyrrolidine
10-18 µg
N-methlypyrrolidine
2.0-3.0 µg
Volatile pyrazines (18)
3.0-8.0 µg
Methylamine
4-10 µg
Other aliphatic amines (23)
3-10 µg
Numbers in parentheses represent individual compounds in a given group.
a
Modified from Hoffmann and Hoffmann (2001).
b
Quantity that passes through a Cambridge glass fiber filter.
c
Estimate.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
Table 3-4. Chemical substances and chemical classes in the particulate
matter of mainstream smoke of cigarettes without filters.a
Constituent
Nicotine
Nornicotine
Anatabine
Anabasine
Other tobacco alkaloids
Bipyridyls (4)
n-hentriacontaine (n-C31H64)
Total nonvolatile hydrocarbons (45)
Naphthalene
Naphthalenes (23)
Phenanthrenes (7)
Anthracenes (5)
Fluorenes (7)
Pyrenes (6)
Fluoranthrenes (5)
Carcinogenic polynuclear aromatic
hydrocarbons (11)
Phenol
Other phenols (45)c
Catechol
Other catechols (4)
Other dihydroxybenzenes (10)
Scopoletin
Other polyphenols (8)c
Cyclotenes (10)c
Quinones (7)
Solanesol
Neophytadienes (94)
Limonene
Other terpenes (200-500)c
Palmitic acid
Stearic acid
Oleic acid
Linoleic acid
Linolenic acid
Lactic acid
Indole
Skatole
Other indoles (13)
Quinolines (7)
Other aza-arenes (55)
Benzofurans (4)
Other O-heterocyclic compounds (42)
Stigmasterol
Sitosterol
Campesterol
Cholesterol
Aniline
Toluidines
Other aromatic amines (12)
Tobacco-specific N-nitrosamines (6)
Glycerol
Numbers in parentheses represent individual compounds identified.
a
Modified from Hoffmann and Hoffmann (2001).
b
Portion trapped on Cambridge glass fiber filter.
c
Estimate. NA= Not available.
For internal use of Philip Morris personnel only.
b
Yield (µg/cigarette smoked)
1.000-3.000
50-150
5-15
5-12
NA
10-30
100
c
300-400
2-4
3-6 c
0.2-0.4 c
0.05-0.1c
0.6-1.0c
0.3-0.5 c
0.3-0.45
0.1-0.25
80-160
60-180c
200-400
100-200c
200-400c
15-30
NA
40-70c
0.5
600-1000
200-350
30-60
NA
100-150
50-75
40-110
150-250
150-250
60-80
10-15
12-16
NA
2-4
NA
200-300
NA
40-70
30-40
20-30
10-20
0.36
0.23
0.25
0.34-2.7
120
41
42
Evaluation of Cigarette Ingredients: Feasibility
yields decreased in the mainstream particulate phase as filter ventilation increased,
from 1.69 mg nicotine/cigarette, in ventilated cigarettes to 0.55 mg/cigarette with
83% dilution (measured by pressure drop).
Nicotine yields in mainstream smoke correlate better with tar (R2 = 0.99) than
carbon monoxide yields (R2 = 0.81) using FTC methods (Taylor, Jr. et al., 2000).
The correlation between tar and carbon monoxide is weaker (R2 = 0.64) using the
Massachusetts method (Taylor, Jr. et al., 2000). Chemists in the Massachusetts
Benchmark Study suggested that vapor phase constituents, such as aromatic
hydrocarbons (e.g., benzene), correlated best with carbon monoxide, whereas
particulate phase constituents, such as aromatic amines (e.g., 1-aminonaphthalene)
correlated best with nicotine or tar, constituents of total particulate matter (Taylor,
Jr. et al., 2000).
3.4.2.3 Carbon monoxide
Carbon monoxide in the vapor phase of smoke makes up 2.8 to 4.6% of the weight
of mainstream smoke (Table 3-3). In 1980, the FTC adopted a new method for
determining carbon monoxide yields using an infrared spectrophotometer to analyze
the post filter gas collected in a plastic sampling bag (Federal Trade Commission,
1980).
3.4.2.4 Water
As the standard benchmark of tar is nicotine-free dry particulate matter, the amount
of water in the TPM should be determined. A measured amount of extracted
particulate matter is injected into a gas chromatograph, and the resulting peak is
compared against a standard curve to estimate moisture in the particulate phase.
The FTC measured particulate matter on a dry basis using the gas chromatographic
methods published by Sloan and Sublett (1965) as modified by Schultz and Spears
(1966) to determine moisture content (Federal Trade Commission, 1967a; Federal
Trade Commission, 1967b).
3.4.2.5 Free radicals
Pryor (1992a) analyzed cigarette smoke using electron spin resonance (ESR)
analysis. The half-lives of vapor-phase free radicals differ from those in tar. The
free radicals in the vapor phase of smoke are small reactive radicals with short
half-lives (Pryor, 1992a). Pryor and Stone (1993) estimated that vapor-phase
cigarette smoke contained approximately 1 x 1015 free radicals per puff, mainly
reactive carbon and oxygen centered radicals that typically have a lifetime of less
than 1 second and include nitric oxide and alkyl, alkoxyl, and peroxyl chemical
species. Radicals identified in the particulate phase are more stable, for example
semiquinone. Kodama and coworkers (1997) identified hydrogen peroxide and
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
43
superoxide free radical in the particulate phase of cigarette smoke which they
ascribed mainly to polyphenols e.g., hydroquinone.
Using electron spin resonance technique, Anderson and coworkers (2002) reported
approximately 1 x 1016 free radicals per cigarette in the vapor phase and
approximately 7 x 1014 free radicals per cigarette in the particulate phase. The
free radical yield also varied by type of cigarette. Cigarettes made from dark, aircured tobacco produced 2.0 x 1016 free radicals per cigarette in the vapor phase
and 1.3 x 1015 free radicals per cigarette in the particulate phase. Cigarettes with
charcoal filters produced 9.9 x 1015 free radicals per cigarette in the vapor phase
and 5.4 x 1014 free radicals per cigarette in the particulate phase (Anderson et al.,
2002).
Mainstream smoke exiting the butt of a cigarette contains nitric oxides, primarily in
the form of nitric oxide, which is a free radical species (Table 3-3) (Brunnemann
& Hoffmann, 1982; Cueto & Pryor, 1994; Hoffmann & Hoffmann, 2001; Sloan &
Kiefer, 1969). Sloan and Kiefer (1969) measured 44 µg of nitric oxide and 1.5 µg
of nitrogen dioxide per 35 mL puff of smoke from a commercial cigarette. The
amount of nitric oxide per puff increased from the second puff to the tenth puff,
whereas the amount of nitrogen dioxide did not change significantly. The nitric
oxide yield, obtained by summing individual puff yields, was approximately double
the 275 µg obtained when the total smoke vapor was trapped and analyzed, implying
that these chemical species have short half-lives.
Pyrolysis of nitrates is the main source of nitric oxide in mainstream smoke
(Brunnemann & Hoffmann, 1982; U.S. Department of Health and Human Services,
1988; Williams, 1980). Cigarettes made from bright leaf tobacco (flue cured)
containing only 0.1% nitrate yield an average of 4 µg nitric oxide per puff, whereas
cigarettes made from nitrate-treated tobacco containing 5.8% nitrate will yield in
excess of 200 µg of nitric oxide per puff (Sloan & Kiefer, 1969). A linear relationship
(r = 0.99) exists between the percentage of nitrate in the tobacco in the range
typically smoked and the yield of nitric oxide in the vapor phase of mainstream
smoke, with an extrapolated residual amount of 3 to 10 µg of nitric oxide in
mainstream smoke per puff that could not be attributed to the nitrate’s nitrogen
(Norman et al., 1983; Unemura et al., 1986; Williams, 1980). For sidestream
smoke the percentage of nitrate in the tobacco blends also positively correlates
(r = 0.98) with nitric oxide yields (Norman et al., 1983; Unemura et al., 1986).
Baker (1999) cited the studies of Norman and coworkers (1983) and Unemura
and coworkers (1986) to support his idea that, in addition to the tobacco nitrate,
“there must be other sources of nitric oxide.” Use of nitrogen-free cellulose
cigarettes demonstrated that oxidation of atmospheric nitrogen generated some
nitric oxide in mainstream smoke (Norman et al., 1983; Unemura et al., 1986).
However, for sidestream smoke, nitrogen oxidation only accounted for approximately
For internal use of Philip Morris personnel only.
44
Evaluation of Cigarette Ingredients: Feasibility
15% of residual nitric oxide. Most of the nitric oxide in sidestream smoke comes
from the oxidation of organic nitrogen compounds, such as amino acids and proteins
(Unemura et al., 1986).
3.4.2.6 Metals
To avoid artefactual contamination, Kalcher and coworkers (1993) preferred
electrostatic precipitators instead of Cambridge filters for metals analysis.
Electrostatic precipitators charge the particles, usually negatively, and deposit them
on a counter electrode, enclosed in a quartz tube thus avoiding contact with metallic
electrodes. Once collected, voltametry is used to analyze the collected particles
for individual metals.
Chortyk and Schlotzhauer (1984) detected the transfer of selenium to smoke either
by spiking the cigarettes with aqueous solutions of selenium or by spraying selenium
onto growing tobacco plants. For high tar reference cigarettes (Kentucky 2R1),
spiked with 0 to 25 µg Se/cigarette, an average of 10% of the added selenium
transferred into the mainstream smoke condensate. Similarly, the average amount
transferred to the mainstream smoke condensate from tobacco (sprayed with
selenium in the field) was 8.9%.
Kalcher and coworkers (1993) measured the distribution of cadmium and lead in
the ash, butt and mainstream smoke of French filtered cigarettes, 70 mm in length,
using a smoking machine modified for trace element analysis. They calculated the
amounts of metals in sidestream smoke by subtraction. Butt or ash retained onehalf of the cadmium. Sidestream smoke lost 40%, and particulate matter of
mainstream smoke transferred 10%. Butt or ash retained 84% of lead. Sidestream
smoke lost 10%, and mainstream smoke transferred 6%, equally divided between
gas and particulate phases.
3.4.3 Separation of Smoke
3.4.3.1 Filtration (particulate and vapor phases)
Many studies partition cigarette smoke into two phases, usually designated
particulate and vapor (gas), using filtration and trap (collection) devices. McRae
(1990) reviewed both historically important and recently used chambers. Dube
and Green (1982) reviewed the traps used to collect mainstream smoke, including
electrostatic precipitators, jet impactors, cold traps, solid adsorbents, solvent traps,
and Cambridge filters.
The Cambridge filter, once produced by the Cambridge Filter Corporation of
Syracuse, New York, served as the standard method of separation in studies of
cigarette smoke (Ogg, 1964; Pillsbury, 1996; Wartman et al., 1959). The FTC
used the original Cambridge Filter Method to separate vapor phase and particulate
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
45
phase constituents (Wartman et al., 1959) (Pillsbury, 1996). Ogg (1964) specified,
and Pillsbury and coworkers (1969) reiterated that the Cambridge filter or other
filters of equivalent construction must collect at least 99.9% of all particles more
than 0.3 µm in diameter at a flow rate of 28 linear feet per minute, having a
maximum pressure drop not exceeding 93 mm water at 28 feet per minute and
containing not more than 5% acrylic type binder.
Dube and Green (1982) described the Cambridge filter as a glass fiber pad stabilized
by an organic binder. The filter was positioned just after the cigarette with no dead
space. The pump was positioned after the filter, suctioning smoke through the
filter and forcing vapor into a plastic collection bag (Guerin, 1996; Pillsbury, 1996).
Investigators define total particulate matter or condensate of cigarette smoke as
the portion of smoke collected on a conventional Cambridge filter (Baker, 1999;
Dube & Green, 1982). The filter pad traps the aerosol particles or liquid phase,
and the vapor or gas phase passes through. Tar per cigarette is obtained by
subtracting the final weight of the filter from its initial weight and dividing this
difference by the number of cigarettes smoked and filtered. The FTC (1967a)
refers to tar as the weight in grams of the total particulate matter collected minus
the weight of alkaloids, as nicotine, and water. In other words, FTC’s tar is nicotinefree dry particulate matter filtered from mainstream smoke (CORESTA, 1991c).
Investigators often use Cambridge filters to define particulate and vapor phases of
cigarette smoke empirically. Most studies do not attempt to characterize the
proportion of the vapor phase trapped on a filter or the proportion of particles that
a filter fails to trap. Particulate and vapor phases separated by Cambridge filters
differ from one another in chemical composition and other physicochemical
properties (Dube & Green, 1982; Hoffmann & Hoffmann, 2001). Baker (1999)
summarized the evidence of the distributions of exogenously radioisotopically labeled
nicotine and N-dotriacontane between particulate and vapor phases in both
mainstream and sidestream smoke, based on nine studies conducted between 1958
Table 3-5. Percent distribution of radioactivity in mainstream and sidestream
cigarette smokes from 14C-labeled nicotine or n-dotriacontane
(based on tobacco consumed).a
Compound
14
C-nicotine
C-dotriacontane
14
a
No. of
Studies
5
4
Mainstream Smoke
Particulate
Vapor phase
phase
19.6 – 23.5
2.2 – 5.3
24.7 – 42.9
0.4 – 13.5
Sidestream Smoke
Particulate
Vapor
phase
phase
52.6 – 61.9
18.5 – 24.3
46.0 – 60.6
9.8 – 13.5
Modified from Baker (1999). n-Dotriacontane is a C32 straight chain alkane found in tobacco and other
plants.
For internal use of Philip Morris personnel only.
46
Evaluation of Cigarette Ingredients: Feasibility
and 1978. Table 3-5 shows that distribution of some substances, including nicotine,
varies between mainstream and sidestream smoke, as well as between the
particulate matter and vapor phases of these types of smoke.
Reliance on Cambridge filters assumes that filtration effectively separates the two
phases and that continued transfer of substances between vapor and the particulate
phase is either very slow or unimportant (Pankow, 2001). Williamson and Allman
(1966) described a third phase of smoke, which indicates that the separation is not
absolute. They defined a semi-volatile phase as the fraction retained on a Cambridge
filter pad at an ambient temperature, which becomes a volatile gas at 100-200 ºC.
Hoffmann and coworkers (2001) classified phenol, N-nitrosoamines, hydrogen
cyanide and low boiling aldehydes in the semi-volatile phase.
Kalcher and coworkers (1993) reported that the Cambridge glass fiber filters were
not ideal for the collection of particulate matter, because they clogged. Also, filters
having a large surface area generate contamination. Caldwell and Conner (1990)
reported an artifact in measuring N-nitrosamines. They showed that some Nnitrosamines formed on the Cambridge filter pad under FTC conditions, as described
by Hoffmann and coworkers (1983) and Hecht and coworkers (1983), when smoke
passed through the filter. This led to significant overestimates of N-nitrosamine
concentrations in cigarette smoke (Caldwell & Conner, 1990). Treating the filter
with vitamin C before smoke collection inhibited the formation of nitrosamines
(Caldwell & Conner, 1990).
Baker (1999) reported the creation of artifacts during the collection and storage of
smoke from reactions occurring in the aging of smoke. Dube and Green (1982)
reported finding cyanohydrins in mainstream smoke collected in water but not
when collected in a cold trap (-79ºC). Cyanohydrins might also form as artifacts
through reactions between hydrogen cyanide and volatile carbonyl components of
the smoke (Baker, 1999; Dube & Green, 1982).
While characterization of a brand or kind of cigarette by tar content may provide
useful chemical and regulatory characterizations, and while analysts have established
the accuracy of tar content as a measure, the biological implications of particulate
or tar content remain unclear. LSRO does not understand from the available
information whether the epidemiologically described adverse human health effects
of cigarette smoking relate to the particulate, vapor, or both fractions of cigarette
smoke.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
47
3.4.3.2 Chromatographic separation
Developments in solvent partition, column chromatography, flame ionization
detection, electron capture detectors and isotope dilution techniques enabled
quantitative identification of cigarette smoke constituents (Gudzinowicz &
Gudzinowicz, 1980). For a review of the analysis of cigarette smoke constituents
in smoke and human tissues (urine, blood, and exhaled breath), see the book by
Gudzinowicz & Gudzinowicz (1980).
Measurements of substances in smoke are not made simultaneously; investigators
generally do not collect and run a single chromatography column to estimate several
components at once. Instead, they measure different substances with different
methods, applied in parallel or in succession (CORESTA, 1991c; Rustemeier et
al., 2002). Problems with the interpretation of data for whole animal studies and
chemical studies can arise, if investigators obtained and analyzed samples with
different devices and methods.
3.4.4 Manufacturers’ Data
In addition to reporting the numbers of cigarettes sold for tax purposes,
manufacturers report the quantities of substances, or the yields from mainstream
smoke, collected and measured using standard procedures (Federal Trade
Commission, 2000b; Government of British Columbia, 2001; Hoffmann &
35
3.5
30
3
25
2.5
20
2
15
1.5
10
1
5
Nicotine (mg)
Tar (mg)
Sales Weighted, Tar and Nicotine Yields:
1968-1998
0.5
0
0
1955 1965 1975 1985 1995 2005
Year
Tar (mg)
Nicotine (mg)
Figure 3.5 Sales-weighted tar and nicotine values for U.S. cigarettes as measured by the FTC
method 1954-1998. Values before 1968 from Hoffmann & Hoffmann (2001). Modified from
Burns & Benowitz (2001).
For internal use of Philip Morris personnel only.
48
Evaluation of Cigarette Ingredients: Feasibility
Hoffmann, 2001; Taylor, Jr. et al., 2000; Tobacco Manufacturers Association
[London], 2003). Data about trends in the numbers and kinds of cigarettes sold
often also are useful.
Based on volume of sales of different cigarette brands, nicotine and tar yields per
cigarette smoked have decreased since the early 1950’s (Fig. 3.5) (Burns &
Benowitz, 2001; Federal Trade Commission, 2000a). The sales-weighted tar yields
reported by the FTC (2000a) represented a 45% decrease from 21.6 mg per
cigarette in 1968 to 12.0 mg per cigarette in 1998. The FTC (2000a) reported
sales-weighted nicotine yields of 1.35 mg/cigarette in 1968 and 0.88 mg/cigarette
in 1998.
The FTC closed its analytical laboratory in 1987, reporting that the Commission
could obtain data from other sources and by other means to verify the accuracy of
tests for tar, nicotine, and carbon monoxide (Federal Trade Commission, 2000a).
The FTC did not reference a quality control program in this notice but did list the
tar, nicotine and carbon monoxide yields of 1294 varieties of cigarettes sold in the
U.S. in 1998. Five of the largest cigarette manufacturing companies in the U.S.
carried out the 1998 testing. The Tobacco Institute Testing Laboratory, a private
laboratory operated by the cigarette industry, conducted most of the tar, nicotine,
and carbon monoxide analyses (Federal Trade Commission, 2000a). A gas
chromatographic method for tar and nicotine was modified to use the method of
Wagner and Thaggard (1979).
3.4.5 Classes of Chemical Substances
The subsections below briefly describe a few categories of chemical substances
found in cigarette smoke, hydrogen ion, phenols, pyridines, and aldehydes. This list
is not intended to be comprehensive but rather to illustrate the chemical complexity
of cigarette smoke. When classified by functional groups, cigarette smoke contained
more than 25 categories of chemical substances (Roberts, 1988).
3.4.5.1 Hydrogen ion
Rodgman (2000), Pankow (2001) and Bevan (1991) reviewed the pH of cigarette
smoke, including analytical methods and interpretation of results. The major hydrogen ion donors remain uncharacterized. Kruszynski (1982) prepared a list of reports discussing methods of pH determination in smoke published between 1930
and 1981. Battig and coworkers (1982) reported that the pH of smoke did not
correlate with the number of puffs, puff interval, puff duration, peak pressure or
puff volume. The pH of cigarette smoke interested investigators concerned that
substances with pKas in the physiological range or substances with zwitterionic
structures might absorb differentially, given changes in hydrogen ion concentration.
For internal use of Philip Morris personnel only.
Cigarettes And Cigarette Smoke
49
3.4.5.2 Phenols
In 1982, Dube and Green estimated that mainstream smoke contained 282 phenols
(Dube & Green, 1982). Phenols mostly partitioned to the particulate phase. Smith
and coworkers (2002) reported the chemical structures of 253 substituted phenols
in cigarette smoke. The estimated yield of phenol was 60-140 µg/cigarette in
mainstream smoke from unfiltered cigarettes with a sidestream/mainstream ratio
of 1.6-3.0. Other major substituted phenols were estimated at mainstream levels
per cigarette of 11-37 µg for cresols and 100-360 µg for hydroquinone (Baker,
1999).
The Massachusetts Benchmark Study showed that the average phenol level of
mainstream smoke from 26 brands was 37 µg/cigarette with a range of 10-64 µg/
cigarette (Taylor, Jr. et al., 2000). The range was greater than any of the other of
the 43 mainstream smoke constituents measured (Gray & Boyle, 2002; Taylor, Jr.
et al., 2000). A further study of smoke constituents from 25 brands representing
58% of the UK market (Tobacco Manufacturers Association [London], 2003) also
observed a wide range of 0.91-46.2 µg/cigarette, mean level of phenol in mainstream
smoke.
3.4.5.3 Pyridines
The four major pyridine alkaloids of tobacco are nicotine, nicotinic acid, nicotinamide
and nornicotine (Bush, 1999). Tobacco pyridines have organoleptic properties and
influence the flavor of cigarette smoke. Pyridine is found primarily in the particulate
phase of cigarette smoke (Taylor, Jr. et al., 2000). However, Brunnemann and
coworkers (1978) detected 21 volatile pyridines in mainstream smoke, including
32.4 µg/cigarette of pyridine and 12.3 µg/cigarette of α-picoline. In their study,
sidestream smoke contained up to 28 times greater concentrations of pyridines
than mainstream smoke. They identified 11 individual pyridine compounds in
mainstream smoke and 12 in sidestream smoke with inhibitory properties.
In 1999, the Massachusetts Benchmark study produced estimates of pyridines in
mainstream and sidestream cigarette smoke from 26 commercial brands of
cigarettes. The mainstream smoke yields of pyridine averaged 19 µg/cigarette
with a range of 13-24 µg/cigarette. For sidestream smoke, the yield was 268 µg/
cigarette with a range of 196-340 µg/cigarette, confirming earlier data that pyridine
concentrations in sidestream smoke were higher than mainstream smoke. Filters
selectively remove volatile pyridines, reducing concentrations by 63% with cellulose
acetate filters and by 69% with charcoal filters (Brunnemann et al., 1978).
Comparisons of volatile pyridines in tobacco to those in cigarette smoke indicated
that most pyridines in smoke arose from pyrolysis and pyrosynthesis (Brunnemann
et al., 1978).
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Evaluation of Cigarette Ingredients: Feasibility
Using a new GC-MS technique, Kulshreshtha and Moldoveanu (2003) reported
that the levels of pyridines in mainstream smoke depended on the type of cigarette.
For full flavored cigarettes, they found pyridine levels as high as 18 µg/cigarette,
compared to 3 µg/cigarette for an ultralight cigarette. Substituted pyridine yields
varied between 0.1 and 5.0 µg/cigarette for full flavored cigarettes to between a
few and 0.2 µg/cigarette for ultralight cigarettes.
3.4.5.4 Aldehydes
The most assayed chemical constituents in mainstream smoke, nicotine, tar and
carbon monoxide occur in milligram quantities per cigarette (Taylor, Jr. et al., 2000).
Of 34 minor mainstream smoke components measured by the Massachusetts
Benchmark Study, the acetaldehyde level of 1.618 mg exceeded the other 33
substances, including the other aldehydes that occur in microgram quantities per
cigarette (Gray & Boyle, 2002; Taylor, Jr. et al., 2000). In 1982, Dube and Green
(1982) estimated that tobacco smoke contained 108 aldehydes. However, the
present literature usually reports data for less than ten (<10) aldehydes, almost
entirely partitioned into the vapor phase of mainstream smoke (Dube & Green,
1982).
Formaldehyde is a chemically reactive substance, which can cross-link proteins
with other proteins or with nucleic acids (Yu, 1998). Cross-linking may be a
mechanism of toxicity (Boor & Hysmith, 1987) (Boor, 1983). Formaldehyde forms
after incomplete combustion of organic materials, including tobacco (Budavari,
1989; Taylor, Jr. et al., 2000). Hypothetically, metabolism of nicotine increases
formaldehyde formation. Animal model information supporting this hypothesis has
been reported (Wisborg et al., 2000). Although generally considered a gas, the
Massachusetts Benchmark Study assigned formaldehyde to the particulate phase,
where many other aldehydes partition (Taylor, Jr. et al., 2000). In contrast,
Hoffmann and Hoffmann (2001) classified formaldehyde and other volatile
aldehydes as part of the vapor phase.
The Massachusetts Benchmark Study reported an average yield of formaldehyde
of 69 µg/cigarette (Taylor, Jr. et al., 2000). The estimated yield for sidestream
smoke was more than tenfold higher, 787 µg/cigarette. The range of the mean
estimated yields of formaldehyde in mainstream smoke exceeded eightfold, from
12 µg/cigarette for a light-filtered cigarette to 105 µg/cigarette for a full-flavored
cigarette (Taylor, Jr. et al., 2000). Seventeen of the 26 brands of produced average
formaldehyde yields below the 60 µg/cigarette reported for Kentucky Reference
cigarettes (1R4F).
Recent studies by Baker et al. (2004a; 2004b), looked at the effect of different
groups of added ingredients (482 total) on 44 smoke constituents. Formaldehyde
levels in smoke increased in nearly all cigarettes that contained a form of sugar in
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Cigarettes And Cigarette Smoke
51
the casing material. In one case an increase of 73% by weight was observed,
compared to smoke from a cigarette without casing. Rustemeier et al. (2002),
also observed increases in smoke formaldehyde from cigarettes containing corn
syrup sugar among other added ingredients.
Acetaldehyde also is a chemically reactive aldehyde. Acetaldehyde is the most
prevalent aldehyde in cigarette smoke and is primarily found in the vapor phase
(Hoffmann & Hoffmann, 2001; Taylor, Jr. et al., 2000). At sufficiently high
concentrations, acetaldehyde is a general narcotic (Budavari, 1989). For many
cigarette brands, yields of acetaldehyde correlate with yields of tar and of carbon
monoxide. The primary region of mainstream smoke acetaldehyde deposition in
humans is the upper respiratory tract including the mouth (Seeman et al., 2002).
Seeman and coworkers (2002) reported that natural tobacco polysaccharides,
including cellulose, are the primary precursors of acetaldehyde in mainstream smoke.
Sugars added as ingredients, including D-glucose, D-fructose and sucrose, did not
produce higher yields of acetaldehyde in mainstream smoke than tobacco on a
weight-for-weight basis (Seeman et al., 2002). Cigarette design apparently
influenced the acetaldehyde content of smoke more than native or added sugars.
Seeman and coworkers suggested that the design factors potentially affecting
acetaldehyde yields included filtration, ventilation and paper porosity (Seeman et
al., 2002).
A third aldehyde, acrolein, also partitions mostly into the vapor phase of cigarette
smoke (Taylor, Jr. et al., 2000). Acrolein can penetrate the upper respiratory
passages and forms a highly reactive zwitterion (Leikauf et al., 2002). Mainstream
smoke yielded approximately 80 µg/cigarette (Baker, 1999). The sidestream to
mainstream ratio was in the range of 8-15. The Massachusetts Benchmark Study
reported 184 µg/cigarette (range 150-218 µg/cigarette) in mainstream smoke (Taylor,
Jr. et al., 2000), and in sidestream smoke the average was higher, 450 µg/cigarette
(Taylor, Jr. et al., 2000). The range of acrolein yields from mainstream smoke
appeared unrelated to cigarette length but related to the type of tobacco. Analysis
of cigarette smoke from puffed, expanded, and freeze-dried tobaccos yielded 105.0,
87.7, and 92.4 µg/cigarette of acrolein, respectively (Gori, 1977; National Institutes
of Health, 1996). Leikauf and coworkers (2002) suggested that aldehydes, such
as acrolein are enriched in sidestream smoke, because of lower combustion
temperatures.
Acetaldehyde, formaldehyde, and acrolein are, in order, the prevalent aldehydes
on a molar basis in cigarette smoke and are found mostly in the vapor phase. Their
reactive properties anticipate biological effects and raise interest in the toxicity of
the vapor phase.
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Evaluation of Cigarette Ingredients: Feasibility
3.4.6 Carcinogens
Scientists working on the chemistry and physics of tobacco smoke searched
intensively for carcinogens in cigarette smoke. A lack of rapid bioassays and a
lack of bioassays with external validity in predicting human carcinogenic effects of
cigarette smoke have hampered overall progress. The sections below briefly
summarize some of the analytical work.
3.4.6.1 Mutagenic activity
Many experts subscribe to a theory of cancer causation called somatic mutation.
Instead of mutations passing heritable changes from a person to progeny, under
somatic mutation heritable changes pass from cell to the cell’s progeny (HerreroJimenez et al., 2000). If more than one mutation alters both growth control and
location control of a cell, uncontrolled growth of cells at abnormal locations could
occur, which is the very definition of a malignancy. If a permanent mutation were
to develop in a critical region of DNA, close to or within the code for an oncogene
or tumor suppressor gene, activation of the oncogene or inactivation of the tumor
suppressor gene might occur (Brambilla & Brambilla, 1997).
Other nongenetic mechanisms of cancer have been proposed. Inhibition of
methylation of regulatory regions of DNA or other mechanisms might lead to
genomic instability (Hoeijmakers, 2001). Abnormal formation of gap junctions
also could cause mutations or serve as a nongenetic mechanism of carcinogenesis.
Substances in cigarette smoke could affect any of these processes. In addition, all
mutagens, and even all DNA adduct forming substances, are not necessarily
carcinogens (Ashby et al., 1989; Elcombe et al., 2002).
The most frequently applied test for assessing ability of cigarette smoke and its
components to induce point mutations is the mutation reversion assay in Salmonella
typhimurium (the Ames test) developed by Ames and coworkers (1973).
Salmonella typhimurium test strains carry a mutant gene, which renders the
bacteria unable to synthesize histidine. Reversion refers to the ability of a chemical
or its metabolite to reverse this mutation and is measured by the ability of the
bacteria to grow in the absence of histidine. Mutation reversion is therefore a
measure of the mutagenic potential of a chemical or substance. With bacterial
strains for this assay investigators can assay cigarette smoke itself as well as the
urine of human smokers for reversion potency. Moreover the assay may be utilized
to assay fractionates of smoke and urine to identify the mutagenic substances.
In 1977, Yamasaki and Ames (1977) reported that urine from cigarette smokers
exhibited mutagenic activity, using the Ames assay. Curvall and coworkers (1987)
reported nearly tenfold higher mutagenic activity in 24 hour samples of urine of
smokers compared to the activity in similar samples from non-smokers. Chepiga
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Cigarettes And Cigarette Smoke
53
and coworkers (2000) reported a correlation between mutagenic activity and tar
delivery in mainstream smoke.
Curvall and coworkers (1984) used the Ames test to estimate mutagens in total
cigarette smoke condensate and its fractions. They reported that the total and the
nonvolatile fraction contained direct-acting base-pair mutagens and indirect-acting
frame-shift mutagens. Although the semi-volatile (SV) fraction was not mutagenic,
two subfractions, (acids and phenols), had base pair mutagens that required no
metabolic activation. The contribution of these subfractions to the total activity of
the SV fraction must be considered low since they contribute only a small portion
of the SV fraction. After removal of protein and peptides from flue-cured cigarettes
by water extraction and protease digestion, Clapp and coworkers (1999) reported
significant reductions in Salmonella reversion activity in smoke condensates. Based
on these results, the authors concluded that protein pyrolysis products contribute to
the mutagenic properties of cigarette smoke condensates. However, in these
condensates they did not examine cell proliferation, regenerative hyperplasia or
chronic irritation.
Other assays estimate potential clastogenic DNA damage or chromosomal
aberrations from cigarette smoke, including sister chromatic exchange (Institute
of Medicine, 2001), micronucleated, polychromatic erythrocytes (Hayashi et al.,
2000), micronucleated hamster lung cells (Massey et al., 1998), and DNA strand
breaks. The comet assay is a popular way of measuring strand breaks (Tice et al.,
1994; Tice et al., 2000). A recent study compared cigarette smoke condensates
from several products with a battery of genotoxicity assays (Eclipse Expert Panel,
2000).
Roemer and coworkers (2002) evaluated the potential effects of three different
groups of added ingredients commonly used in cigarette manufacturing. Each
group was added at low and high levels to test cigarettes, and Salmonella reversion
potency of the particulate phase of the cigarette smoke was measured, using five
Ames test strains. Control cigarettes with the same construction and blend, but
without the addition of the added ingredients, and Kentucky Reference Cigarette
IR4F, served as controls. Within the limitations of the sensitivity and specificity of
the Ames test, the in vitro mutagenicity of cigarette smoke did not appear to be
increased by the added ingredients (Roemer et al., 2002). However, due to the
experimental design of testing cigarettes, using only large numbers of added
ingredients grouped together, the effect of single ingredients could not be assessed.
3.4.6.2 Polycyclic aromatic hydrocarbons
More than 500 polycyclic aromatic hydrocarbons have been identified in cigarette
smoke and some are classified as carcinogens by regulatory organizations (Rodgman
& Green, 2002). Hoffmann and Hoffmann (2001) list ten carcinogenic polycyclic
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Evaluation of Cigarette Ingredients: Feasibility
aromatic hydrocarbons found in cigarette smoke, with benzo[a]pyrene B[a]P
classified as carcinogenic on human evidence and the remaining nine on laboratory
animal studies (Hoffmann & Hoffmann, 2001). B[a]P is found in the particulate
phase of mainstream smoke (Taylor, Jr. et al., 2000). The average yield of B[a]P
in the mainstream smoke of 26 U.S. brands was 22.2 ng/cigarette with a range
from 5.6 to 41.5 ng/cigarette (Gray & Boyle, 2002; Taylor, Jr. et al., 2000). The
type of tobacco may relate to the B[a]P level. The lowest levels of B[a]P generally
were obtained with light or ultra light brands and the highest levels were obtained
with traditional, full-flavored cigarettes (Gray & Boyle, 2002; Taylor, Jr. et al.,
2000). Rustemeier and coworkers (2002) report that added ingredients incorporated
into U.S. blend test cigarettes resulted in a 50% decrease in polycyclic aromatic
hydrocarbons in mainstream smoke.
3.4.6.3 Nitrosamines
Nitrosamines occur in green tobacco leaves (Adams et al., 1983; Taylor, Jr. et al.,
2000). A portion of the nitrosamines in mainstream smoke directly transfers from
tobacco to the particulate phase. However, the largest portion of nitrosamines
forms during the smoking process (Adams et al., 1983; Taylor, Jr. et al., 2000).
Nicotine, a tertiary amine, is the major alkaloid in tobacco. Some minor alkaloids
are formed by the demethylation of nicotine and structural rearrangement:
nornicotine, anatabine, and anabasine (Brunnemann et al., 1996; Hecht et al.,
1994). Seven major tobacco specific nitrosamines (TSNAs) have been identified
(Hecht & Hoffmann, 1989). Studies on four of these have generated more data:
N’Nitrosonornicotine (NNN), 4-(N’-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone
(NNK), N’Nitrosoanabasine (NAB), and N’-Nitrosoanatabine (NAT). Interest in
the TSNAs relates to their biological properties. Two of the TSNAs, NNN and
NNK, are hypothetically causes of human cancer in smokers (Hecht et al., 1994).
Hecht and coworkers (1994) developed biomarkers for NNN and NNK.
TSNA levels markedly increase during the curing of tobacco (Djordjevic et al.,
1989; Hecht & Hoffmann, 1989; Sasson et al., 1985; World Health Organization,
1986). TSNAs are the most prevalent of all nitrosamines in cigarette smoke (Hecht
et al., 1994). During curing, microbes can convert nitrate to nitrites, which can
combine with alkaloids in the tobacco to form TSNAs. During cigarette combustion
pyrosynthetic nitrosation of the tobacco alkaloids generates some, but not all, TSNAs
(Brunnemann et al., 1996; Fischer et al., 1990; Hecht & Hoffmann, 1989). Adams
and coworkers (1984) added nitrate to cigarette tobaccos and obtained significant
increases in TSNA levels in the mainstream smoke. In contrast, after the addition
of endogenous nitrate to tobacco, Fischer and coworkers (1990) obtained
inconclusive results for NNN and NNK. Thus, there are data conflicts about the
source of nitrate for the formation of TSNAs during smoking (Rodgman & Green,
2002). Microwave treatment reduces TSNAs levels in cured tobacco leaf,
apparently by inhibiting microbes (Blackwell, 2000; Institute of Medicine, 2001).
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Cigarettes And Cigarette Smoke
55
However, Djordjevic and coworkers (1989) could not establish whether the increase
in TSNAs during the curing process related to biochemical processes within the
leaf or to microflora activity. In addition, atmospheric nitrogen can apparently
serve as a source of nitrogen to produce nitrosamines and TSNAs through oxidation
of NO to NO2. Norman and coworkers (1983) supported this pathway by reporting
that nitrogen free, cellulose cigarettes produced approximately 50 µg/cigarette of
NO.
3.4.6.4 Interpretation of carcinogenicity and mutagenicity data
For some investigators, the presence of animal carcinogens in cigarette smoke,
particularly lung carcinogens, is sufficient evidence to explain the biological effects
of cigarette smoke, at least the carcinogenic effects of cigarette smoke (Hecht,
1999). Stemming from extensive investigations, the suggestion has been that
polycyclic aromatic hydrocarbons and TSNAs explain the cancer observed in
epidemiological studies of cigarette smokers (Hecht et al., 1983). For others,
however, the carcinogenic substances in cigarette smoke need to provide a
quantitative explanation of observed biological effects (Rodgman & Green, 2002;
Tricker, 2001). General skepticism exists about the relevance of data from animal
models of carcinogenesis to human cancer (Freedman & Zeisel, 1988). In addition,
Fowles and Bates (2000) in their ranking of all carcinogenic substances in cigarette
smoke, using regulatory estimates which were essentially upper bounds on potency,
found that the most potent substance, 1,3-butadiene, which is found in the vapor
phase, had a cancer risk approximately equivalent to 0.001 cigarettes smoked per
day over a lifetime. Other substances would contribute little to the overall estimate,
and the modeling assumptions force additivity onto the calculation, eliminating the
possibility of synergy. Similar calculations with polycyclic aromatic hydrocarbons
and TSNAs imply that their quantitative potencies do not account for the cancer
observed in epidemiological studies of cigarette smokers.
Extensive literature is available about the toxicity and tumorigenic properties of
cigarette smoke condensate components using experimental test systems (Table
3-6) (Institute of Medicine, 2001; World Health Organization, 1986). Classes of
chemical substances found in the particulate (tar) phase of mainstream smoke
have been targeted as primary causes of human cigarette-induced lung cancer.
These substances include B[a]P, other polycyclic aromatic hydrocarbons, NNK,
and other TSNAs, and they have been assayed in cell culture and animal models.
Using these test systems as surrogates for humans, the substances have been
classified as carcinogens (D’Agostini et al., 2001; Rodgman & Green, 2002).
However, negative or quantitatively inconclusive results over the last fifty years,
particularly with whole cigarette smoke, illustrate the difficulties of inducing an
animal disease like human lung cancer (D’Agostini et al., 2001; Hoffmann &
Hoffmann, 2001; Roberts, 1988; Witschi et al., 1997b).
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Evaluation of Cigarette Ingredients: Feasibility
Table 3-6. Carcinogens in cigarette smoke a
Agent
PAHb
Benz (a) anthracene
Benzo (b) fluoranthene
Benzo (j) floranthene
Benzo (k) floranthene
Benzo (a) pyrene
Dibenz (a,h) anthracene
Dibenzo (a,l) pyrene
Dibenzo (a,e) pyrene
Indeno (1,2,3-cd) pyrene
5-Methlychrysene
Heterocyclic hydrocarbons
Furan
Quinoline
Dibernz (a,h) acridine
Dibernz (a,j) acridine
Dibenzo (c,g) carbazole
Benzo (b) furan
N-nitrosamines
N-nitrosodimenthylamine
N-nitrosoethylmethylamine
N-nitrosodiethylamine
N-nitrosodi-n-propylamine
N-nitroso-di-n-butylamine
N-nitrosodiethanolamine
N-nitrospiperidine
N-nitrosodiethanolamine
N-nitrosonornicotine
4-(methylnitrosamino)-1(3-pyridyl)-1-butanone
Aromatic amines
2-Toluidine
2,6-Dimethylaniline
2-Naphthylaniline
4-Aminobiphenyl
N-Heterocylic amines
AaC
IQ
Trp-P-1
Trp-P-2
Glu-P-1
Glu-P-2
PhIP
Adehydes
Formaldehyde
Acetaldehyde
Concentration
in smoke
1986 IARC category
Laboratory animal
Human
20-70 ng
4 -22 ng
6-21 ng
6-12 ng
20-40 ng
4 ng
1.7-3.2 ng
Present
4 -20 ng
0.6 ng
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
18-37 ng
1-2 ng
0.1 ng
3-10 ng
0.7 ng
Present
Sufficient
-c
Sufficient
Sufficient
Sufficient
Sufficient
2-180 ng
3-13 ng
ND-2.8 ng
ND-1.0 ng
ND-30 ng
3-110 ng
ND-9 ng
ND-68 ng
120-3700 ng
80-770 ng
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
30-337 ng
4 -50 µg
1-334 ng
2 -5.6 ng
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
25-260 ng
0.3 ng
0.3-0.5 ng
0.8-1.1 ng
0.37-0.89 ng
0.25-0.88 ng
11-23 ng
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Possible
70-100 µg
500-1400 µg
Sufficient
Sufficient
Limited
Insufficient
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Probable
Cigarettes And Cigarette Smoke
57
Table 3-6. Carcinogens in Smoke (continued)a
Agent
Volatile hydrocarbons
1.3-Butadiene
Isoprene
Benzene
Styrene
Misc. Organic compounds
Acetamide
Acrylamide
Acrylonitrile
Vinyl chloride
DDT
DDE
Catechol
Caffeic acid
Methyleugenol
nitromethane
2-nitropropane
Nitrobenzene
Ethyl caramate
Ethylene oxide
Propylene oxide
Inorganic compounds
Hydrazine
Arsenic
Beryllium
Nickel
Chromium (only hexavalent)
Cadmium
Cobalt
Lead
Polonium-210
a
b
c
Concentration in
smoke
1986 IARC category
Laboratory Animal
Human
20-75 µg
450-1000 µg
20-70 µg
10 µg
Sufficient
Sufficient
Sufficient
Limited
35-56 µg
Present
3-15 µg
11-15 ng
800-1200 µg
200-370 µg
100-360 µg
<3 µg
20 ng
0.3-0.6 µg
0.7-1.2 µg
25 µg
20-38 µg
7 µg
12-100 ng
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
24-43 ng
40-120 µg
0.5 ng
ND-600 ng
4 -70 ng
7-350 ng
0.13-0.2 ng
34-85 ng
0.03-1.0 pCi
Sufficient
Inadequate
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Insufficient
Sufficient
Limited
Sufficient
Probable
Sufficient
Inadequate
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
Inadequate
Inadequate
Sufficient
Modified from Hoffmann et al. (2001).
Abbreviations: AaC, 2-amino-9H-pyridol [2,3-b] indole; DDT, 1,1,1-trichloro-2,2-bis(pchlorophenyl)ethane; DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene; Glu-P-1, 2-amino-6methyl[1,2-a:3',2''-d]imidazole; Glu-P-2, 2-aminodipyrido[1,2-a:3'2''-d]imidazole; IQ, 2-amino-3methylimidazo [4,5-b]quinoline; ND, not detected; PAH, polynuclear aromatic hydrocarbons; PhIP, 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido [4,3b]indole; Trp-2, 3-amino-1methyl-5H-pyrido [4,3-b]indole.
Not reviewed for carcinogenicity.
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Evaluation of Cigarette Ingredients: Feasibility
Carcinogenic substances are not limited to the particulate phase. The vapor phase
of mainstream smoke includes chemical substances classified as carcinogenic,
including acetaldehyde, isoprene, benzene, styrene and 1-3-butadiene (Hoffmann
& Hoffmann, 2001; World Health Organization, 1986). Indeed, some scientists
question whether the active substances in cigarette smoke are limited to the
particulate phase and have developed as alternative hypotheses, either whole smoke
or vapor (Witschi et al., 1997a). To test these hypotheses, both in vitro and in
vivo studies have been designed to test the toxicity of whole smoke or vapor
phase, instead of particulate (tar) phase.
Using a strain of mouse that develops lung adenomas, Witschi and colleagues
published a series of studies that test the inhalation of a combination of mainstream
and sidestream smoke regarding its potency to induce lung carcinomas (Witschi et
al., 1995; Witschi et al., 1997b; Witschi, 1998; Witschi et al., 1999; Witschi et al.,
2000; Witschi, 2000b).
Witschi’s protocol is novel. It involves a five-month exposure to cigarette smoke
followed by a four-month recovery period (Institute of Medicine, 2001). It also
involves a mixture of mainstream and sidestream smokes. The lung tumors seen
in this mouse model, adenomas, are thought to resemble human lung tumors which
have become more prominent, as smokers have switched to filtered and light
cigarette brands. Witschi and coworkers reported that exposure of the mice to the
gas phase of this experimental smoke produced lung tumor multiplicity at a similar
rate to whole smoke (Witschi et al., 1997a; Witschi, 1998). Witschi and coworkers
(1999; 2000) also showed that chemopreventive agents known to protect against
tumors induced by substances found in the particulate phase did not protect against
their experimental cigarette smoke. Other laboratories recently replicated Witschi’s
A/J mouse model experiments (D’Agostini et al., 2001). The relevance of the
system to human lung cancer must be confirmed experimentally.
3.4.7 Other Biological Properties
No particular scientific reason exists to focus attention on the carcinogenic effects
of cigarette smoke, to the exclusion of other health effects. The rationale appears
to lie in public attention and policy objectives. Other endpoints, such as ciliatoxic
effects, which seem a useful model for COPD, appear equally useful candidates
for research (Riveles et al., 2003). Therefore, the search for an animal model of
emphysema has absorbed attention (Wright & Churg, 2002b).
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59
3.5 SUMMARY
In studying the potential health effects of any substance, an early and essential
step is the definition of what toxicologists call “the study substance” or “the test
material.” If an ingredient added to a cigarette changed the chemical and physical
composition of the resulting smoke, then the ingredient might contribute to an adverse
health impact already caused by smoking the cigarette. In this circumstance, cigarette
smoke becomes the test material, and the added ingredient augments or modifies
the test properties of the cigarette smoke.
Cigarette smoke is a complex and variable mixture. LSRO has explored the
chemical and physical composition of smoke from cigarettes and thus, how scientists
could measure the influence of added ingredients on smoke. This chapter also
lays a foundation to understand what manufacturers might practically accomplish
by testing the effect of added ingredients on the chemistry and physical composition
of smoke.
Most of the data about cigarette smoke come from mechanically generated smoke,
often intended to mimic at least in some average way, human smoking patterns,
but not from studies of inhaled human cigarette smoke. Because humans draw
mainstream smoke immediately into their pulmonary trees, an inaccessible location,
scientists have to infer the properties of inhaled mainstream smoke from the study
of these mechanically generated smokes. Mechanically generated smoke may
have a different chemical composition from the smoke inhaled by humans, depending
on cooling, dilution, and other processing. Thus, scientists need to exercise caution
in interpreting these studies. In addition, cigarette smoke inhaled by humans contains
variable proportions of sidestream smoke.
Even when the mechanical generators better mimic human smoking patterns with
respect to number of puffs, puff intervals, inspiration rates, and other properties,
which they generally cannot do, these machines will still need to duplicate other
physiological factors such as impaction within the branching pulmonary tree,
hydration from fluid lining of the pulmonary cavity, and so forth. In addition there
is high variation within the human smoking population in the way cigarettes are
smoked. Should mechanically generated smoke ever improve on its ability to
duplicate human smoking and the variation between individuals, it would be not be
unexpected to still find high variation in the physical characteristics and analytical
yields of chemical substances in cigarette-to-cigarette comparisons. Yields change
as smoking proceeds down the tobacco rod and paper, from which the smoke is
generated.
Many confounding factors also contribute to variation in the content of cigarette
smoke. Tobacco growing, cigarette engineering, and control of cigarette
manufacturing, will affect the compositions of brands of cigarettes and the
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60
Evaluation of Cigarette Ingredients: Feasibility
constituents of their smokes. Added nontobacco ingredients, such as menthol, can
affect cigarette composition and to a measurable degree, alter smoke composition.
The scientific literature does not document the effects on smoke composition of
many added ingredients.
Cigarette smoke is a variable, heterogeneous mixture of several thousand organic
and inorganic chemical constituents. The overall high variance in the chemistry
and physics of cigarette smoke does not mean that the human health effects of
cigarette smoking (or the testing of nontobacco ingredients added to the cigarettes)
are not subjects amenable to scientific study. It does mean that scientific methods
that depend on a high precision of data cannot be employed.
Some substances in cigarette tobacco undergo pyrolysis, whereas other substances
undergo complex chemical reactions in the smoke. The chemical substances added
to tobacco will undergo the same processes. Because of the amounts of ingredients
added to cigarettes, a relatively high level of exposure to the combustion products
of added ingredients is possible. Therefore, LSRO needs to understand how added
ingredients change smoke chemistry and composition. Such testing is readily
performed but quantitative measurement of components of cigarette smoke will
require robust experimental controls. The use of reference cigarettes and reference
smoking conditions is required for purposes of reproducibility and internal
comparison.
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DOSIMETRY
OUTLINE
4.1
INTENT & PURPOSE
4.2
HUMAN SMOKE
4.2.1 Cigarette Consumption
4.2.2 Time Weighted Average, Haber’s Rule, and Pack-Years
4.2.3 Human Smoking Behavior
4.2.4 Smoking Compensation
4.3
DOSIMETRY OF CIGARETTE SMOKE
4.3.1 Structure of the Human Respiratory Tract
4.3.2 Absorption of Smoke
4.3.3 Distribution of Smoke in the Lung
4.3.4 Smoke Elimination From the Lung
4.3.5 Models of Smoke Dosimetry
4.4
BIOLOGICAL MONITORING OF SMOKING EXPOSURE
4.4.1 Nicotine
4.4.2 Cotinine
4.4.3 Carbon Monoxide
4.4.4 Other Substances
4.5
EXPERIMENTAL ANIMAL EXPOSURE TO CIGARETTE
SMOKE
4.5.1 Methods for Experimental Animal Exposure to Cigarette
Smoke
4.5.2 Carbon Monoxide Pharmacokinetics in Animal Species
4.5.3 Absence of Animal Models of Human Health Effects
4.6
SUMMARY
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4
CIGARETTE SMOKE: EXPOSURE AND
DOSIMETRY
4.1 INTENT & PURPOSE
The terms exposure and dose express different but related concepts. An
environmental exposure is a potential dose because not all of the exposed material
is absorbed or otherwise made available for potential biological activity (Byrd &
Cothern, 2000). For cigarette smoking, pulmonary exposure is the relevant route.
For inhalation, exposure at any moment in time is the quantity of a chemical substance
just outside of the membranes of the pulmonary system, and dose is the quantity of
the same substance that has moved inside the membranes. From this perspective,
the absorbed amount would be the same as the dose. Transit through the pulmonary
membrane is not a simple process. Some portion of a chemical substance may not
be available to the circulation but may remain adhered to, or within, the membranes
of the pulmonary system.
Biological measures of the absorbed amount of a substance in biological fluids,
such as integrated measures of metabolites in urine, contrast usefully with
simultaneous measures of exposure to the same substance, either in individuals or
in populations. As explained in the previous chapter, cigarette smoke is a complex
mixture of many thousands of chemical substances. Thus, relating the inhalation
exposure to cigarette smoke to biological measures of dose is scientifically difficult.
The purpose of understanding exposure in this context is ultimately to allow the
subsequent establishment of an exposure-response relationship for external
observers to measure. The driving forces in human health effects are dose and
time (Rozman, 1998). Dose bears a complex, often nonlinear relationship to
exposure. Time also has complex relationships to exposure, as time has several
component relationships bearing on chronic health effects. In epidemiology, these
relationships relate to variables recorded for each subject, including chronological
year of birth, age of initiation of exposure, duration of exposure, and age at
observation or death. In toxicology, each substance will have at least three time
scales: pharmacokinetic, pharmacodynamic, and frequency of administration. All
three scales interact with dose and species to produce a complex situation for
interpretation in chronic health effects (Rozman & Doull, 2000). For inhalation
studies dose is not easily observed or measured.
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Cigarette Smoke: Exposure And Dosimetry
63
Cigarettes contain a psychoactive drug, nicotine (U.S. Department of Health and
Human Services, 1988). Smoking of cigarettes permits self-administration of
nicotine. Cigarette smoking also exposes smokers to chemical substances besides
nicotine. Because nicotine contributes to increasing smokers’ exposure to cigarette
smoke, this chapter describes the pharmacokinetics of nicotine. To understand the
feasibility of testing ingredients added to cigarettes, scientists need to understand
the factors contributing to smoke exposure and dosimetry, of the mixture of all
substances together, as well as the individual substances in the smoke.
4.2 HUMAN SMOKING
4.2.1 Cigarette Consumption
Human smoking and smoke exposure are complex processes, which include the
decision to smoke, puffing behavior, inspiration into the lung, coating of pulmonary
surfaces, and the absorption, metabolism, distribution, and elimination of substances
in the smoke. Human consumption of a cigarette involves ignition of the cigarette
and inhalation of smoke as the cigarette decreases in length, through a series of
oral inspirations (puffs). Smokers usually inhale a breath containing cigarette smoke
more deeply and more rapidly than a normal inhaled breath (Pearson et al., 1985).
Cigarette smoke is held briefly in the mouth and throat during inspiration of a puff
(Hinds et al., 1983). Subsequently, the smoker inhales the smoke into the respiratory
tract, mixed with ambient inhaled gases after the puff is completed. The residual
constituents are gradually exhaled, and the breathing pattern quickly returns to
baseline.
Smoking machine yields of cigarette constituents, such as carbon monoxide,
generated under standard FTC conditions, do not accurately predict human
biomarker measurements (Benowitz, 2001; Gori & Lynch, 1985; Woodward &
Tunstall-Pedoe, 1993). Wide variation in human smoking behaviors may explain
some of this difference. Inhaled volumes of cigarette smoke vary between 5 mL
and 75 mL per puff.
4.2.2 Time-Weighted Average, Haber’s rule, and Pack-Years
The usual epidemiological measure of smoke exposure, the pack-year, implies that
exposure to substances in smoke does not change over the smoker’s lifetime.
However, the smoker changes, even if the brand of cigarette and the smoker’s
smoking behavior do not change. The structures of the body tissues, including the
respiratory tract, change with age and change even more with chronic exposure to
smoke. Chronic cigarette consumption compromises ciliated cells in the respiratory
tract. Mucus producing cells and metaplastic, squamous-like cells replace ciliated
cells (Dalhamn & Rylander, 1971; Kensler & Battista, 1966). Removal of airway
secretions becomes compromised, while simultaneously hyperplasia of the mucus
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Evaluation of Cigarette Ingredients: Feasibility
producing cells and glands increases. Chronic exposure to cigarette smoke activates
inflammatory and immune cells, contributing to progressive destruction of lung
tissues.
Duration of exposure and intensity of exposure to cigarette smoke both contribute
to the development of disease (Doll & Peto, 1978). Measures such as the number
of cigarettes smoked per day, the depth of smoke inhalation and the tar and nicotine
content of the cigarette help to reveal the intensity of cigarette exposure.
Epidemiologists usually elicit patterns of cigarette consumption from questionnaires
or interviews, with the usual uncertainty surrounding retrospective self-assessments
(Kuper et al., 2002). Biological markers can provide more accurate estimates of
the smoking intensity.
Haber’s rule is identical to the standard measure of smoke exposure, pack-years,
both mathematically and biologically (smoking intensity in packs/day multiplied by
duration of smoking in years). Haber’s rule also is identical to time-weighted
average (TWA), a frequently used measure of occupational exposure. The TWA
is the air concentration of a substance, multiplied by its duration in air (Atherley,
1985). Fritz Haber established Haber’s rule in the process of making observations
about chemical warfare agents (Witschi, 2000a). Some scientists believe that the
rule best fits acute toxicity. For linear systems, no reason exists to suppose that
Haber’s rule will not extend in time or expand according to a power law. Later
experiments established these relationships for carcinogenic substances (Druckrey,
1967).
Haber’s rule commonly is understood to apply only to acute effects and only in its
simplest form, C x t = k, where C is concentration, t is time, and k is a constant. In
its expanded, power law version, the exponents applying to concentration and time
can emphasize or diminish the importance given to exposure concentration and
exposure duration for any substance in generating an observed toxic effect, Ca x tb
= k (Institute of Medicine, 2000). If a > 1 and b > 1, applications of the unmodified
form of Haber’s rule (C x t = k) to high concentration, prolonged duration data
yield upper bounds on concentration-duration relationships. For some substances
the unmodified form may be inappropriate. Some toxicologists prefer using Haber’s
rule and applying safety factors, when working with chronic data. Working with a
pack-year relationship merits caution, because packs per day times years of smoking
experience may not yield a valid estimate of exposure.
In this context, the number of cigarettes or packs of cigarettes smoked per unit of
time, e.g., cigarettes per day or packs per year, is an exposure rate. An exposure
rate equates to an air concentration during a period of time. The number of packs
per day, multiplied by the number of years a smoker has smoked at that rate, is a
measure of total exposure. For chronic exposure, the standard inhalation intake
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Cigarette Smoke: Exposure And Dosimetry
65
rate has units of mg/kg/day. This rate is usually calculated, not measured (Byrd &
Cothern, 2000). It is a product of concentration, inhalation rate, absorption factor,
body weight, and an exposure factor, as follows;
air inhalation intake rate = (C x IR x AF x EF)/BW
where:
C = contaminant concentration (mg/M3)
IR = inhalation rate (M3 /day)
AF = absorption factor (no units)
EF = exposure factor (no units)
BW = body weight (kg)
For cigarette smoking the absorption factor is known to be less than 1.0. (See
subsection 4.3.2, below.) The exposure factor is a product of exposure frequency
(events/year) and exposure duration (years/lifetime). Constant exposure over a
lifetime generates an EF = 1. For cigarette smoking, exposure frequency is equivalent
to packs smoked per day, and exposure duration is the same as the number of
years of smoking at that rate. So, introduction of pack-years into the standard
inhalation intake rate calculation changes the units and the physical assumptions
that go into the calculation.
Standard estimation of inhalation exposure assumes that concentrations in the
external air will equilibrate with internal concentrations in the pulmonary air space.
For cigarette smoking, this assumption cannot hold. Either for the gas or particulate
phases, nonequilibrium circumstances prevail. Instead, the smoker rapidly inhales
a series of puffs with little volume of air between the end of the cigarette and the
surface of the lung.
Rozman’s theory of toxicity supports the use of the power law version of Haber’s
rule (Ca x tb = k) (Rozman, 2000). It suggests that mortality and morbidity from
cigarette smoking, like other chronic health effects, should relate to both exposure
rate and duration of exposure (Rozman, 1998). However, either exposure rate or
duration of exposure may contribute more to a toxic effect. Even so, for cigarette
smoking, an estimate of exposure rate should normalize chronic human exposureresponse relationship data.
The potential inaccuracy of using pack-years is obvious (Kuper et al., 2002).
Epidemiologists usually obtain exposure information about smoking from
questionnaires or interviews. Retrospective, self-reported data will inaccurately
characterize the cigarette exposure of an individual smoker. Measures of cigarette
consumption cannot easily account for variable smoking, for example, in bars or at
parties. However, replacing pack-years as an estimate of exposure rate in
epidemiological surveys will not be easy. Cigarette smoking does not provide a
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Evaluation of Cigarette Ingredients: Feasibility
constant exposure but if fluctuations occur on a time scale very much shorter than
the evolution of disease, such fluctuations may be unimportant. The chief difficulty
with pack-years is estimating it.
Recently, the application of Haber’s rule to cigarette smoking has undergone scrutiny.
Leffondré and coworkers (2002) used data from a case control study and Cox’s
model to study smoking status, intensity, duration, cigarette-years, age at initiation,
and time since cessation, using time-dependent variables and goodness of fit as
their primary criteria. They found (a) that using cigarettes per day and duration
separately improved fits with their model, compared to Haber’s rule and (b) that
colinear effects declined, when they estimated the effects of time since cessation
or age at initiation, if they used Haber’s rule.
4.2.3 Human Smoking Behavior
Human smoking behavior influences puff volume, puff duration, peak puff flow
velocity (the maximum rate of mainstream smoke flow from cigarette to smoker),
interval between puffs, number of puffs per cigarette, and residual butt length left
at completion of smoking the cigarette. The puff profile graphically represents the
rate of flow of mainstream smoke during a puff. Puff profiles can be used to
calculate puff volume (International Organization for Standardization, 2000). Figure
4.1 shows the puff profiles of three subjects and a puff profile generated by standard
smoking machine settings.
The Health Consequences of Smoking. Nicotine Addiction (1988) summarized
data about puffing behavior, as a common measure of smoke exposure. The
summary cited 29 separate studies of 8 to 517 subjects carried out between 1978
and 1986. The means (ranges) of the indices were:
Puffs per cigarette: 11 (8-16; n=27 studies),
Puff duration: 1.8 seconds (1.0-2.4 seconds; n=27 studies),
Puff volume: 43 mL (21-66 mL; n=18 studies),
Peak puff flow: 36 mL per second (28-48 mL per second; n=6 studies),
Inter-puff interval: 34 seconds (18-64 seconds; n=21 studies),
Total smoking time: 346 seconds per cigarette (232-414 seconds; n=13 studies),
Total smoke inhalation volume per cigarette: 591 mL (413-918 mL; n= 5 studies).
Bentrovato and coworkers (1995) reported that puff volumes declined progressively
with a lower average rate of mainstream smoke flow from the cigarette to the
smoker (peak puff flow) than the Surgeon General’s report (1988). (See Figure
4.2) Bentrovato and coworkers found 18 mg of tar per cigarette delivered to the
mouths of adult smokers.
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67
Figure 4.1 Kinetics of human puff flows for three smokers. Modified from Bentrovato, et al., (1995).
Figure 4.2 Average puff volumes as a function of sequential puff number. Error bars represent one
standard deviation. Modified from Bentrovato, et al., (1995).
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Evaluation of Cigarette Ingredients: Feasibility
The taste of cigarette smoke influences smoking (Huber et al., 1990; Nil & Battig,
1989). Ingredients added to cigarettes can affect taste. Extensive government
reports discuss the factors contributing to initiation and maintenance of smoking,
and they cite a few studies about the sensory experience of smoking, including
human perceptual parameters, such as flavor and aroma, which added ingredients
might alter (U.S. Department of Health and Human Services, 2001b) (U.S.
Department of Health and Human Services, 1994) (U.S. Department of Health
and Human Services, 2001a). Sensory cues, such as cigarette flavor and aroma,
also might support the maintenance of smoking, as cues, through learned behavior.
Smoking pairs a distinctive aroma with a pharmacological effect (Rose et al.,
1993; Westman et al., 1996).
No individual smoker exhibits a constant pattern of cigarette smoking. An individual’s
behavior changes, even during the smoking of a single cigarette (Bentrovato et al.,
1995; Creighton & Lewis, 1978; Pillsbury, 1996). Smokers tend to draw larger
puff volumes initially and taper down by the last puff (Adams, 1976; Reeves &
Dixon, 1995). The decreases in puff volume correlate with decreases in puff
duration, while smoking a single cigarette (Reeves & Dixon, 1995). As illustrated
in Figure 4.2, average puff volume in one study decreased from approximately 60
mL for the first puff to less than 20 mL for the eighteenth puff (Bentrovato et al.,
1995; Polydorova, 1961). Inhalation also contributes to variation in cigarette smoking.
Depth of inhalation has been estimated by various methods: self-report, chest wall
expansion, impedance magnetometers, and inductance plethysmography (US
Consumer Product Safety Commission, 1993).
Variations in puff profiles lead to the notion that no two persons smoke the same
brand of cigarette in an identical manner (Bentrovato et al., 1995; Creighton &
Lewis, 1978; Nil & Battig, 1989). Among smokers of filtered cigarettes in France,
men took fewer puffs per cigarette than women, but men had greater durations
and volumes of puffs (Hee et al., 1995). Similar gender-specific findings were
reported for smokers in the U.S., but the total volume of cigarette smoke inhaled
per day did not differ significantly by gender (Djordjevic et al., 1998).
Nicotine binds to receptors on dopamine-releasing cells in the ventral tegmental
area of the brain’s mesolimbic system, creating a reinforcing sense of satisfaction
(Clarke, 1990; Corrigall et al., 1994) (Figure 4.3). Whether smokers consume
cigarettes in amounts and ways, such that they obtain a constant level of satisfaction,
is not clear. Individual smoking behavior, which varies, does determine the delivery
of cigarette smoke and nicotine to the respiratory tract (Gori & Lynch, 1985).
Benowitz and Jacob (1994) measured nicotine uptake with deuterium labeled
nicotine and found an average uptake of 2.3 mg per smoker. However, these
investigators decided that a prestudy period of abstinence influenced selfadministration. They recommended the continued use of a standard 1 mg per
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Cigarette Smoke: Exposure And Dosimetry
69
cigarette for nicotine delivery. Similarly, CNS levels of nicotine will vary among
individuals in response to the same delivery, and satisfaction will vary among
individuals with the same extent of receptor binding.
Figure 4.3 Schematic representation of the nicotinic-acetylcholine receptor, illustrating nicotine
binding. Reprinted with permission from (Schapira et al., 2002)
The delivered dose of nicotine from smoke affects smoking behavior. Benowitz
and coworkers (1983) studied 272 subjects who smoked different brands of
cigarettes. Smokers of low yield cigarettes achieved concentrations of blood cotinine
similar to smokers of high yield cigarettes. Cotinine is a biomarker of nicotine
exposure. The data are consistent with a biological feedback mechanism involving
nicotine. The data also demonstrate compensation for lower nicotine yields. As
nicotine yields declined, smokers inhaled more smoke in poorly understood ways.
Investigators have documented that at least partial compensation occurs (Lee, 2001).
4.2.4 Smoking Compensation
Benowitz (2001) and Scherer (1999) used mathematical equations to define the
extent of compensation based on proportional changes in biomarker levels and
machine-determined yields of a constituent, before and after a change in brand of
cigarette. One possible explanation of compensation is that an increase in the
number of cigarettes consumed will compensate for the reduced amount of nicotine
in the cigarette (Russell et al., 1980). Another option is a change in smoking
behavior, so that the smoker obtains more nicotine per cigarette (Lee, 2001). Still
another is the smoker blocking holes in cigarette papers, to reduce dilution with air.
In addition, smokers could engage in all of these behaviors (increase cigarette
consumption and smoke to obtain more nicotine per cigarette by inhaling deeper
and blocking air holes).
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Compensation need not be absolute. Burns and coworkers (2001) used data from
an American Cancer Society cohort of 174,997 U.S. male smokers, which included
brand and number of cigarettes smoked over a 12-year period from 1960 to 1972,
and found a relationship between nicotine content and cigarette consumption. For
each milligram decline in machine-measured nicotine yield per cigarette, the number
of cigarettes smoked increased by 0.85 per day. Among 169,610 subjects, who
switched brands of cigarettes in follow-up surveys, the number of cigarettes smoked
per day increased by 2.3 cigarettes per day for each mg decline in nicotine yield
(Burns et al., 2001).
Stepney (1980) analyzed the results of fifteen studies about switching from brands
with medium nicotine deliveries (machine deliveries of 1-2 mg) to brands with
decreased nicotine delivery. In twelve of the fifteen studies, the number of low tar
and nicotine cigarettes smoked increased, compared with the number of medium
tar and nicotine cigarettes smoked. These data produced a correlation of R = 0.59
between the percent reduction in nicotine delivery and percent increase in the
number of cigarettes smoked per day. Regression analysis predicted that a
reduction of 50% in nicotine delivery would result in a 9% increase in consumption.
From a reanalysis of these results, Lee (2001) also concluded that brand switching
showed only modest increases in number of cigarettes smoked per day relative to
major decreases in delivery of tar and nicotine.
Garfinkel and coworkers (1979) reported data from a large prospective American
Cancer Society study of 28,561 U.S. male smokers, 60% of whom smoked a
lower yield brand of cigarette in 1972 than in 1959. Persons who increased
consumption did so by ten cigarettes per day. Garfinkel and coworkers (1979)
concluded that this increase did not relate to the yield of tar and nicotine. Of those
who smoked less than one pack per day in 1959, 55% increased the number of
cigarettes smoked per day in 1972 after changing to cigarettes with decreased tar
and nicotine. However, the majority of other smokers consuming less than one
pack per day in 1959, who selected cigarettes of the same or increased content of
tar and nicotine in 1972, also increased the number of cigarettes they smoked daily
by 1972. Lee (2001) cited these data to show that reduced tar and nicotine did not
change the number of cigarettes smoked per day.
Smokers could compensate for reduced tar and nicotine by puffing more frequently,
by inhaling deeper, and/or by blocking ventilation holes, impeding the dilution of
smoke in the tobacco rod (Kozlowski et al., 2001b; Sutton et al., 1978; Sutton et
al., 1982). All three behaviors increase smoke yields (National Institutes of Health,
2001). Changes in puff volume might explain why a 40% reduction in cigarette tar,
obtained from smoking machine data, would result in only a 15% reduction in
smoker’s tar exposure (Lee, 2001).
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71
According to Scherer (1999), changing puff volume is most probably the dominant,
if not only mechanism of compensation. Scherer (1999) reviewed sixty studies
involving a total of more than 2000 smokers, to see whether low-yield cigarettes
are smoked more intensely than high-yield cigarettes. He summarized the studies
as compensation for tar (four studies), taste (six), irritation (one), draw resistance
(five), nicotine content (29), and no compensation (eight). Puustinen and coworkers
(1987) reported the eight volunteers who smoked cigarettes yielding 4-5 mg tar
and 0.32 mg nicotine had greater puff duration (2.2±0.2 sec), greater puff volume
(36±4 mL) and took more puffs per cigarette (18.4±1.4). The smokers inhaled a
greater volume of smoke per cigarette (649±69 mL) compared to their earlier
brand of cigarette, containing 14-15 mg tar and 0.97 mg nicotine (1.8±0.2 mL,
26±4 mL, 13.6±1.0 mL, and 345±46 mL, respectively).
Lynch and Benowitz (1987) obtained plasma cotinine measures from 197 smokers
at two time points, six years apart. For those who switched to lower yield cigarettes,
the number of cigarettes smoked decreased by 6.6 cigarettes (20%), from an
average of 32.9 to 26.3 per day. Plasma cotinine concentration increased by ~5%
for those who smoked cigarettes with similar yields (n=104), decreased by ~20%
for those who smoked cigarettes with lower yields (n= 62), and increased by ~23%
for those who switched to higher yield cigarettes (n= 31). For those who switched
to lower yield cigarettes, total daily nicotine yields (nicotine yield per cigarette
multiplied by the total number of cigarettes smoked per day) declined by ~50%.
Because Lynch and Benowitz (1987) did not measure puff profiles, Benowitz (2001)
estimated the intensity of smoking of study subjects by dividing the plasma cotinine
concentration by the number of cigarettes smoked per day. Smokers who switched
to lower yield cigarettes compensated by smoking more intensely, because their
plasma concentrations of cotinine, relative to the number of cigarettes smoked,
closely resembled levels obtained when the higher yield cigarettes were smoked
previously.
Peach and coworkers (1986) measured urinary nicotine metabolites of 599 male
smokers in a multi-site, randomized heart disease prevention project. The number
of cigarettes smoked per day declined during the study for all subjects. Smokers
excreted similar amounts of urinary nicotine metabolites, regardless of tar yield,
consistent with compensation.
Benowitz (2001) summarized five studies of up to ten day durations and five longer
term studies of five weeks to nine months durations, in which smokers were assigned
a test cigarette and group mean compensation ranged from 20 to 100%, as defined
by biomarkers (blood concentrations of cotinine or carboxyhemoglobin, expired
carbon monoxide, or urinary nicotine metabolites). Guyatt and coworkers (1989),
Robinson and coworkers (1983) and Zacny and Stitzer (1988) showed that some
smokers increased the number of cigarettes smoked per day but that the primary
means of compensation was accomplished by increased intensity of smoking. The
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Evaluation of Cigarette Ingredients: Feasibility
intensity of smoking could be increased by increasing puff volume, increasing peak
flow, decreasing the inter-puff interval, and/or increasing the number of puffs per
cigarette (Guyatt et al., 1989; Zacny & Stitzer, 1988). Some subjects blocked
ventilation holes (Guyatt et al., 1989).
Thus, two reviewers, Lee (2001) and Benowitz (2001) generally agree that some
compensation occurs but that a switch to a lower yield cigarette does not necessarily
lead to an increase in the number of cigarettes smoked per day. Benowitz (2001)
further concludes that such a switch probably will alter smoking behavior in the
direction of extracting more nicotine from the cigarette, compensating to some
degree. Burns reached a similar conclusion in a report of the U.S. Consumer
Product Safety Commission (1993), suggesting that changes in design (draw
resistance and filter perforation) could alter cigarette smoking.
Mechanisms of compensation for decreased cigarette yield, not involving
consumption of more cigarettes, have not been fully characterized. Smokers can
achieve higher nicotine yields through poorly understood smoking behaviors.
Pritchard and Black (1984) reported a decrease in the total body and thoracic
deposition of tar in 23 healthy male smokers who switched from a high tar (16-17
mg), to a low tar (8-9 mg) cigarette for six months, using cigarettes labeled with
radioactive 123I-iodohexadecane, which has a half-life of thirteen hours and
undergoes little pyrolysis during cigarette combustion. At baseline, and at one,
three and six months following the switch to the lower tar cigarette, subjects smoked
the labeled cigarette in their usual manner. Changing to the lower tar cigarette
significantly reduced total body deposition of labeled particles by 25% at one month
and more than 30% by six months (Table 4-1). However, deposition in the head
and abdomen remained the same. Pritchard and Black (1984) suggested that a
large part of this deposition would be in the mouth and throat, perhaps reflecting
modifications in the smoking pattern to compensate for differences experienced
smoking the lower tar cigarette.
Table 4-1. Regional deposition of tar 24 weeks after switching to a lower tar cigarette (mean ± SE).a ±
Region
Total Body
Extra Thoracic (head, abdomen)
Thoracic
Tracheobronchial airway
Vascularized pulmonary tissue
a
b
Deposition (%)b
Higher-tar
Lower-tar
(16-17mg)
(8-9mg)
±
±
100.0 ± 6.8
67.1 ± 5.1
13.9 ± 3.4
15.6 ± 3.4
86.1 ± 6.8
51.5 ± 4.2
51.1 ± 3.4
33.8 ± 3.0
35.0 ± 4.2
18.1 ± 2.1
Modified from Pritchard and Black (1984).
Expressed as a percentage of the total deposited from the higher tar cigarette.
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4.3 DOSIMETRY OF CIGARETTE SMOKE
When people inhale cigarette smoke, they expose their respiratory tracts to both
the gas and particulate phases of the smoke. In addition to absorption of smoke
constituents on the surface of the respiratory tract, many chemical substances
circulate, further exposing internal tissues and organs. The scientific community
has made progress in identifying the chemical substances in cigarette smoke. (See
Chapter 3 of this report.) Quantitative assessment of human exposure is limited.
4.3.1 Structure of the Human Respiratory Tract
Some background information will aid the discussion of absorption and deposition
of smoke constituents. Hollander and Stober (1986) based their descriptions on a
task force report (Bates et al., 1966). A naso-pharyngeal (extra-thoracic)
compartment begins at the anterior nares and extends beyond the anterior and
posterior pharynx to the epiglottis. The respiratory tract divides into two regions:
the tracheobronchial tree (the upper airway) and the pulmonary parenchyma (the
vascularized tissue where gas exchange occurs). The tracheobronchial
compartment consists of the trachea and the bronchi. These airways branch and
subdivide over and over, approximately 25 times in the human, to reach fine divisions
in the terminal bronchioles.
Ciliated epithelia characterize the tracheobronchial compartment, greatly increasing
the surface area. Both glandular and nonglandular secretions bathe the lining of
the airway (Bates et al., 1966; Hollander & Stober, 1986). The cilia move and
transport secreted mucus and deposited particles up the tracheobronchial tree and
out of the lungs. The pulmonary compartment consists of the nonciliated respiratory
bronchioles, which extend from the terminal bronchioles, and their supporting
vascularized tissue. The peripheral pulmonary compartment includes alveolar ducts,
atria, alveoli attached to the respiratory bronchioles, and alveolar sacs. The
vascularized air sacs of the alveoli serve as the interface where gases exchange
between the airway and the perfusing pulmonary capillary compartment (the
parenchymal tissue).
Leading from the trachea, the structure of the tracheobronchial tree is asymmetric
(Yeh et al., 1976). A parental airway tube divides into two, uneven daughter
branches, a major and a minor branch, distinguished by their differences in diameter.
The airways in the human lung are more divided by branching, than the airways of
dogs, rats and hamsters. In humans and dogs, angles of the major and minor
branches increase progressively down the tracheobronchial tree. Data for variation
in human tracheobronchial tree branch angles, daughter to parent tube diameter
ratios, and daughter tube length to diameter ratios are available, as are data for
some laboratory mammals. Yeh and coworkers (1976) suggest that species
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Evaluation of Cigarette Ingredients: Feasibility
differences in the structures of pulmonary lobes may explain the findings of uneven
deposition of particles in respiratory tracts of different animals.
4.3.2 Absorption of Smoke
Absorption refers to the transport of a chemical or substance from the site of
administration into the systemic circulation. In the case of inhaled cigarette smoke,
constituents will transfer from the pulmonary air space of the respiratory tract
onto its surface. Potentially, components of the smoke transfer further into the
circulation through this highly vascularized tissue. Both the gas and particulate
phases of smoke reach, and react with, the surface of the lung. Localized surface
reactions lead to some of the biological effects of smoke. In addition to coating of
lung surfaces, smoke includes unreacted chemical fragments created by pyrolytic
and pyrosynthetic reactions associated with cigarette combustion. These fragments
can react covalently with cellular elements of the lung, instead of each other.
Smoking studies sometimes use an alternative term for absorption, deposition. In
deposition a fraction of inspired smoke is not expired (Brain & Valberg, 1979). By
definition, the processes of absorption and deposition overlap. For cigarette smoking,
the particulate fraction of captured smoke, deposited on a filter, can be weighed
before and after inhalation, leading to an experimental measure of deposition. The
mass entering and exiting an airway also can be calculated from aerosol
concentrations and flow rates (Subramaniam et al., 2003). Several processes lead
to deposition in the respiratory tract, including impaction, sedimentation and diffusion
(Martonen, 1992; Yeh et al., 1976). Changes in breathing patterns alter deposition.
An increased velocity of inhaled particulates increases deposition by impaction
(Yeh et al., 1976).
Cigarette smoke is a significant environmental source of radioactive polonium-210
(210Po) (Little et al., 1965). Polonium-210 is a naturally occurring, alpha particle
emitting element, with a half-life of 138 days, generated from decay of its parent
lead-210 with a half-life of twenty years (Little & McGandy, 1968). In addition to
postmortem examination of lung tissues, early studies of 210Po provided evidence
that components in cigarette smoke were absorbed into lung tissues and into the
systemic circulation. Little and coworkers (1965) found that smokers had 210Po in
their lung tissue equivalent to the amount naturally occurring in seven packages of
cigarettes. They considered this deposition low, because their smoking subjects
consumed at least one-half pack of cigarettes per day. Because they found only
small amounts of 210Po in lung parenchyma, they suggested that the lung cleared
210Po, which absorbed into the circulation rapidly (Little & Radford 1967). Smokers
had blood concentrations of polonium-210 2.5 times higher than nonsmokers.
Consistent with clearance from the lung into the circulation, blood concentrations
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
75
of 210Po declined an average of 14% three-to-four days after smoking cessation
and by 20.5% at 11 to 14 days after cessation (Little & Radford, Jr., 1967).
Minty and coworkers (1984) investigated whether cigarette smoking would increase
the permeability of the pulmonary epithelial tissue. Five male nonsmokers breathed
an aerosol containing [99mTc]diethylenetriamine pentacetic acid ([99mTc]DTPA)
for two to three minutes, twice at baseline and on days one and three during the
experimental period when they smoked ten cigarettes per day for three days. A
probe scintillation detector was placed over the right upper zone of the chest to
detect [99mTc]DTPA. Values were corrected for increasing radioactivity in blood
and non-lung tissue. The half-time elimination of [99mTc]DTPA decreased by 26%
to 53% for subjects by the third day of smoking. Smoking increased the permeability
of pulmonary epithelial tissue, which permitted greater absorption of the aerosol
from lung into blood (Minty et al, 1984).
No precise estimate of particulate deposition from inhaled cigarette smoke exists.
Many factors could affect the deposition of smoke particulate, including smoking
behavior, smoking experience, brand of cigarette, humidity, body size, age, gender,
disease status, and previous injury, depth of inhalation, residence time in the lung,
exercise, and life style. Table 4-2 (World Health Organization, 1986) shows estimates
of the fraction of inhaled tar deposited in the respiratory tract from seven studies
of exhaled particles conducted between 1923 and 1984. The method for measuring
particle capture varied among these early studies. Subjects retained most of the
inhaled tar (74% to 97%). However, one early study found 47% tar retention by
fluorophotometric measures of 11 subjects (Hinds et al., 1983). All 13 measures
of deposition by Hinds and coworkers (1983) in five female smokers were less
than 50%, and their average of 40% was significantly less (p< 0.01) than the 57%
average deposition for six male subjects in the study.
Table 4-2. Some estimates of inhaled tar deposition using different
exhaled particle collection methods.a
RETAINED (%)
RANGE
73-98
70-90
(5-sec inhalation)
94-99
(30-sec inhalation)
b
79-97
22-89
22-75
a
b
c
d
CAPTURE TECHNIQUE
AVERAGE
88
82
Electrostatic precipitation
Balloon
97
Balloon
96
87
74
47
Cold trap
Filter
Vacuum-assisted filterc
Vacuum-assisted filterd
Modified from World Health Organization (1986).
Not reported.
Data from Polydorova (1961)
Data from Hinds et al. (1983)
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Evaluation of Cigarette Ingredients: Feasibility
Particulate deposition may be important. Based on an assumption of 50% deposition,
Gurney (1998) calculated that over a 40-year period, more than 6 kg of particulate
matter would be deposited in the lung of a one-pack per day smoker, who smoked
cigarettes containing 20 mg of tar. The potential tumorigenic activity of particulate
deposition has been studied using particulate matter painted onto skin surfaces of
animal models (Coggins et al., 1982; Dontenwill et al., 1972; Dontenwill et al.,
1976). This is thought to create an analogy to the deposition of particles on the
surface of the lung. Both lung and skin are epithelial surfaces.
Later studies suggest less deposition of cigarette smoke, approximately 15 to 50%
of the inhaled particulate. Hollander and Stober (1986) suggested that up to 6% of
smoke constituents might deposit in the tracheo-bronchial compartment and between
22% and 32% in the pulmonary region. Theoretically, only 15% to 30% of
mainstream smoke particles of 0.2 µm average diameter would deposit in the
human respiratory tract, based on the deposition of similar sized nontobacco particles
inhaled by mouth or nose during typical low-activity conditions of 750 cm3 tidal
volume and 15 breathes per minute (Hinds et al., 1983; National Council on
Radiation Protection and Measurements, 1997). (See Figure 4.4.)
Figure 4.4 Relative deposition of particles in a lung model
90%
Nasopharyngeal
Tracheobronchial
Pulmonary
Total
80%
70%
Deposition
60%
50%
40%
30%
20%
10%
0%
0.01
0.06
0.2
0.6
1
2
Particle Diameter (µm)
Figure 4.4 Percent deposition of particles as a function of particle size under conditions of 770 cm3
tidal volume, 13 breaths per minute and functional residual capacity of 0.4 total lung capacity.
Data from National Council on Radiation Protection and Measurements (1997).
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
77
Using a Weibel symmetric lung model under conditions similar to that of a smoker
(750 mL tidal volume, 5-second inhalation, 3-second pause, and 2-second expiration),
and assuming that the growth of saturated saline droplets represented the
hygroscopic characteristics of cigarette smoke particles, Muller and coworkers
(1990) calculated 31% to 53% deposition for mainstream particles and 6% to 28%
deposition for sidestream particles.
Schultz and coworkers (2000) reviewed the physical mechanisms of particle
deposition in the respiratory tract. Determinants of inhaled particle deposition in
the respiratory system include particle size, shape, density, charge and
hygroscopicity. These factors affected growth of particles (agglomeration) in the
humidified respiratory tract (Kaufman et al., 1996; National Council on Radiation
Protection and Measurements, 1997; Sethi & Rochester, 2000). Martonen (1992)
and others (Chen & Yeh, 1990) thought the behavior of mainstream cigarette smoke
in the lung related to particle cloud motion, an effect produced by high particle
number (~ 3x109 per cm3) and mass (~0.0001 g per cm3) densities of the smoke.
Particle cloud motion could explain why mainstream cigarette smoke would deposit
at sites that differ from the sites of deposition expected for a dilute aerosol of
similar sized particles (Martonen, 1992; Phalen et al., 1994).
The change in the size of smoke particles as they age may influence the amount
deposited. The respiratory tract has high humidity, estimated at 99.5 per cent
(Ferron, 1977). Subsequent coagulation of mainstream smoke caused by high
humidity in the lungs might increase particle diameters, acquiring the physical
characteristics of a smoke cloud with high concentrations of particles, exhibiting
larger diameters, perhaps up to six or seven µm (Keith, 1982). In theory, a greater
portion of larger particles would deposit than particles of 0.2 µm diameter, the
initial average diameter of mainstream cigarette smoke particles (Figure 4.4).
Disagreements exist about the maximum growth of smoke particles in the humid
conditions of the respiratory tract and the effect of particle growth on deposition
(McRae, 1990).
Ventilation parameters, such as tidal volume, rate of cigarette smoke inhalation,
dilution with inspired air, and timing of inhalation, can influence deposition. Whether
the breathing pattern is controlled or spontaneous may be an important consideration
for comparative studies (Martonen et al., 2000). Lung anatomy and the size of
study subjects, including morphological differences in the shape and size of the
larynx and tracheobronchial airways, will alter cigarette smoke particle deposition
in the respiratory tract (Yeh & Schum, 1980). Geometric parameters, such as
airway segment diameters, lengths, branching angles and angles of inclination to
gravity, vary between individuals and contribute to the complexity of lung anatomy.
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78
Evaluation of Cigarette Ingredients: Feasibility
The vapor pressure and solubility of chemical substances in cigarette smoke
determine their partitioning between the vapor and particulate phases in smoke
(Pankow, 2001). Figure 4.5 illustrates direct deposition of a substance from the
vapor phase of a smoke aerosol onto a tissue and evaporation of the substance
from the particulate phase into the vapor phase with subsequent deposition onto
the tissue. Unlike ingestion, substances absorbed from the respiratory tract do not
undergo first pass metabolism in the liver.
Figure 4.5 Pankow’s depiction of four processes for molecular absorption of smoke aerosol
particle into respiratory tract tissue. Reprinted with permission and modified from (Pankow,
2001). Copyright 2001 American Chemical Society.
Dalhamn and coworkers (1968a; 1968b) published data on the disappearance of
carbon monoxide, acetaldehyde, isoprene, acetone, acetonitrile, toluene, and total
particulate matter from cigarette smoke by mouth-only and inhalation exposures
(Table 4-3). They exposed volunteers to smoke from unfiltered cigarettes yielding
about 30 mg of tar each. Smoke from a two-second, 35 mL puff was delivered
into the mouth of the subject and either blown out by the subject without inhaling,
or inhaled and then blown out. Inhalation of smoke resulted in disappearance of
more than 85% of all volatile compounds and for 54% of the carbon monoxide,
compared with only 3% disappearance of carbon monoxide by the mouth-only
exposure. The authors stressed the importance of using exposure methods that
approximate actual smoking conditions (Dalhamn et al., 1968a). For example,
exclusive mouth breathing inhalation narrows the theoretical range of deposition of
smoke particles to approximately 15% to 20%, compared to up to 30%, based on
methods that include nasal inhalation (National Council on Radiation Protection
and Measurements, 1997).
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
79
Table 4-3. Human deposition of cigarette smoke components after a two-second,
35 mL puff and inhalation or mouth-only exposure.a
Compound
Carbon monoxide
Acetaldehyde
Isoprene
Acetone
Acetonitrile
Toluene
Particulate matter
Subjects
(n)
5
10-16
10-16
10-16
10-16
10-16
10-16
Deposition after
inhalation
(% ± SD)
54 ± 12.7
99 ± 1.2
99 ± 0.6
86 ± 5.5
91 ± 4.1
93 ± 4.3
96 ± 3.1
Deposition after mouthonly exposure
(% ± SD)
3 ± 0.7
60
20
56
74
29
16
a Modified from Dalhamn et al. (1968a; 1968b). Reprinted with permission from Heldref
Publications.
4.3.3 Distribution of Smoke in the Lung
Limited data indicate that approximately half of inhaled cigarette smoke deposits
in the middle zone of the lung, 25% in apical regions and a little more than 25% in
base regions. The base regions receive the major portion of the inhaled breath.
Martonen (1992) reported that localization of smoke particle deposits in airway
bifurcations in ex vivo human cast models, specifically the carinal ridges, resembles
the location of lung carcinomas in the upper tracheobronchial tree. Smoke particles
do not show a uniform dispersion along tubular airways in human cast models,
instead the particles concentrate in the posterior segments (Martonen et al., 1987;
Martonen, 1992). (See Figure 4.6)
Figure 4.6 Schematic diagram of
cigarette smoke deposition in a
cast model of the human
tracheobronchial tree.
Reprinted with permission and
modified from (Martonen et al.,
1987).
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80
Evaluation of Cigarette Ingredients: Feasibility
Compared with resting ventilation, Pearson and coworkers (1985) detected more
deposition of cigarette smoke particles in the apical and central regions of the lung
during smoking for eleven habitual smokers. For total deposition, the mid-to-lower
zone of the lungs collected most of the technetium99m sulfur colloid particles in the
experimental cigarette smoke (apex: 25%; mid zone: 47%; base: 27-28%), similar
to the overall pattern for nonsmoking breaths (Pearson et al., 1985). Longer
inhalation and breath holding might contribute to deposition of mainstream smoke
in the lower airways (Muller et al., 1990).
During elective surgery or autopsy, Little and coworkers (1965) obtained fresh
bronchial and parenchymal tissue (whole lung or single lobes) from thirty-six patients,
including twenty-five who had smoked for 20-60 years and measured 210Po in
selected sections of tissue. Centrally located parenchymal tissue was available
for twelve cigarette smokers. The concentration of 210Po in central specimens
was approximately twice that of the peripheral samples and generally two orders
of magnitude greater in the bronchial epithelium than in parenchyma or lymph
nodes. The average concentration of 210Po was highest at segmental bronchial
bifurcations. No 210Po was detected in segmental bronchi or upper-lobe bifurcations
of nonsmokers.
Churg and Stevens (1992) inspected lung tissue samples from autopsies of five
smokers (30-90 pack years) and five age and gender matched nonsmokers, using
a scanning microscope equipped with a wavelength dispersion x-ray spectrometer.
They detected irregularly shaped particles (0.5 to 0.8 µm) containing calcium, carbon
and oxygen in lung segments of smokers (23±10% of all particles) but rarely in
nonsmokers (0.1±0.2%). It is not known whether these particles were deposited
intact from smoke (e.g., calcium carbonate) or formed from smoke and tissue
interactions (e.g., calcium oxalate). A portion of the particle burden in smoke may
translocate to the lung interstitium and remain indefinitely.
Boykin and coworkers (1993) labeled filtered research cigarettes by injecting 1-2
µCi of [14C]dotriacontane along the tobacco rod. They administered smoke from
these cigarettes, using FTC protocol through tracheostomies to mechanically
ventilated dogs. After exposure to two radiolabeled cigarettes, they sacrificed the
dogs and systematically analyzed tissues for the isotope. The tracer deposited in
regions of the lung in direct relation to tissue mass, the time smoke was delivered
relative to inspiration, and animal posture. When they delivered a bolus of smoke
at the start of an inhalation, peripheral deposition of 14C averaged twice the amount
of central deposition. The distribution of 14C was more uniform when the bolus of
smoke was delivered at mid-inhalation. A supine posture significantly increased
deposition in the upper lobes compared to an upright position (Boykin et al., 1993).
In contrast, Brain and Valberg (1979) suggested that for small mammals, deposition
of aerosols was generally greater in the apex than at the base of the lung, regardless
of whether the animal was standing, prone or inverted.
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Cigarette Smoke: Exposure And Dosimetry
81
The tar and nicotine content of smoke may not affect the location of deposition.
Phelps et al. (1984) exposed female rats to [14C]dotriacontane labeled smoke from
three types of cigarettes that varied in tar and nicotine yields, as measured by the
FTC smoking machine protocol. Although the composition of the smoke varied,
patterns of deposition were similar for all three groups. The major portion of the
inhaled smoke penetrated the upper airways and deposited deep in the lung (Phelps
et al., 1984). The cigarettes yielding high tar and low nicotine generated significantly
greater external deposition on the rat body than the other cigarettes that yielded
low tar and high nicotine or high tar and high nicotine, suggesting that animals did
not maintain nose-seal throughout the exposure and the results may have been
confounded.
Yeh and Schum (1980) noted that for a given inspiratory flow rate, deposition in
the region of the mouth increased as the particle size increased. Figure 4.4 illustrates
the effect of particle size on regional deposition (National Council on Radiation
Protection and Measurements, 1997).
Compensation may alter smoke distribution. Pritchard and Black (1984) reported
a decrease in the total body and thoracic deposition of tar in 23 healthy male
smokers who switched from a high tar (16-17 mg), to a low tar (8-9 mg) cigarette
for six months, using cigarettes labeled with radioactive [123I]-iodohexadecane,
which has a half-life of thirteen hours and undergoes little pyrolysis during cigarette
combustion. At a baseline, and at one, three and six months following the switch to
the lower tar cigarette, subjects smoked the labeled cigarette in their usual manner.
Changing to the lower tar cigarette significantly reduced total body deposition of
labeled particles by 25% at one month and more than 30% by six months (Table 41). However, deposition in the head and abdomen remained the same. Pritchard
and Black (1984) suggested that a large part of this deposition would occur in the
mouth and throat, perhaps reflecting modifications in the smoking pattern to
compensate for differences experienced in smoking the lower tar cigarette.
4.3.4 Smoke Elimination From the Lung
Smokers eliminate inhaled cigarette smoke particles in their lungs by exhaling or
coughing, by swallowing particles cleared by the mucocilliary elevator in their lung,
or by absorbing particles into their systemic circulation. For the most part, cigarette
smoke particles have a nonaqueous, liquid composition, so systemic absorption
should play a major role. Initially, when particle and vapor phase components
come into physical contact with the epithelial surface of the lung, they encounter
the respiratory tract lining fluids. The mucus fluid can entrap particles, which
mucocilliary movement or phagocytosis then eliminates. The epithelial fluid lining
the lung is mobile. Materials deposited in one area of the lung can transport to
other areas. The fluid undergoes active secretion and resorption. Components
deposited in the epithelial fluid may concentrate or dilute.
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82
Evaluation of Cigarette Ingredients: Feasibility
Albert and coworkers (1971) described a typical pattern of elimination of radioactive
198Au particles (1.5-4.1 µm) and 99mTc particles (2.3-7.9 µm) in aerosol for human
smokers and nonsmokers, detected by serial tracheal and chest measurements.
Elimination consisted of two distinct phases. A period of rapid elimination was
complete within a few hours, and a second slower phase took 3 to 24 hours.
Considerable variability in elimination rates existed among human subjects,
regardless of smoking habit. However, the size of the test particles apparently
varied between subjects. Some smokers cleared particles rapidly compared to
nonsmokers. Albert and coworkers (1971) suggested that inter-subject variability
relates to the rate of the second phase mucocilliary elimination of smaller-sized
test particles (1.6 µm and 1.9 µm) from the lower portion of the bronchial tree.
The test particles were markedly larger than cigarette smoke particles, which
ranged from 0.18 µm to 0.7 µm diameter. Pritchard and Black (1984) used [123I]iodohexadecane as a tracer in a similar study of smokers and also characterized
lung elimination as biphasic. Duration of phases was similar for twenty-three
smokers of high tar (16-17 mg) cigarettes and twenty-seven smokers of lower tar
(8-9 mg) cigarettes. The first phase had a half-life of approximately two hours,
and the second phase had a half-life of 18 eighteen hours.
Smoking status influences the rate of smoke particle elimination from the lungs.
Albert and coworkers (1975) studied the short-term effects of cigarette smoking
on the elimination of radioactive test aerosols containing particles ranging from 2.7
to 4.7 µm in size. Six smokers and nine nonsmokers inhaled radioactive (198Au or
99mTc) aerosol for one minute at a rate of fourteen breaths per minute and a volume
of approximately one liter per breath. After an interval of several hours, the subjects
repeated this process with a different radioactive (198Au or 99mTc) aerosol. Then,
all subjects smoked from two to five cigarettes, inhaling once every thirty seconds,
with approximately a ten-minute interval between cigarettes. Cigarette smoking
decreased the time required for 90% bronchial elimination by approximately 30%
(an accelerated rate of elimination from 292 to 201 minutes for nonsmokers and
from 257 to 173 minutes for smokers). Albert and coworkers (1975) speculated
that an increased rate of mucous elimination from distal ciliated airways during the
test smoking session may have accelerated the rate of bronchial elimination, possibly
related to the effect of cigarette smoke on mucous production and ciliary elimination.
The location of deposition in the respiratory tract might contribute to elimination
rate. Ilowite and coworkers (1989) pooled data from earlier studies of mucociliary
elimination using [99mTC]sebecate, [99mTC]iron oxide and [99mTC]albumin to
determine inter-subject variation from activity time plots and the ratio of centralto-peripheral airway counts. The elimination of deposited aerosol was faster, the
more central the location of the deposit (larger ratio). The coefficients of variation
for particle retention at 120 minutes were 31%, 30% and 18% for sebacate, iron
oxide and albumin, respectively.
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
83
Disease status influences the elimination of particles. Moller and coworkers (2001)
measured the elimination of ferromagnetic tracer particles (1.35 µm diameter) by
magnetopneumography for 300 days after controlled voluntary inhalation in 39
healthy adults and 42 patients. They described three distinct, sequential phases of
elimination. In phase one, patients with chronic obstructive bronchitis rapidly cleared
bronchial areas within 48 hours. This phase did not necessarily occur in healthy
young nonsmokers. In phase two, particles redistributed but did not move from the
lung during a translocation period lasting up to six weeks. In phase three, the
remaining particles slowly cleared from the alveolar space. Nonsmokers with
sarcoidosis (n=10) or idiopathic pulmonary fibrosis (n=5) had significantly longer
alveolar elimination times than nonsmokers who were healthy (Moller et al., 2001).
The half-life of alveolar elimination for healthy nonsmokers was 124±66 days.
Alveolar elimination increased by 5.7±1.3 days per pack-year (p<0.01) in cigarette
smokers. In addition to smoking status and disease, age influenced particle
elimination. Alveolar elimination was faster for smokers younger than 40 years of
age (n=13) 220±74 days and slower for smokers who were older 459±334 days
(n=9).
Albert and coworkers (1971) also studied whether smoking and disease might
contribute to variation in the elimination of radioactive aerosols from the lungs.
They calculated the weighted average elimination, representative of the average
residence time for particles deposited in, and cleared from, the bronchial tree, by
dividing the area under the elimination curve, minus the residual activity remaining
in the lung, by the total amount of bronchial elimination (Albert et al., 1971). Total
elimination times averaged 126±14 minutes for the healthy nonsmokers (n=18),
170±18 minutes for the healthy smokers (n=19) and 238±33 minutes for patients
with bronchitis (n=6), significantly longer than for nonsmokers. Several smokers
with bronchial deposition levels of 90 to 100% had pronounced delays in the
initiation of elimination. Patients with bronchitis had delays in elimination of up to
five hours.
Coggins and coworkers (1980) showed that rats exposed for twelve weeks to
whole cigarette smoke had marked loss of cilia at tracheal bifurcations compared
to control rats (Figure 4.7a & Figure 4.7b). Based mainly on these data, Martonen
(1992) speculated that dedicated bifurcation zones could slow mucociliary transport
in response to cigarette smoke particles.
For internal use of Philip Morris personnel only.
84
Evaluation of Cigarette Ingredients: Feasibility
Figure 4.7a Ciliated epithelium at a tracheal bifurcation in an unexposed rat. SEM x 1044.
Reprinted with permission from (Coggins et al., 1980).
Figure 4.7b Deciliation at a tracheal bifurcation in a rat exposed to cigarette smoke. SEM x 1044.
Reprinted with permission from (Coggins et al., 1980).
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
85
4.3.5 Models of Smoke Dosimetry
In addition to the biological model of the rat, used by Coggins and coworkers, and
others, investigators have employed isolated organs, physical models, and
mathematical models of the human lung to understand the dosimetry of cigarette
smoke. For example, Mitchell (1975) simulated normal breathing patterns in an
intact excised human lung at room temperature within eight hours of death. By
counting particles of diluted cigarette smoke inhaled and exhaled by the lung, he
determined that deposition of particles increased as residence time of particles in
the lung increased. Morawska and coworkers (1998) calculated that particle
residence time in the human lung could vary from four seconds (one breath in and
out) to approximately 24 seconds, assuming 3 L of alveolar gas exchanged at a
rate of 7.5 L/min-1. Phalen and coworkers (1994) reported no difference in
deposition efficiency of sidestream cigarette smoke between different sized silicone
models representing the tracheobronchial tree of young adults and children of four
and seven years of age. They calculated deposition efficiency as the deposition in
the cast model divided by the total amount measured in the cast, airways, and
filters through which the smoke exited the model.
Aerosol deposition models first were developed to aid in establishing exposure
limits for radioactive aerosols (Phalen & Oldham, 2001). To characterize the
deposition of cigarette smoke following inhalation, investigators must define attributes
of the aerosol, the morphology of the respiratory system, and ventilatory conditions
(Martonen et al., 2000). Martonen (2001) advised that models of particle deposition
include the naso-oral cavity for four reasons: (1) Humans are naso-oral breathers
(unlike laboratory rodents). (2) The naso-oro-pharynx compartment filters inspired
air. (3) The ratio of nasal to oral airflow may affect the aerosol mass delivered to
deeper airways. (4) The ability to heat and humidify inspired air varies between
the nose and mouth.
Yeh and Schum (1980) developed a model of human tracheobronchial airways
based on detailed morphometric measurements. A silicone rubber replica cast
was made by injecting liquid rubber into the lung of a deceased man who died of a
myocardial infarction and whose lungs showed no abnormalities. After the silicone
had cured in situ, the lung tissue was removed. Detailed measurements were
obtained of the airway diameters, lengths, angles of branches and inclination to
gravity. Using a similar approach to the Findeisen-Landahl computational method,
their lung model predicted the deposition of airborne particles in different regions
of the lung during different breathing patterns. They also wrote a computer program
using geometrical data from the cast and estimated inhaled particle deposition for
portions of the lung (i.e., lobe, or whole lung).
The National Council on Radiation Protection and Measurements (NCRP) developed
a model in 1997 to predict dosimetry of particles inhaled by radiation workers and
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86
Evaluation of Cigarette Ingredients: Feasibility
the general public, based on the model of Yeh and Schum (1980), but modified to
include more recent data (Yeh et al., 1996). Deposition predictions agreed
reasonably well with data from nose breathing and mouth breathing (1997). Yu and
Diu (1982) compared the Yeh and Schum model (1980) with models developed by
Weibel (1963), Olson and coworkers (1970), and Hansen and Ampaya (1975).
They determined that total deposition curves for nose breathing and mouth breathing
did not differ substantially among these models and also compared reasonably well
with other experimental data. For particles of 0.2 µm in diameter, they obtained
agreement among models for regional deposition fractions for both nose breathing
and mouth breathing.
The NCRP suggested that predictions from the Yeh and Schum model resembled
predictions of the 1971 model developed by Horsfield and coworkers (1971). The
NCRP model (Yeh et al., 1996) predicts slightly lower nasopharyngeal deposition
for particles with diameters < 1-2 µm than a 1994 model by the International
Commission of Radiological Protection (ICRP), which was based on a model by
Egan and coworkers (National Council on Radiation Protection and Measurements,
1997). Table 4-4 compares the 1997 NCRP model to earlier models. Additional
background information is available about methods to assess particle deposition in
the respiratory tract (Schultz et al., 2000).
Table 4-4. Percentage of inhaled particles (diameter 0.2 µm) predicted to deposit
in human lung regions by three models.
Naso-pharyngeal
DEPOSITION (%)
Yeh and Schumb
ICRPa
1966
1980
0
0
NCRPc
1997
1.8
Tracheobronchial
2.7
3.3
4.9
Pulmonary
28.1
14.8
16.2
Total
30.8
18.1
22.9
a
b
c
ICRP: International Comission on Radiological Protection (1966) using 750 cm3 tidal volume and
15 breaths per minute.
Yeh and Schum (1980) using 750 cm3 tidal volume, 15 breaths per minute, and a functional residual
capacity of 40% total lung capacity.
NCRP: National Council on Radiation Protection and Measurements (1997) using 770 cm3 tidal volume,
13 breaths per minute, and a functional residual capacity of 40% total lung capacity.
Biomarkers of chemical substances in cigarette smoke intrinsically measure neither
exposure nor dose. However, measures of biomarkers easily convert into exposure
estimates, through calculating the exposures that should have led to those biomarker
levels (Scott et al., 2001). Most studies of constituents of cigarette smoke in
smokers measure substances in blood, urine, or expired air. Some typical substances
For internal use of Philip Morris personnel only.
Cigarette Smoke: Exposure And Dosimetry
87
measured include thiocyanate, nicotine, cotinine, carbon monoxide and benzene, or
metabolites of nicotine, carbon monoxide and benzene, in blood or urine. Smokers
have higher internal concentrations of these substances than nonsmokers (Table
4-5) (Jarvis et al., 1984).
Table 4-5. Comparison of exposure biomarkers in nonsmokers and active smokers.a
Carbon Monoxide
(ppm in expired air)
Carboxyhemoglobin
(% in whole blood)
Nicotine (mg/mL)
Plasma
Saliva
Urine
Cotinine (ng/mL)
Plasma
Saliva
Urine
Thiocyanate (µmol/L)
Plasma
Saliva
Urine
a
NONSMOKERS
(n=100)
5.6
SMOKERS
(n=94)
21
0.9
4
0.9
4.8
8.3
15
673
1750
1.5
1.7
5
275
310
1391
50.8
1300
74.8
123
2450
155
Modified from Jarvis et al. (1984).
4.4 BIOLOGICAL MONITORING OF SMOKING
EXPOSURE
4.4.1 Nicotine
Nicotine in cigarette tobacco transfers intact during smoking (Schmeltz et al., 1979).
The boiling point for nicotine is 247ºC. Thus, nicotine distills into smoke in advance
of the burning cone, before it can undergo pyrolysis (Darby et al., 1984). The
particulate phase of smoke contains most of the nicotine in smoke (Houseman,
1973; Jenkins, Jr. et al., 1971; Jenkins, Jr., 1990). Nicotine is a useful model for
substances that transfer in smoke. Nicotine also is a tracer for the particulate
fraction of cigarette smoke.
Tobacco contains approximately 1.5% nicotine by weight and a cigarette contains
approximately 0.8 to 1.0 g of tobacco. So, cigarettes have approximately 12-15
mg of nicotine each. Tobacco contains several alkaloids, including nicotine,
nornicotine, anabasine, myosmene, nicotyrine and anatabine. The alkaloids other
than nicotine usually constitute approximately 10% of the alkaloid content (Piade
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Evaluation of Cigarette Ingredients: Feasibility
& Hoffmann, 1980). However, some varieties of tobacco have more nornicotine
than nicotine (Schmeltz & Hoffmann, 1977).
The pKa of nicotine is 8.0. At the pH of blood (approximately 7.4), nicotine is 31%
unionized. Smoke from flue-cured tobaccos is acidic, so little nicotine absorbs in
the mouth (Gori et al., 1986). However, most of the nicotine in smoke absorbs
rapidly in the human lung. Skin also absorbs nicotine base well. Nicotine absorbs
poorly in the stomach but well from the small intestine. Blood concentrations peak
approximately 1 hour after the oral administration of nicotine in capsules (Jenner
et al., 1973).
The pharmacokinetics of nicotine absorbed from cigarette smoke has been reviewed
(Benowitz et al., 1990). Peak blood concentrations of nicotine range between 10
and 50 ng/ml. Benowitz and Jacob (1984) found an approximately 40 mg daily
intake of nicotine per smoker, or 2 mg per cigarette for a one pack a day smoker,
approximately one-half of the value obtained by Feyeraband (1985). In a study
with deuterium labeled nicotine, Benowitz, et al. (1991) found that smokers inhaled
a dose of 2.3 mg of nicotine. However, these investigators suggested that a prestudy
period of abstinence increased subjects’ inhalation and that a lower dose would be
more appropriate.
Blood or urine nicotine levels are poor indicators of nicotine absorption (Benowitz
et al., 1982). Little nicotine binds to plasma proteins. The initial half-life of elimination
is approximately 2 hours and the volume of distribution is approximately 180 liters
(Benowitz & Jacob, III, 1994). However, nicotine elimination is complex, and it
changes with blood flow and smoking (Benowitz et al., 1990). Total clearance
was approximately 1300 ml/min. Kidney, liver, heart, and brain had the highest
concentrations of nicotine in rabbits and adipose tissue the lowest, after 24-hour
intravenous infusions. In rats, kidney, liver, and brain had the highest concentrations
of nicotine and adipose tissue the lowest (Benowitz et al., 1990). The
pharmacokinetics of nicotine after smoking resembles the pharmacokinetics after
rapid intravenous administration. At normal urinary pH, little nicotine reabsorbs
(Borzelleca & Lowenthal, 1967).
In the dog, the liver metabolizes most absorbed nicotine, and most nicotine undergoes
metabolism (Turner et al., 1975). Human renal elimination of unmetabolized nicotine
accounts for only 5-10% of total absorbed dose. Urinary nicotine concentrations
correlate poorly with nicotine intake. Human liver extracts most nicotine and
metabolizes it to cotinine and nicotine-N-oxide in a ratio of the two metabolites of
approximately 17:1. (Mice have a ratio of approximately 1:1, and rats approximately
1:2, which may make these common laboratory animals poor models for some
purposes.)
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Armitage and coworkers (1974; 1975) used cigarettes labeled with [14C] nicotine
to show that smokers retain as little as 29% or as much as 92% of the nicotine in
mainstream smoke, perhaps depending on how deeply they inhaled.
By subtracting the nicotine in exhaled air from the amount in mainstream smoke,
obtained by duplicating the subjects’ puffing dynamics with a smoking machine,
Frost and coworkers (1998) calculated 35% and 40% deposition of nicotine in the
mouth after zero and ten seconds, respectively. Deposition of nicotine increased
to more than 80% of smoke nicotine after a shallow inhalation of 70 mL and to
90% and 100% after typical inhalation volumes of 500 and 700 mL.
Nicotine concentrations in body fluids and tissues are relatively specific for exposure
to tobacco. Some foods, such as tomato, cauliflower and tea contain nicotine. Of
these, the highest concentrations of nicotine were measured in instant tea (up to
285 ng/g wet weight) (Davis et al., 1991). However, the influences of diet on
biological measures of nicotine metabolites are negligible (Davis et al., 1991).
Because smoking involves multiple exposures to nicotine, nicotine accumulates in
the body leading to persistent levels of blood nicotine for 24 hours each day.
Measures of peak arterial nicotine concentration ranged from 8 to 93 ng/mL in
smokers (Armitage et al., 1975; Gourlay & Benowitz, 1997; Henningfield et al.,
1993). Blood concentrations of nicotine related to the volume of the puffs inhaled.
In a review of the literature, Schneider and coworkers (2001) reported a low value
for plasma concentrations of nicotine in cigarette smokers of 10-37 ng/L and a
peak value of 19-50 ng/mL, indicating wide variation. Varying cigarette smoke
puff volume from 15 to 60 mL influenced the concentration of plasma nicotine.
Nicotine concentration increased as puff volume increased (Zacny et al., 1987).
In contrast, plasma nicotine was not influenced by varying inhalation volume from
20% to 60% of vital capacity or by holding the breath from 5 to 21 seconds, suggesting
that the inhaled cigarette smoke puff volume is the most important contributor to
plasma nicotine concentrations.
The two major metabolites of nicotine are nicotine-N-oxide and cotinine. (For
cotinine, see section 4.4.2 below). Both metabolites undergo additional metabolism.
Unmetabolized, renal elimination of nicotine accounts for approximately 9% of an
absorbed human dose, the nicotine-N-oxide metabolite accounts for approximately
4% , and 17% of absorbed nicotine has an unknown elimination pathway (Benowitz
et al., 1990).
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4.4.2 Cotinine
Human liver converts approximately 70% of nicotine to cotinine, and cotinine
undergoes extensive metabolism. Little cotinine binds to plasma proteins. Despite
extensive tubular reabsorption, renal elimination of unmetabolized cotinine accounts
for approximately 17% of the total produced. The elimination half-life of cotinine
is approximately eighteen hours and the volume of distribution is approximately
ninety liters. Urinary flow and pH have little influence on urinary cotinine elimination,
and urinary cotinine concentrations correlate well with cotinine production. These
characteristics make cotinine a good biomarker of nicotine exposure over
intermediate, and day-long periods of exposure (Galeazzi et al., 1985).
Cotinine concentrations in saliva and plasma correlate with cigarette smoke uptake
in both active and passive smokers (Jarvis et al., 1984). Curvall and coworkers
(1990) confirmed these correlations in two experiments that monitored the increase
in cotinine in plasma, saliva and urine as indices of nicotine exposure. The plasma
and salivary concentrations of cotinine demonstrated a positive linear association
with the quantity of nicotine infused, r ≥ 0.97. Individual urinary cotinine excretions,
which did not reach a plateau level within five hours of a one-hour infusion, did not
correlate well with nicotine dose. However, Andersson and coworkers (1997)
found significant differences (p < 0.01) between salivary cotinine concentrations
and cigarette consumption by Swedish smokers. For smokers consuming 11(±2),
18(±2) and 28(±6) cigarettes per day and receiving an estimated 11(±3), 19(±3)
and 30(±4) mg nicotine per day, salivary cotinine concentrations were 174(±86),
238(±99) and 330(±105) ng/mL, respectively.
The National Health and Nutrition Examination Survey (NHANES) measured the
levels of cotinine in the urines of nonsmokers representative of the U.S. population.
Between 1988 and 1991, the median level was 0.20 ng/mL (Pirkle et al., 1996).
By 1999-2000 the level decreased to 0.059 ng/mL. Smokers defined within the
study as current smokers of greater than five cigarettes per day have urine cotinine
levels of at least 10 ng/mL.
4.4.3 Carbon Monoxide
The vapor phase of cigarette smoke contains carbon monoxide (Scherer, 1999).
Breath (expired) carbon monoxide serves as a marker for deposition of the vapor
phase constituents of cigarette smoke (US Consumer Product Safety Commission,
1993). Hemoglobin binds carbon monoxide, serving as a sink for absorbed carbon
monoxide and complicating the picture of deposition and pharmacokinetics.
Because of carbon monoxide’s relatively short half-life, Benowitz (2001) suggested
that measurements of expired carbon monoxide reflected only recent exposure
(several hours). Physical activity affects the rate of carbon monoxide elimination.
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Wald & Howard (1975) calculated, based on the mathematical modeling of data
gathered by Coburn and coworkers (1965), that the half-life of carbon monoxide
varies from four hours during sedentary activity to approximately one hour during
vigorous physical activity. Carbon monoxide is not specific to cigarette smoke.
Other sources could influence exposure, including air pollution. Humans produce
small amounts of carbon monoxide endogenously, accounting for carbon monoxide
normally found in exhaled air and blood. An advantage of using expired carbon
monoxide as a biomarker of cigarette exposure is that the method is automated.
Results are immediately available (Secker-Walker et al., 1997).
Bentrovato and coworkers (1995) measured the expired air of thirteen adult smokers,
immediately prior to their 5th through 9th cigarettes of the day, for four consecutive
days. The carbon monoxide concentrations of their expired air varied from 6 ppm
to 44 ppm (mean 25 ppm) and correlated poorly (R2 = 0.44) with the yields of
carbon monoxide per cigarette as measured by a smoking machine programmed to
duplicate the human smoking profiles (Bentrovato et al., 1995). Thus, oral delivery
of carbon monoxide does not predict uptake from smoke. Secker-Walker et al.
(1997) examined the association between self-reported cigarette consumption and
expired carbon monoxide in pregnant women. Data were collected from 521
women at their first visit to a prenatal clinic for underserved women. For the
second visit at 36 weeks, 365 of the women returned. The correlations between
cigarette consumption (number of cigarettes per day) and expired carbon monoxide
are 0.65 for the first visit and 0.70 for the second (p < 0.001 for both). Self-reports
suggested that the participants provided crude estimates of cigarette consumption.
Varying puff volume from 15 to 60 mL influenced the concentration of carbon
monoxide in expired air, which increased with increasing puff volume (Zacny et
al., 1987).
The carbon monoxide concentration of exhaled air is closely related to the carbon
monoxide saturation of the hemoglobin. Carbon monoxide binds reversibly to
hemoglobin to form carboxyhemoglobin in a tighter complex than the oxygenhemoglobin complex. Therefore, carboxyhemoglobin compromises oxygen transport
by hemoglobin.
Smokers have higher concentrations of carboxyhemoglobin than nonsmokers
(Behera et al., 1991). A diurnal rhythm is evident in smokers, with higher
concentrations during the day (Wald & Howard, 1975). The concentration of
carboxyhemoglobin ranges from 3% in nonsmokers to 17% or higher in smokers
(Behera et al., 1991). Under experimental conditions, inhalation of 7,000 to 24,000
ppm carbon monoxide by 18 healthy men for three to five minutes followed by
exposure to 232 ppm carbon monoxide for up to 130 minutes, achieved
carboxyhemoglobin levels of 16% to 23% with no significant oxygen deprivation
symptoms (Benignus et al., 1987). In contrast, accidental exposure to approximately
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Evaluation of Cigarette Ingredients: Feasibility
500 ppm of carbon monoxide for 150 minutes or less resulted in complaints of
illness from 160 of 184 exposed persons, whose carboxyhemoglobin levels were
30% or less (Burney et al., 1982). Increasing exposures result in symptoms of
headache, dizziness, muscle weakness, visual problems, nausea, cardiovascular
dysfunction, CNS derangement, coma and death (Burney et al., 1982; Crocker &
Walker, 1985). For children less than 15 years of age, carboxyhemoglobin levels
of 24% or higher are associated with syncope (Crocker & Walker, 1985). Although
patients with serious carbon monoxide poisoning have carboxyhemoglobin values
exceeding 25% (Sloan et al., 1989), carboxyhemoglobin concentration is not
necessarily indicative of a prognosis (Burney et al., 1982; Raub et al., 2000).
Behera and coworkers (1991) measured blood carboxyhemoglobin in 58 healthy
nonsmoking men, 25 asymptomatic male cigarette smokers and 20 symptomatic
male smokers having persistent episodes of coughing for more than one month.
Blood samples were drawn from smokers exactly 10 minutes after the subject
finished smoking his own cigarette in his usual manner. Carboxyhemoglobin levels
were 3.72 ± 1.15% for nonsmokers, 15.33 ± 1.98% for smokers, and 17.24 ±
3.41% for symptomatic smokers. For smokers, the self-assessed depth of inhalation
was directly proportional to the blood carboxyhemoglobin level (p < 0.05). Because
of its short half-life, measures of carboxy-hemoglobin reflected recent exposure,
similar to measures of carbon monoxide in expired breath.
4.4.4 Other Substances
Cyanide occurs in the vapor phase of cigarette smoke, as a combustion product of
tobacco, humans metabolize it primarily to thiocyanate. Thus, blood thiocyanate is
a biomarker of cigarette smoke exposure. Blood thiocyanate concentrations
positively correlated with the number of cigarettes smoked per day when expressed
relative to ideal body weight (Gardner et al., 1984). Longitudinal studies tracking
changes in repeated measures of blood thiocyanate for one individual are supportive
of periods of smoking cessation and/or resumption of smoking (Pettigrew & Fell,
1972).
Blood thiocyanate concentration may not be useful for verifying whether a person
is a smoker or nonsmoker, because of a considerable overlap between the groups.
Cyanides are also found in common foods, such as leafy vegetables and nuts,
which contribute to the high variability of serum thiocyanate concentrations among
individuals (Borgers & Junge, 1979; Pettigrew & Fell, 1972; World Health
Organization, 1986).
Concentrations of benzene in the homes of smokers exceed benzene concentrations
in the homes of nonsmokers. The average overnight air concentration of benzene
in homes with one or more smokers increased two-fold or more (Wallace et al.,
1987). Higher indoor benzene concentrations in homes with smokers are consistent
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Cigarette Smoke: Exposure And Dosimetry
93
with the hypothesis that cigarette smoke represents a source (Wallace et al., 1987).
Jo and Pack (2000) recently used breath analysis to estimate exposure to benzene
associated with active smoking involving five different brands of cigarettes, two
each from Korea and the U.S. and one from Japan (Table 4-6). The five subjects
ranged from 22 to 24 years old. The average breath concentration of benzene
before smoking was 17.6 µg/m3 (range 15.9 to 19.2 µg/m3) and one minute after
smoking was 69.9 µg/m3 (range 58.1 to 81.30 µg/m3). Thus, breath after smoking
was three to five times higher in benzene, depending on the brand, than before
smoking.
Table 4-6. Breath concentrations (µg/m3) of selected smoke constituents by smokers (S) and
nonsmokers (NS) [Data given as unweighted geometric averages]a
Location
New Jersey
California,
Los Angeles
California,
Antioch/Pittsburg
a
b
c
d
e
Falld
Summer
Winter
Winter
Spring
Sc
150
66
26
29
11
NS
188
76
23
85
40
Benzeneb
S
NS
21
5.3
-e
15
2.4
14
1.6
Spring
19
49
14
Season
n
0.8
Ethyl-benzene
S
NS
3.9
2.0
2.0
0.5
2.3
1.1
2.4
0.6
3.2
0.5
2.0
0.3
S
1.3
1.0
0.6
0.8
1.9
Styrene
NS
0.6
0.3
0.2
0.2
0.1
1.1
0.2
Modified from Wallace et al. (1987). All comparisons of concentrations between smokers’ and
nonsmokers’ geometric means differed significantly (P < 0.001). Reprinted with permission of
Heldref Publications.
Measurements for benzene may have been elevated due to permeation of exhaust fumes into vanmounted spirometer.
S = smokers; NS = nonsmokers.
Measurements during this visit may have been elevated due to permeation of exhaust fumes into
van-mounted spirometer.
Not reported.
Estimates suggest that the most important source of benzene exposure for smokers
is their cigarette consumption (1989). Benzene concentrations in the exhaled breath
of smokers were six- to seven-fold higher than for nonsmokers (Wallace et al.,
1987). Smokers’ breath styrene concentrations were four-fold higher, and
ethylbenzene concentrations were three-fold higher, compared with nonsmokers
(Table 4-6). Breath concentrations of benzene correlated with the number of
cigarettes smoked. As shown in Figure 4.8, the benzene concentrations varied
from 9.4 ug/m3 for nonsmokers to 30 µg/m3 for smokers of 40 cigarettes on the
day of measurement. For heavy smokers (>50 cigarettes per day) breath benzene
was 47 µg/m3. Breath analysis of benzene also generated estimates of absorption
efficiency, biological half-life, and exposure to passive smoke. Individuals smoking
33 cigarettes per day inhale approximately 2.0 mg per day of benzene compared to
a nonsmoker, who inhales approximately 0.2 mg per day.
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Evaluation of Cigarette Ingredients: Feasibility
Figure 4.8 Average benzene concentrations in breath as a function of number of cigarettes smoked
by cigarette smokers. Values are unweighted geometric means. The numbers of subjects in each
category were 10, 37, 47, 37, 20, and 7, respectively.
Copyright 1987 from Archives of Environmental Health, by Wallace, et al. Reproduced with
permission of Heldref Publications.
The Massachusetts Tobacco Control Board requires test data on the urinary
concentration of a benzene metabolite, trans, trans-muconic acid, using a method
described by Melikian and coworkers (1993), or a similar method approved by
their Department of Health, in smokers of each brand.
4.5 EXPERIMENTAL ANIMAL EXPOSURE TO
CIGARETTE SMOKE
4.5.1 Methods for Experimental Animal Exposure to Cigarette
Smoke
Thomas and Vigerstad (1989) cite many early reports of exposing animals to whole
cigarette smoke, in contrast to fractions of the smoke. Historically, investigators
preferred rodents, including rats, mice, hamsters and guinea pigs (Coggins, 1998;
Witschi et al., 2000) (Wright & Churg, 2002a; Yamato et al., 1996). Under similar
experimental exposure conditions, different animal species responded differently
to cigarette smoke (Fishbein et al., 2000). Studies of exposure of animals to
cigarette smoke have not replicated the diseases observed in human smokers in
epidemiological surveys (Coggins, 2001).
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95
Phalen and coworkers (1984) reviewed four methods of inhalation exposure for
animal experiments, whole body, head only, nose or mouth only, and lung by
intubation or tracheostomy. A common problem with voluntary breathing methods
is that animals prefer to breathe air and reject cigarette smoke. They hold their
breath and/or cough, when exposed to smoke (Battista et al.,1973). Whole body
exposure contaminates the animals’ skin, fur and eyes with smoke (Kuller et al.,
1989). The animals ingest these contaminants during grooming, making the exposure
pathway different and dosimetry difficult.
Whole body cigarette smoke exposure systems modified from those developed by
Escolar and coworkers (Escolar et al., 1995) (Figure 4.9) were used in murine
models of acute pulmonary oxidative stress and chronic emphysema (Cavarra et
al., 2001b; Cavarra et al., 2001a). Whole body cigarette smoke exposure systems
have also been developed for use in murine models of smoke-induced lung tumors
from machine-generated sidestream smoke (D’Agostini et al., 2001; Teague et
al., 1994; Witschi, 2000b). In whole body studies, urinary cotinine may not be a
meaningful indicator of smoke exposure, because oral ingestion during grooming
contributes to nicotine exposure and metabolite level (Vanscheeuwijck et al., 2002).
Figure 4.9 Schematic representation of whole body exposure of rats to cigarette smoke. A cage
containing three animals and without filter cover was placed in a chamber of 0.42 m3. The chamber
had three orifices of 0.75 cm diameter (A) and six of 3 mm (B). A lit cigarette that was connected
to a compressor was introduced through one of the 0.75 cm orifices (C); air generated by two
compressors was introduced through the remaining 0.75 cm orifices (D). The air flowed out of the
chamber through the 3 mm orifices. Copyright 1995 from Experimental Lung Research, by Juan
de Dios Escolar, et al. Reproduced with permission of Taylor & Francis, Inc., http://
www.routledge-ny.com
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Evaluation of Cigarette Ingredients: Feasibility
The head only method of exposure has the advantages of minimal skin contamination,
uniform individual exposure, conservation of smoke, and difficultly in avoiding
inhalation. However, restraint adds stress to the design. The most popular method
for delivering cigarette smoke to experimental rodents is by nose only exposure.
Rodents are obligate nose breathers. Major advantages are the reduction of skin
contamination (compared to whole body exposure) and the ability to pulse smoke
exposure at rates similar to human puff patterns. The smoke is also easier to
contain and less smoke is used than the whole body or head only methods (Battista
et al., 1973; Phalen et al., 1984). Disadvantages include technical difficulties in
sealing the face/nose, and labor intensiveness (Phalen et al., 1984).
Matulionis (1984) tested four early models of nose only exposure systems designed
for use with rats, mice or hamsters. For the same species, responses varied between
the different exposure systems. Nose only systems lend themselves to measuring
smoke concentrations and processing smoke before exposure, including cooling
and dilution of smoke with air (Baumgartner & Coggins, 1980).
Animal models present an opportunity to control experimental exposure variables,
such as smoke concentration and duration. However, to date no animal model has
replicated the major features of human smoking-related diseases. In addition,
exposure of animals to whole cigarette smoke has a major, perhaps insurmountable
problem, carbon monoxide poisoning.
4.5.2 Carbon Monoxide Pharmacokinetics in Animal Species
Like canaries in coal mines, small mammals like mice and rats have higher metabolic
rates in relation to their body size than humans. Studies exposing rats to diluted
cigarette smoke for 2 hours a day over 2 weeks reported COHb levels reaching 20
to 40% placing considerable stress on the rat (Carmines et al., 2003). They also
reported the need to interrupt the 2 hour exposure with a 30 minute smoke free
period to allow COHb dissociation due to concerns of potential CO related mortality
in the rodent. Smaller animal species, such as rodents are considerably more
sensitive to CO levels than humans. For human sensitivity to carbon monoxide,
see the preceding Section 4.4.3. No lasting and important functional effects occur
at exposures that generate carboxyhemoglobin up to 17%. Exposure-response
relationships for carbon monoxide and various biological effects have been reviewed
for other species (Benignus et al., 1990).
Efforts to understand the toxicology, physiology, and pathology of acute carbon
monoxide poisoning have proceeded for more than a century (Penney, 1990). Even
so, whether the effects of carbon monoxide result from hypoxia secondary to
carboxyhemoglobinemia or direct entry into critical organs and cells, where it binds
key components, remains unknown (Winneke, 1992). Further, the overall literature
about the exposure-response relationship between carboxyhemoglobin and carbon
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monoxide effects, including human behaviors, is inconsistent (Benignus et al., 1990).
Across many species, the exposure-response relationships are nonlinear. Besides
mortality when carboxyhemoglobin levels exceed 20%, effects on the
cardiopulmonary system, metabolism, and the central nervous system are most
prominent. Short of mortality, the most exposure sensitive morphological indicator
is damage to the white matter in the central nervous system. Carbon monoxide
induces acidosis and a prominent hypotension, which compromises blood flow and
worsens the acidosis.
4.5.3 Absence of Animal Models of Human Health Effects
The notion of biological continuity between species forms the basis of any effort to
obtain an animal model or to extrapolate results between species (Winneke, 1992).
However, the idea that animal models, particularly chronic bioassays in rats and
mice, predict the human health effects from smoking represents confusion between
regulatory policy needs and scientific needs for animal models. Regulatory policy
is based on the notion that carcinogenic results in animal models can be directly
applied to humans. This policy forms an inadequate basis for scientific research,
which requires a replication of the modeled phenomenon in all essential aspects, to
achieve predictivity. Otherwise, research findings in an animal model cannot provide
useful scientific information (Bukowski et al., 2001).
Some studies have implicated isolated substances in lung cancer induction based
on their presence in cigarette smoke and their pulmonary carcinogenicity in lab
animals (Hecht, 1999). It is implied that studies of animal exposures to whole or
fractionated cigarette smoke are predictive of the disease observed in human
smokers. A statement that a particular substance, typically a polycyclic aromatic
hydrocarbon or tobacco-specific nitrosamine, causes lung cancer in laboratory
animals provides important information when put in an appropriate context. The
quantitative presence of the substance in cigarette smoke should be related to the
carcinogenic potency of whole cigarette smoke, or, should qualitatively explain
disease properties. In reality, potencies of the identified carcinogens in cigarette
smoke sum to a small portion of the quantitative potency of smoking (Fowles et al.,
2000; Menzie Cura & Associates, 1999). No single or group of chemicals in
smoke has been identified which can explain the full range of human health effects
observed in epidemiological studies of cigarette smokers.
Studies of exposure of animals to cigarette smoke have not replicated the range of
diseases observed in epidemiological surveys of human smokers (Coggins, 2001).
Most studies of the effects of cigarette smoke in animal models have concentrated
on carcinogenic effects. One example is the Witschi model developed to study the
induction of pulmonary adenomas by cigarette smoke (Witschi et al., 1997b; Witschi,
1998). Witschi et al. (1997b; 1999; 2000; 2002a) developed a smoke exposure
procedure using the A/J strain mouse model to screen potential chemopreventive
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Evaluation of Cigarette Ingredients: Feasibility
agents. The authors subjected strain A/J mice for five months through wholebody exposure to a mixture of mainstream and side stream smokes, better replicating
human smoking conditions (Witschi et al., 1997b). After a recovery period of
exposure to room air for four months he found excess pulmonary adenomas. Later,
Witschi exposed female A/J mice to unfiltered or HEPA-filtered smoke and found
that the volatile or gas phase of smoke caused as many tumors as full smoke
(Witschi et al., 1997a; Witschi, 1998).
An increase in adenomas in mice is not a compelling finding. Cigarette smoke
apparently accelerates the appearance of a mouse tumor that usually occurs at a
high incidence in strain A/J. However, pulmonary adenomas have acquired new
prominence in studies of the epidemiology of cigarette smoking. The proportion of
lung adenocarcinomas to squamous cell carcinomas of the lung has recently
increased among cigarette smokers. Some investigators link this trend to an
increased use of cigarettes with filters (Hoffmann et al., 1996). The argument is
two-fold: (1) Lower nicotine yields increase smoking intensity, leading to more
smoke deep in the peripheral lung, a site more prone to develop adenocarcinomas.
(2) Smoke from filtered cigarettes produces more of a lung-specific carcinogen, 4(N’-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone) (NNK).
4.6 SUMMARY
This chapter outlines the exposure, and some of the dosimetry, of human smoking.
The previous chapter covered the properties of cigarette smoke, and the following
chapter explains some of the adverse human health effects observed in
epidemiological studies. Thus, the three chapters form a logical progression. Other
than this linkage, the chapter should discourage sophisticated exposure or dose
measurements as a way of characterizing the relative risk of a cigarette with an
ingredient compared to an identical cigarette without the same ingredient. However,
simple measurements, such as smoking rates (the number of cigarettes smoked
per unit of time) or biomarkers of smoking exposure, given some smoking rate,
hold promise.
Dose and duration of exposure are the primary determinants of the mortality and
morbidity associated with cigarette smoking. The relationship between inhalation
exposure and dose is scientifically challenging. Exposure can either be calculated
or measured. Dose often is inaccessible, and direct measurement is not possible.
For a mixture with thousands of chemical components, the analysis of dose of
cigarette smoke is particularly daunting. However, biomarkers of exposure often
come closer to dose estimates. In addition, some information is available about the
human absorption, distribution, and elimination of whole smoke or the particulate
fraction of cigarette smoke. Smoking affects the condition of the body. Therefore,
exposure changes over time.
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No two individuals smoke cigarettes in the same way. A smoker’s pattern of
smoking differs while smoking the same cigarette. Smoking behavior is affected
by yield of nicotine, tar, taste, irritation, and draw resistance. Smoking behavior
influences smoke substance delivery to the respiratory tract. When cigarettes are
altered through engineering design, smokers may compensate to obtain the same
nicotine yield. Compensation appears to involve small increases in the number of
cigarettes consumed and large changes in inhalation.
Monitoring of chemical substances to which an individual is exposed provides some
information and may be achieved with biomarkers of smoke exposure, such as
cotinine or carboxyhemoglobin. Experimental animal exposure has several
disadvantages: (1) the animal models do not replicate the diseases that smokers
have, (2) the animals die of carbon monoxide poisoning, when exposed to whole
smoke in ways that mimic human exposure and (3) the animal’s life span limits the
long term latency period necessary for production of some of the observed human
health effects.
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5
ADVERSE HUMAN HEALTH EFFECTS
OUTLINE
5.1
INTENT & PURPOSE
5.2
PREMATURE MORTALITY FROM CIGARETTE SMOKING
5.2.1 Communication of Premature Mortality
5.2.2 Changes in Cigarette Related All-Cause Mortality
5.2.3 Detection Bias
5.2.4 Mathematical Cancer Models
5.2.5 Interactions with Other Carcinogens
5.3
CIGARETTE-RELATED CAUSES OF PREMATURE MORTALITY
5.3.1 Cardiovascular Diseases
5.3.2 COPD
5.3.3 Cancer
5.3.4 Mortality From Other Diseases
5.3.5 Infant Death
5.3.6 Morbidity and Other Health Effects
5.3.7 Disease and Exposures not Covered in This Report
5.3.8 Relationship of Risk Analysis to Safety Assessment
5.4
SUMMARY
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5
ADVERSE HUMAN HEALTH EFFECTS
5.1 INTENT & PURPOSE
The feasibility of testing an ingredient added to cigarettes relates to the potential
health effects of the ingredient, which could arise from any of three processes: (1)
inhalation of the ingredient or its pyrolysis products, (2) changes the ingredient
induces in the chemical and physical structure of the surrounding matrix of cigarette
smoke, and (3) changes the ingredient induces in smoking behavior. Changes in
smoking behavior could affect smokers’ health indirectly through, for example, the
consumption of more cigarettes or deeper inhalation.
To exert direct health effects after inhalation, an ingredient would have to survive
pyrolysis and either transfer intact in cigarette smoke or have a pyrolysis product
that would transfer into smokers’ lungs. The presence of an ingredient could
change the adverse outcomes of cigarette smoking, either by creating a new
outcome, directly related to the presence of the ingredient or its pyrolysis products
in the smoke, or by changing the known outcomes of smoking. This chapter
summarizes the adverse human health effects of cigarette smoking.
For LSRO, one question remains preeminent. Will ingredients added to cigarettes
in some way change the human health risks already associated with cigarette
smoking? If so, how will testing allow detection of (and reduction in) the amounts
of those ingredients? Many studies document the adverse health effects of cigarette
smoking. Detailed descriptions and analyses of these studies fall beyond the scope
of this report. Instead, this chapter refers readers elsewhere for more detailed
information (National Institutes of Health, 1997; Wilson & Crouch, 2001).
LSRO finds that epidemiological information has provided almost all of the available
information about the human health effects of cigarette smoking. In LSRO’s view,
tests that predict the results of epidemiological studies of human cigarette smoking
are unavailable. Animal models of human health effects are also unavailable.
Even so, epidemiological studies also have limitations.
Epidemiology is a crude instrument to detect differences, like the differences
expected between a population smoking cigarettes without an ingredient and a
similar population smoking nearly identical cigarettes with the ingredient. Because
the primary approach, comparison of populations depends on stochastic measures,
statistical considerations limit the kinds of possible comparisons. Small differences
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Evaluation of Cigarette Ingredients: Feasibility
lie beyond practical means; they require unmanageably large populations. Studies
that seek to measure outcomes that require long durations of observation also
appear impractical; members of the study populations would migrate and/or change
their smoking habits. Epidemiological data seldom, if ever, directly inform mechanism
or causation, although they can disprove hypotheses. Instead, scientists require a
surrounding context of information from different sources. Epidemiological studies
generate associations.
Other reasons exist to conduct more kinds of studies than human epidemiological
studies. For example, many screening toxicology studies are established parts of
regulatory practice, essentially a form of conventional wisdom. Despite their lack
of predictivity for human health effects of cigarette smoking, these tests are thought
predictive of general health effects. If an ingredient added to a cigarette transfers
into human lungs in smoke, as some will, screening to detect novel effects will
come into play, using the standard methods of inhalation toxicology. Decisions to
employ these methods will be matters of scientific judgment. LSRO can devise no
science-based rationale to apply these methods in uniform ways to all ingredients
added to cigarettes, nor should they be uniformly excluded.
5.2 PREMATURE MORTALITY FROM CIGARETTE
SMOKING
Overall, chronic cigarette smoking causes premature mortality. Premature mortality
means that, on average, a group of persons with a defined characteristic has a
different, shorter average life span than persons without this characteristic (Cohen,
1991). For example, a population of smokers lives for fewer years than an otherwise
similar population of nonsmokers. Data from many epidemiological studies supports
the idea that chronic cigarette smokers have shorter life spans than nonsmokers
(Doll et al., 1994; Doll & Peto, 1976; National Institutes of Health, 1997). No
credible epidemiological studies contradict this conclusion. Premature mortality is
most easily determined by taking otherwise similar persons (with similar incomes,
body weights, etc.) of approximately the same age and dividing the death rate of
the smokers by the death rate of nonsmokers. If this ratio exceeds 1.0 (100%), then
subject to statistical qualifications, the smokers experienced premature mortality.
The best way to avoid the adverse health effects of cigarette smoking is never to
begin smoking. However, once smokers begin, the second best health protective
approach is to cease smoking as soon as possible. After smokers cease smoking,
all-cause premature mortality begins to decline (Kuller et al., 1991). The rate and
extent of this decline is influenced by a number of factors including the number of
cigarettes smoked and the duration of smoking. Whether this decline depends on
the age of cessation is unknown. Thus, smoking cessation remedies some, but not
all, of the increased incidence of premature mortality. Chronic obstructive pulmonary
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Adverse Human Health Effects
103
disease (COPD) outcomes decline little, if at all, after smoking cessation (Davis &
Novotny, 1989).
5.2.1 Communication of Premature Mortality
To understand the effects of an ingredient, LSRO relies most on a perspective of
premature mortality. The most important objective is that the ingredient should not
contribute to, or increase premature mortality experienced relative to smoking the
same cigarette without the ingredient. The ingredient also should not contribute to,
or increase, any of the diseases or causes of death associated with premature
mortality from cigarette smoking, including cardiovascular diseases, COPD, and
cancer. Evidence that the ingredient does not contribute to, or increase, one of
these diseases is not sufficient. An ingredient might be shown hypothetically, to
decrease carcinogenesis, but might increase one of the other diseases associated
with premature mortality from cigarette smoking. Thus, a meaningful “no detectable
change” in premature mortality has appeal in the context of ingredients added to
cigarettes.
Risk assessors argue about the best way to communicate premature mortality.
Expressions vary, particularly depending on the population and time of application.
In this chapter, LSRO refers to the current U.S. population. Cigarette smoking
would cause little premature mortality in a human population with a short life
expectancy. As life expectancy of the U.S. population increased over the past
century, medical authorities have eliminated some previously notable causes of
death, mostly infectious diseases (Amler & Eddins, 1987). The list of most prominent
preventable sources has changed.
Each year the Centers for Disease Control and Prevention (CDC) present a tabulation
of nationwide death certificate reports (Hoyert et al., 2001). The National Center
for Vital Statistics processes this information and reports total mortality in the United
States, age-adjusted death rates, as well as enumerating life expectancy at birth and
leading causes of death (Hoyert et al., 2001). This information provides the backbone
for other calculations that rank the importance of all the risk factors for diseases.
The CDC follows guidelines set by the World Health Organization (WHO) that
require member organizations to classify and code deaths using the International
Statistical Classification of Diseases and Related Health Problems (ICD) (World
Health Organization, 1992). WHO guidelines allow mortality statistics from all
participating countries to be compared, by installing a uniform format for the reporting
of cause of death on a death certificate in the form of an ICD code. The WHO
continually revises the ICD guidance. Revisions can lead to some discrepancies in
the data from year to year, because the definition of a disease may change or
because the code for a disease may change. Thus, part of the CDC’s annual vital
statistics report must address the similarity between mortality statistics from year
to year, due to changes in the definitions of diseases.
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Evaluation of Cigarette Ingredients: Feasibility
The basic way to capture statistics about the cause of death is through death
certificates (Hoyert et al., 2001). The issuer of a death certificate determines the
disease or injury that lead to death, or the circumstances of the accident or violence
which produced the fatal injury (National Center for Health Statistics, 1991). The
CDC tabulates data from all death certificates and creates a rank-ordered list of
causes of deaths (Hoyert et al., 2001). Age-adjusted death rates are also critical
to the analysis of the data presented in the cause of death tables, because those
rates correct for the effects of age. The age-adjusted data represent a more
accurate way to indicate relative incidences of diseases and examine changes in
incidence of death from specific diseases over time.
Mathematical formulae are applied to the vital statistics data to attribute mortality
to risk factors (Centers for Disease Control and Prevention, 1993). This information
is important, because the causes of all diseases are multifactorial. For example, to
determine what number of heart disease deaths to attribute to smoking, the CDC
uses a formula based on smoking prevalence and relative risks for heart disease
that compares current and former smokers with never-smokers. The result is
known as the Smoking-Attributable Mortality (SAM) (Centers for Disease Control
and Prevention, 2002b). SAM is made up of three types of deaths: death from
smoking-related diseases for adults aged 35 years or older, deaths from smokingrelated diseases for infants and deaths from cigarette-related fires (Centers for
Disease Control and Prevention, 1999).
The CDC uses data on the prevalence of current and former smokers (Centers for
Disease Control and Prevention, 1997). By obtaining data from the Behavioral
Risk Factor Surveillance System, combined with relative risk estimates from the
American Cancer Societies Cancer Prevention Study II (1982-86), the number of
deaths from smoking-related diseases from Vital Statistics Data, the number of
deaths associated with cigarettes from the National Fire Protection Association and
population estimates from the U.S. Census, the CDC can prepare estimates of the
number of deaths caused by smoking for each state. This information is part of a
national database called the Smoking-Attributable Mortality, Morbidity and Economic
Costs database (SAMMEC) (Centers for Disease Control and Prevention, 1999).
Cohen (1991) suggested that the traditional measure of risk assessors, events per
million persons (or events per million persons per year) may generally distort
comparisons between risks. He developed loss of life expectancy (LLE) as an
alternative expression instead. For cigarette smoking, he estimated the risk of
smoking one to two packs of cigarettes per day over a lifetime for men at
approximately -6.8 years lost life expectancy per smoker (Cohen & Lee, 1979). A
more recent calculation independently supported this estimate: approximately -6.6
years LLE for one or more packs per day per lifetime male smoker (Lew &
Garfinkel, 1987). A similar government-approved statistic is person years of life
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Adverse Human Health Effects
105
lost (YPLL). Calculations of YPLL show how many productive life years, premature
mortality takes. The CDC estimates YPLL due to smoking, by multiplying ageand sex-specific smoking attributable mortality (SAM) by remaining life expectancy.
YPLL figures for the 1995-1999 period indicate that SAM translated into a loss of
3,332,272 YPLL for men and 2,284,113 YPLL for women (Centers for Disease
Control and Prevention, 2002a). These figures include smoking-attributable fire
deaths but exclude any deaths due to second-hand smoke.
The precision or usefulness of the LLE, or YPLL, may be debated. The method
used by Federal agencies to calculate premature mortality, inevitably generates
some double counting. Smoking related premature mortality is currently set in
excess of 400,000 deaths per year in the U.S., or an annual rate of approximately
1.4 X 10-3 (Centers for Disease Control and Prevention, 2002a), others disagree
and set the death toll lower at approximately 250,000 deaths per year, for an annual
rate of approximately 9 X 10-4 (Choi & Pak, 1994; Siegel et al., 1994; Sterling et
al., 1993). Whatever the most precise estimate, the seeming avoidable and selfinflicted nature of these deaths and the associated morbidity makes them nearly
intolerable to the public health community.
5.2.2 Changes in Cigarette Related Mortality
Two large-scale U.S. prospective studies, Cancer Prevention Study-I (CPS-I) and
Cancer Prevention Study-II (CPS-II), conducted under the auspices of the American
Cancer Society (ACS), examined death certificates of white middle-class smokers
and nonsmokers (Thun et al., 1995; Thun et al., 1997). Between the 1960s to the
1980s, when ACS conducted the two studies, overall premature mortality doubled
among women and continued approximately the same among men. However, the
diseases contributing to mortality changed. Lung cancer surpassed coronary heart
disease as the major cause of death in the second study (Thun et al., 1997). Thus,
the exact magnitude of premature mortality, and the precise magnitude of the major
contributions to it, varied from study to study, even when the studies were conducted
under analogous study conditions. However, the conclusion that cigarette smoking
causes premature death is consistent over all epidemiological studies.
In addition, causes of death can change from study to study. For example, from
CPS-I to CPS-II, an increase in smoking-related deaths from cancers and COPD
more than offset a decrease in deaths from vascular diseases in male smokers of
all ages (Thun et al., 1995). Premature mortality of male smokers increased from
1.7 to 2.3 between CPS-I and CPS-II (Thun et al., 1995). Table 5-1 gives a
summary of smoking related mortality taken from Smoking and Tobacco Control
Monograph No. 8 (National Institutes of Health, 1997) which includes the CPS I
and II studies as well as the Kaiser Permanente study (Friedman & Tekawa,
1997). Numbers of deaths for the four major smoking-related diseases represent
approximately 50% of the total smoking related mortality in all three of these studies.
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13551
9899
405
CPS I
CPS II
Kaiser
208
6232
4921
Woman
613
16131
18472
Total
53
1781
1035
Men
54
1014
157
Woman
107
2795
1192
Total
18
422
284
Men
10
303
56
Woman
COPD
28
725
340
Total
107
2722
6068
Men
50
1161
1248
Woman
CHD
157
3883
7316
Total
11
476
960
Men
20
423
537
Woman
Stroke
31
899
1497
Total
53
51
56
major SRDs
% death from 4
For internal use of Philip Morris personnel only.
Kaiser Permanente (Friedman & Tekawa, 1997) data are age specific (35 and older) smoking-related deaths due to all smoking-related causes including the four
major SRD’s. Data is based the number of deaths from male and female current smokers.
Cancer Prevention Study (CPS-I and CPS-II) (Thun et al., 1997) data represent the age adjusted death rates from all cause mortality as well as the data for the
individual four diseases. The figure for total mortality (all cause mortality) consists of all men and women listed as current smokers that died of a smokingrelated disease.
Summarizes smoking related deaths as reported in Smoking and Tobacco Control Monograph No. 8 (National Institutes of Health, 1997) which includes data
from three epidemiological studies. Values are given for total smoking-related mortality as well as figures for the four major smoking-related diseases (SRDs);
lung cancer, chronic obstructive pulmonary disease (COPD), coronary heart disease (CHD) and stroke.
Men
Lung Cancer
STUDY
Total Mortality
Table 5-1 Summary of Smoking-Related Deaths
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Evaluation of Cigarette Ingredients: Feasibility
Adverse Human Health Effects
107
Studies in other countries have confirmed these broad conclusions and show that
the effects of cigarette smoking on premature mortality do not vary greatly with
culture (Forey et al., 2002). For example, Yuan and coworkers (Yuan et al., 1996)
showed that middle-aged male smokers of 20 or more cigarettes per day in Shanghai,
China, had a 60% greater risk of death relative to lifelong nonsmokers. More
generally, the seven countries study, specifically examined cross-cultural factors in
the mortality effects of cigarette smoking by looking at the pooled mortality
experience of men who smoked ten or more cigarettes per day followed for twentyfive years. They found that the association between all cause mortality (1.8; 95%
confidence interval, 1.7-1.9) did not vary across the studied countries (Jacobs, Jr.
et al., 1999). These investigators attributed a few instances of reversal of the
association of the four causes of premature mortality within one of the studied
countries, to the statistical effects of random variation associated with inevitably
smaller numbers of subjects within one country, occasionally low prevalence of
disease among never smokers, and multiple comparisons.
Because cigarette smoking amplifies the background incidence of at least three of
the diseases that contribute to premature mortality, hypothetically cigarette smoking
might accelerate aging, prematurely harvesting deaths. Two approaches show
that this hypothesis is not true. Cigarette smoking exerts an overall adverse effect
and does not merely accelerate normal mortality experience. First, the overall
mortality experience of smokers changes relative to that of nonsmokers, as a cohort
ages and becomes deceased. The smokers die earlier. The diseases associated
with cigarette smoking have greater prominence among smokers within a cohort
that has aged sufficiently that almost all of its members have died. Second,
epidemiologists have studied the effects of smoking cigarettes among the elderly.
A small study of individuals age 65 or older in communities of three U.S. states
(Massachusetts, Iowa and Connecticut) with significant prevalence of smoking
showed that the premature mortality effects of smoking extend to older ages
(LaCroix et al., 1991). A recent study in France confirmed these findings. Deceased
elderly persons smoked more cigarettes than those still living during an eight-year
follow-up, and smoking was associated with mortality among current smokers
(Tessier et al., 2000).
Gender-specific effects of cigarette smoking remain uncertain. Cultural factors
clearly account for differences in the prevalence of smoking rates between men
and women in different countries and in cohorts born at different times. So, men
and women experience different adverse health effects of cigarette smoking. Some
investigators have looked for gender-specific target organ effects. A ten year
follow-up of 117,557 U.S. women who initially described their smoking habits
revealed no association between smoking of 25 or more cigarettes per day and
breast cancer (London et al., 1989). Such studies help to define the specificity of
the adverse human health effects of cigarette smoking.
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Evaluation of Cigarette Ingredients: Feasibility
When investigators controlled for age, duration of smoking, and smoking rates between
men and women, gender-specific sensitivity appeared elusive. Prescott and
coworkers (1998) examined a Danish cohort and found both total and cause-specific
mortality similar in male and female smokers relative to nonsmokers. After excluding
non-inhalers, female smokers had a slightly higher relative incidence of respiratory
and vascular diseases than male smokers, but no gender differences were found in
the relative incidence of cancers. However, nonsmoking Danish women had lower
mortality rates of respiratory and vascular diseases, so the investigators noted that
their results should be interpreted with caution (Prescott et al., 1998). Further,
these investigators did not adjust for gender specific differences in body size.
Most investigators agree that smoking cessation produces desirable health effects.
Yet, the effects of smoking cessation remain poorly understood, and different studies
produce conflicting results. In the Multiple Risk Factor Intervention Trial, cigarette
smoking associated weakly with approximately one-half of all deaths, and smoking
cessation rapidly eliminated deaths from all causes associated with cigarette
smoking, compared to continued smoking (Kuller et al., 1991). However, smoking
cessation did not yield diminished lung cancer incidence in this cohort during the
ten-year follow-up period (Kuller et al., 1991). Further, Enstrom and Heath (1999)
found the effects of smoking cessation in CPS-I to have less of an effect than
commonly believed. Of 51,343 male Californians enrolled in CPS-I in 1959 and
followed for 38 years, who had largely ceased smoking, the all cause death rate
dropped from 20.67 to 18.68 for smokers and from 10.51 to 9.46 for never smokers.
Of 66,751 women the all cause death rate increased from 9.54 to 10.14 for smokers
and decreased from 6.95 to 6.44 for never smokers.
5.2.3 Detection Bias
Little ambiguity usually exists about whether a person died or continued to live, or
the date their death occurred. Thus, in principle, data about premature mortality is
associated with less uncertainty than data about cause of death, which requires
some determination, usually taken from a death certificate. The uncertainty arising
from the number of deaths is associated with the counting of objects. Inconsistencies
in diagnosing specific disease and assigning cause of death may result in detection
bias, which is the preferential detection of disease in individuals within a specific
group (Feinstein & Wells, 1974). Mortality risk assessments are often based on
diagnoses. Hence, ultimately detection bias may compromise the validity of risk
assessments calculated from death certificate based data.
Upon death of an individual, the immediate cause of death (the final disease injury or
complication directly causing death), the underlying cause of death (the malady or
injury which began the events that led to the immediate cause of death) (National
Center for Health Statistics, 1991), and other significant conditions contributing to
death but not resulting in the death of an individual (U.S. Department of Health and
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Adverse Human Health Effects
109
Human Services, 1981) are noted. Data from Grulich et al. (1995) suggest that
death certification and coding errors resulted in an approximately 46% increase in
reported prostate cancer mortality in England and Wales between 1970 and 1990. In
cases where individuals have multiple diseases (Hoel et al., 1993), the validity of
death certificate data may be further compromised (Smith Sehdev & Hutchins, 2001).
Calculations of mortality risk from disease on exposure are often derived almost
entirely from information about the underlying cause of a disease. The contribution
of smoking to lung cancer is one such calculation (Sterling et al., 1992).
Determinations of risk based almost solely on information about underlying cause
of disease presuppose that when lung cancer is recorded as the underlying cause
of death, it is duly noted for all groups of individuals, regardless of varied smoking
habit. If the presupposition is not upheld, a “cause of death attribution bias” is said
to have occurred (Sterling et al., 1992). A cause of death attribution bias could
result from assumptions made by a physician, the lack of further investigation by a
physician if a decedent is known to have been a smoker or performance of a more
intense search for other possible causes of death when a non-smoker is diagnosed
with lung cancer. Detection bias of this type could result in an overestimation of
the risk of death from smoking (Sterling et al., 1992).
Sterling et al. (1992) analyzed data from the 1986 National Mortality Followback
study and the 1954-1962 Dorn study and determined that the relative frequency of
recording lung cancer as an underlying cause of death was lower for known never
smokers and higher for known smokers. No cause of death attribution bias could be
attributed to smoking for other diseases associated with cigarette smoking, such as
cardiovascular and cerebrovascular disease. However, a sex specific bias has been
reported for the diagnosis of COPD, with men more likely to be provisionally diagnosed
as COPD than women, whose symptoms are more likely to be attributed to asthma
(Chapman et al., 2001). For never smokers, other cancers were more often listed as
underlying cause of death and lung cancer as contributory, whereas the reverse was
true for current and former smokers (Sterling et al., 1992). Since knowledge of
smoking history may affect a physician’s diagnosis, epidemiological data may, as well,
be affected by physician’s diagnostic bias (Feinstein & Wells, 1974). This diagnostic
bias would result in excess attribution of mortality risk of lung cancer for smokers.
Sterling et al. (1992) proposed that reasonable estimates of the effect of this bias
on the relative risk is to, in using this data, treat each individual for whom there is
mention of lung cancer as having lung cancer as an underlying cause of death.
Detection biases may also occur in live subjects. McFarlane et al. (1986) showed
decreasing gradients of non-detection of lung cancer according to amount of
cigarettes smoked by the patient. Lung lesions detectable by radiology were more
likely to signal lung cancer in smokers than in non-smokers (McFarlane et al.,
1986). Feinstein and Wells (1974) investigated whether knowledge of the smoking
habits of a patient influenced diagnosis of lung cancer during life. To determine
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Evaluation of Cigarette Ingredients: Feasibility
the relationship between extent of use of clinical diagnostic test and the detection
gradient, the search rate for a pap smear of lung sputum (the number of patients
for whom a pap smear test had been ordered divided by the number of patients in
that category) was determined. The data showed a significant increase in search
rate as the amount of cigarette smoking increased. Whether a cough was absent
or present, the search rate increased with amount of smoking. In this study, pap
smears were more likely to be ordered for men than women. Their data suggest
that the stronger the smoking habit of an individual, the greater the probability of
lung cancer diagnosis within a lifetime (Feinstein & Wells, 1974).
5.2.4 MATHEMATICAL CANCER MODELS
Premature death associated with cigarette smoking does not result from a
generalized increase in all causes experienced in the U.S. population. Instead,
three principal causes of death account for most smoking-related premature mortality
among smokers: cardiovascular diseases, cancer, and COPD. All of these diseases
occur in nonsmokers. COPD comes closest to being a disease characteristic of
smoking (U.S. Department of Health and Human Services, 1984). Moreover,
cigarette smoking essentially increases the incidence of diseases that would
otherwise occur at lower incidence.
Susceptibility to the diseases attributed to cigarette smoke varies with age, even in
nonsmokers (Thun et al., 1997). However, little information exists about the effects
of age of initiation of smoking, other than younger age of initiation often results in
increased duration of smoking. In addition, the expression of different smokingrelated diseases occurs at different ages in any cohort with similar smoking
experience. For example, the incidence of coronary heart disease increases first
among smokers, later they experience a rise in lung cancer incidence, and still
later, the incidence of COPD increases (Thun et al., 1997). Since cohorts living at
the same time experience similar historical events, the incidence of smoking also
varies with the chronological year of observation.
Besides duration of smoking, adverse health effects also increase with exposure
rate. The more cigarettes consumed per unit of time, the greater the resulting
premature mortality. Thus, the incidence of premature mortality reflects trends in
two factors, duration and smoking rate. Multiplying average daily cigarette
consumption by duration of smoking generates an estimate, which epidemiologists
sometimes define as cumulative exposure. Toxicologists refer to the same measure
of duration of exposure, times intensity of exposure, as Haber’s rule (Rozman &
Doull, 2001). However, definitions of cumulative exposure and Haber’s rule are
equivalent, and both make several unwarranted assumptions (Wilson & Crouch,
2001). Right now, separate analyses of smoking rate and smoking duration may
lead to a better understanding of adverse health effects than the assumption of
cumulative exposure (Leffondre et al., 2002).
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111
Human mortality results from exposure to multiple chemical substances in cigarette
smoke and results in multiple causes-of-death (Fuchs et al., 1996). However, the
scientific community has produced little information about mathematical models
that describe overall cigarette-associated mortality. Instead of models of overall
cigarette-associated mortality, research has focused on models of cigaretteassociated lung cancer (Doll, 1971; Doll & Peto, 1978; Greenfield et al., 1984;
Moolgavkar & Knudson, Jr., 1981; Wilson & Crouch, 2001). The disease
characteristics of lung cancer aid the development of quantitative models. Subjects
with the disease die rapidly and are seldom misdiagnosed (Feinstein, 1967).
Prevalence is usually well documented (Thun et al., 1997). The medical community
seldom fails to record a case, in part because of the efforts of tumor registries.
Even so, lung cancer is a complex of several diseases. Changes in adenocarcinoma
incidence may differ from changes in bronchiolar carcinoma incidence.
Many carcinogenesis models suppose that each human being begins life with a
certain capacity for damage. Damaging events, invisible “hits” or “bets,” arrive at
some rate. These models are based on the ‘Gamblers Ruin’ stochastic process
that supposes that a person starts with some capital and places bets at a steady
rate, which are won or lost, until bankruptcy occurs (ruin) or a certain amount of
money is won. Mathematicians have thoroughly worked out many variations of
the model (Herrero-Jimenez et al., 1998). Gambler’s Ruin is also intuitive with
many practical applications. In the case of cancer modeling, the bets only have a
losing payoff with smoking causing an increase in the rate of betting.
In their classic studies, Sir Richard Doll and his colleagues gathered data about
lung cancer among British physicians who smoked (Doll & Peto, 1976). In general,
lung cancer prevalence increased as the exposure increased. Smoking more
cigarettes within the same time period or smoking for a longer time increased the
odds ratio for lung cancer. Doll (1971) developed the multistage model to explore
the general effects of age on carcinogenesis. Doll and Peto (1978) used this
model to describe overall lung cancer incidences by age in the British physicians
cohort. Lung cancer incidences increased in proportion to the square of dose
rates (number of cigarettes per day). This attempt to fit a mathematical model to
the lung cancer data from human epidemiological studies of cigarette smokers did
not comply with Haber’s rule. However, Doll and Peto excluded the highest dose
rate group (40 cigarettes or more per day), which showed a lower increase in lung
cancer incidences than expected from this relationship. Doll and Peto (1978)
pointed out some serious systematic biases in the relationship.
Lung cancer incidences in the cohort increased as fourth to fifth power of age,
even among smokers. This result is consistent with four to five stages in the
progression of events to identifiable lung cancer. Smoking appeared to amplify the
background (spontaneous) incidence of lung cancer. Other investigators have
attempted to specify which stages smoking alters, including Brown and Chu (1987),
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Evaluation of Cigarette Ingredients: Feasibility
who believed that smoking primarily affects the first and last stages. Freedman
and Navidi (1990) criticized the multistage model, based on data about lung cancer
among cigarette smokers. For data from three studies they showed that lung
cancer incidence declined after smoking cessation, a result at odds with predictions
of the multistage model.
Other groups applied alternative mathematical models to the same data from the
British physicians study. Moolgavkar and his colleagues developed a different
conceptual model, sometimes called the MVK model, which has two mutational
steps with an intermediate proliferation of mutated cells, as modified by the first
mutation (Moolgavkar & Knudson, Jr., 1981). Analysts have extensively explored
the MVK model and the concepts that it incorporates, such as effects of carcinogens
on cell proliferation (Greenfield et al., 1984). In the basic MVK model, the
population of intermediate cells can expand or contract and these intermediate
cells are the targets for the second mutational step. While this model limits the
number of stages to two, it resembles a multistage model in some ways. In particular,
the overall process still is a Markov chain, in which the first step is obligatory for
the second. Mathematically, the MVK model is more flexible than the multistage
model, making it possible to fit incidence data, for example, from pediatric cancers.
The MVK model also makes the consideration of temporal patterns of exposure
(age and duration) much easier.
Overall, a two mutation model with a cell proliferation compartment fit the data
from the British physicians study well (Moolgavkar et al., 1989; Moolgavkar et al.,
1993). Moolgavkar and coworkers assumed a quadratic model of dose rate, taken
from Doll and Peto (1978). Thus, their model does not comment on the dose rateresponse relationship. However, their work reemphasizes the important impact of
the daily dose of smoke. If the model is an accurate portrayal of biological events
in the cohort, age has an independent and prominent effect on lung cancer incidence,
independently from the effect of duration of smoking. In addition, their model
showed prominent effects of cigarette smoke on the first mutation rate and the
proliferation of intermediate stage cells, but not on the second mutation rate. This
result is consistent with many possibilities, such as a loss of the ability of intermediate
stage cells to respond to potential mutagens in cigarette smoke.
Wilson and Crouch (2001) utilized a multistage model approach, much like that of
Doll and Peto (1978) and applied it to a different source of data about smoking
outcomes, the Cancer Prevention Study-I (CPS-I) and Cancer Prevention StudyII (CPS-II), (Thun et al., 1995; Thun et al., 1997). CPS-I took place between
1959 and 1965 and included more than one million U.S. residents and eleven million
person-years of observation. CPS-II took place between 1982 and 1986 but has
not received reporting as thorough as CPS-I. In part, Wilson and Crouch had to
infer data for CPS-II from follow-up reports to CPS-II which extend the observation
period to 1988 (Thun et al., 1997) and to comparisons of CPS-I to CPS-II.
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Like Doll and Peto, Wilson and Crouch (2001) found that lung cancer incidence
followed approximately the same power law of age dependence in men and women
up to an age of 80, but Wilson and Crouch found differences in several parameters,
including cigarette potency, between men and women (Wilson & Crouch, 2001).
They found a different shape for the dose rate-response relationship: a linear function
of cigarettes per day at low doses, and a square root of cigarettes per day at higher
doses. Thus, the shape Wilson and Crouch found differs from the shape found by
Doll and Peto (1978). It agrees with Haber’s rule at dose rates below 20 cigarettes
per day, and it fits higher dose rates in CPS-I and CPS-II well. Wilson and Crouch
provide a detailed explanation about the limitations on the low dose shape they
found. Because the data they modeled collapsed smokers of 1 to 9 cigarettes per
day into one group, it is not possible to dissect the low dose response in any greater
detail. In essence, Wilson and Crouch imposed a linear dose rate-response
relationship on the data. Further, Wilson and Crouch (2001) remain circumspect
about the shape of the dose rate-response relationship for more than 40 cigarettes
per day, based on the same rationale.
5.2.5 Interactions with Other Carcinogens
Several efforts to quantitatively model human lung cancer prevalence developed
during the processes of studying other substances, which interact with cigarette
smoking, including asbestos, radon and beverage alcohol consumption. For example,
lung cancer from exposure to asbestos is extremely rare in the absence of cigarette
smoking (Selikoff & Hammond, 1979). Chase and coworkers (1985) remodeled
the same dose rate data that Doll and Peto summarized and applied it to data about
cigarette smokers exposed to asbestos. They found that asbestos modified the
effects of cigarette smoking on lung cancer incidence.
Unlike asbestos, radon is a carcinogen for the human lung. Moolgavkar and
coworkers (1993) applied a more exact version of the MVK model to the British
physicians study. They applied this model, essentially as a modifier, to data from a
study of Colorado uranium miners to explore the interaction between radon and
cigarette smoke. Surprisingly, both radon and cigarette smoke increased the first
mutation rate and the intermediate cell division rate, but neither changed the second
mutation rate. Joint exposure to radon and cigarette smoke resulted in a more than
additive but less than multiplicative increase in lung cancer incidence. The initial
age of exposure significantly increased radon and/or smoke induced lung cancer
incidence in this model. This work confirms other observations that fractionation
of radon dose increases lung cancer incidence, suggesting a nonlinear dose-response
relationship. In the MVK model, duration of exposure is more important than exposure
rate in increasing cancer incidence through effects on the proliferation of the
intermediate cell compartment. This effect also would apply to cigarette smoke.
Smoking and beverage alcohol also interact, but the effects of each substance on
consumption behaviors, exposures, and tissue sensitivities of the other substance
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have proven difficult to dissect (Bien & Burge, 1990). In addition, some of the
interactions apparently involve socioeconomic variables, such as socio-economic class
(Cummins et al., 1981; Williams & Horm, 1977). Extensive covariance of consumption
occurs; heavy drinkers tend to be heavy smokers. Epidemiologists have made some
efforts to differentiate the effects of beverage alcohol consumption in the absence of
smoking (Longnecker, 1995). These efforts depend on correction for the effects of
cigarette smoking. However, if the relationship between dose rate and response is not
linear, most of these efforts will have failed. In a nested case-control study, Murata
and coworkers found an effect of beverage alcohol consumption on lung cancer
independent of cigarette smoking but a “synergistic” effect of the two substances on
cancers of the upper aerodigestive tract and bladder (Murata et al., 1996).
5.3 CIGARETTE-RELATED CAUSES OF
PREMATURE MORTALITY
5.3.1 Cardiovascular Diseases
Some medical authorities cite heart attack, stroke and diabetes as the major smokingrelated cardiovascular diseases (Amler & Eddins, 1987). Others cite stroke and
cardiomyopathy as major subdivisions of smoking-related cardiovascular diseases
(National Institutes of Health, 1997). Currently, scientific experts disavow any
such summarization, as these subdivisions imply unproven disease mechanisms.
Regardless of the division, cardiovascular diseases are one of the major causes of
premature deaths among the smoking population.
Thirty percent of all deaths for Americans are attributed to coronary heart disease
(CHD) (Hoyert et al., 2001). Smoking is associated with both a two- to four-fold
increase in the risk of CHD and an elevated risk of sudden cardiac death (Lakier,
1992). CHD related changes induced by smoking include alteration of serum
lipids, changes in platelet aggregation, and damage to the vascular wall (Krupski,
1991). In addition, smoking increases the risk of coronary artery vasospasm (Sugiishi
& Takatsu, 1993). Patients with existing CHD who are smokers have more severe
disease than nonsmokers, as demonstrated by more frequent and longer episodes
of myocardial ischemia (Barry et al., 1989). Women who smoke have a higher
risk of CHD, myocardial infarction, and angina pectoris (Willett et al., 1987).
Smoking also relates to the incidence of atherosclerosis, transient ischemic attacks,
intermittent claudication and aortic aneurysm (Freund et al., 1993).
CHD and type 2 diabetes have many common causal factors (Jarrett & Shipley,
1988; Perry et al., 1995), however there is some debate over the association, if any,
between diabetes and smoking. Recently conflicting reports citing smoking as an
independent and modifiable risk factor for diabetes have been published (Rimm et al.,
1995; Wannamethee et al., 2001; Wilson et al., 1986). Further epidemiological evidence
is required before a causal link between smoking and diabetes can be established.
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In a meta-analysis of 32 studies, Shinton and Beevers (1989) showed that cigarette
smoking status increased the overall incidence of stroke by fifty percent. Because
stroke is a major contributor to overall mortality in the U.S., even small increases
in ischemic disease significantly increase excess mortality. Thus, cigarette smoking
contributes to overall U.S. mortality by increasing stroke, as documented in a major
longitudinal U.S. health study, the Framingham Study, in which smoking two packs
or more per day independently increased the incidence of stroke two-fold (Wolf et
al., 1988). In the meta-analysis, cigarette-related mortality varied by infarction
subtype. The relative incidence of subarachnoid hemorrhage was 2.9 (Shinton &
Beevers, 1989). Younger smokers exhibited greater effects of cigarette-related
mortality, showing a nonlinear exposure-response relationship with higher
prevalence at higher pack-years (Shinton & Beevers, 1989). The risk of ischemic
stroke declines rapidly within two to four years after smoking cessation (Wolf et al.,
1988).
5.3.2 COPD
Smoking is a significant cause of pulmonary disease and death in the U.S. Studies
have shown increased mortality among smokers from pneumonia, influenza, asthma,
and emphysema. Smoking is the single most important risk factor for chronic
obstructive pulmonary disease (COPD) (Sherman, 1992) and is the primary
determinant of COPD-associated mortality (Kuller et al., 1989). Although the
prevalence of smoking has declined among American adults, a concomitant decrease
in mortality from COPD has not occurred (Davis & Novotny, 1989). The residual
risk of COPD mortality from smoking persists after cessation.
Cigarette smoking contributes to pulmonary disorders such as chronic bronchitis
and emphysema. It also relates to other forms of interstitial lung disease, such as
desquamative interstitial pneumonia, respiratory bronchiolitis-associated interstitial
lung disease, and pulmonary Langerhans’ cell histiocytosis (Hartman et al., 1997;
Ryu et al., 2001). Smoking leads to more severe asthma outcomes (Ulrik & Lange,
2001). Smoking exacerbates influenza infections and other infectious pulmonary
diseases (Finklea et al., 1969; Petitti & Friedman, 1985).
5.3.3 Cancer
Smoking contributes to the risk of many cancers. Organs in direct contact with
smoke are at greatest risk. A 26-year follow-up health study of almost a quarter of
a million U.S. veterans revealed that more than 50% of cancer deaths among
smokers and 23% of cancer deaths among former smokers were attributable to
cigarette smoking (McLaughlin et al., 1995). Mortality rates from these cancers
have increased for smokers, and the risk of lung cancer has increased more rapidly
for women than for men. Lung cancer may be the greatest cause of excess
mortality among male and female smokers (Shopland et al., 1991). Lung cancer
death rates among smokers increased from 26 to 155 per 100,000 among women
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and from 187 to 341 per 100,000 among men (Thun et al., 1995). Cigarette smoking
is the predominant risk factor for lung cancer, and shows a dose-response relationship
with all major lung cancer cell types (Kabat, 1996).
In addition to its position as a risk factor for lung cancer, smoking is also associated
with cancers of the pharynx, esophagus, stomach, pancreas, kidney, urethra, and
bladder, with the strongest risk associated to cancers of the mouth and larynx
(Kuller et al., 1991). Smoking was associated with more than half of the deaths in
women from cancers of the oral cavity, esophagus, pancreas, larynx, lung, and
bladder (Stellman & Garfinkel, 1989). Some forms of adult leukemia also associate
with smoking (Brownson et al., 1993). There is also a reported increased risk of
cervical cancer associated with smoking (Slattery et al., 1989).
5.3.4 Mortality From Other Diseases
The 1989 Surgeons General report showed that out of an estimated 390,000 smoking
related deaths, 52,900 were due to smoking related conditions other than
cardiovascular diseases, COPD, or cancer (U.S. Department of Health and Human
Services, 1989). In a cross-sectional analysis of premature death, Amler and Eddins
(1987) stated among the 338,022 tobacco related deaths in 1980, an estimated
1,897 deaths were due to peptic ulcer disease. The ratio of mortality rates for
peptic ulcer disease ranges from 3.0–4.6 in smokers when compared to the nonsmoking population (Doll et al., 1994; Korman et al., 1983) and a greater relapse
rate of ulcers of the stomach and duodenum (Lane & Lee, 1988; Sontag et al.,
1984). A 26 year follow-up of the US veterans study (1954-1980) (Hrubec &
McLaughlin, 1997) listed 50 cause of death groups. Diseases that show excess
risks associated with cigarette smoking are asthma (2.3), tuberculosis (1.6), cirrhosis
(1.5) and chronic nephritis (1.3). The figures in parentheses show the relative
risks based on smokers compared to never smokers for the entire follow-up period
(1954-1980). These conditions are often grouped together in studies of tobacco
related mortality as the numbers of deaths involved are relatively low when
compared to the three smoking related conditions; cancer, COPD and cardiovascular
disease.
5.3.5 Infant Death
An indirect effect associated with smoking is the negative effect of maternal smoke
on perinatal and postnatal mortality. A 1980 report by the Surgeon General (1980)
lists many adverse outcomes of pregnancy associated with maternal smoking,
including fetal growth restriction, increased spontaneous abortion, greater incidence
of bleeding during pregnancy, premature and prolonged rupture of amniotic
membranes, abruptio placentae and placenta previa. Smoking related increases in
childhood behavioral and psychiatric illness (Brook et al., 2000), blood pressure
(Blake et al., 2000) and respiratory disorders (Gilliland et al., 2001) have been
reported. Children from mothers who smoked during the pregnancy have more
hospital admissions, compared to non-smoking mothers (Harlap & Davies, 1974).
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Based on combined data from 2000-2001, 19.8% of women were reported to have
smoked during pregnancy (National Institute on Drug Abuse, 2001). Among these
mothers, the perinatal mortality (PNM) rate exceeds that of non-smokers. Kleinman
(1988) assessed Missouri vital statistics records, which included data from 360,000
live births, 2500 fetal deaths and 3800 infant deaths. Among women in their first
pregnancy, the relative risk of fetal mortality associated with maternal smoking
was 1.36 (95th CI, 1.16-1.59) for less than 1 pack per day smokers and 1.62 (95th
CI 1.34-1.97) for those smoking more than 1 pack per day. A similar dose-related
increase was observed for neonatal mortality. The Ontario Perinatal Mortality
study included data from 51,490 births during 1960-1961 (Meyer & Tonascia, 1977).
It showed that PNM increased significantly (p<0.00001) as maternal smoking
increased. Meyer (1977) showed that most smoking associated PNM related to
anoxia or unknown causes (fetal deaths) or premature deliveries (neonatal deaths).
These deaths are strongly associated with a smoking related increase in the incidence
of bleeding during pregnancy, abruptio placentae, placenta previa or premature
and prolonged rupture of the membrane.
Maternal smoking and sudden infant death syndrome (SIDS) interrelate. The
Ontario Perinatal Mortality study showed that sudden infant deaths were strongly
associated with the frequency and level of maternal smoking during pregnancy
(p<0.001). In a prospective study of 24,986 births, the odds ratio for SIDS in
children from smokers was 3.5 (95th CI, 1.4-8.7), in comparison to children of nonsmokers (Wisborg et al., 2000). The incidence of SIDS was dose related (p<0.05).
Reports link SIDS to smoking related changes in respiration and cardiovascular
control (Browne et al., 2000; Milerad & Sundell, 1993).
5.3.6 Morbidity and Other Health Effects
Using estimates from 1980 of approximately 350,000 attributable deaths per year
and 1,500,000 years of life span lost before age 65 as indicators, parallel rough
estimates are that, as a population, smokers experienced approximately ten days
of hospitalization per year of life span lost or fifty days of hospitalization per death
(Amler & Eddins, 1987). Days of hospitalization do not function well as an indicator
of health status. Efforts, primarily by economists, to produce weighting factors for
various indicators that yield more satisfactory expressions are controversial. Quality
adjusted life years (QALYs) weight the number of years of future life span a
person will spend in each state of health, with perfect health as one and death as
zero. Leaving aside the problems of polling people about their preferences for
various states of health and assigning numerical weights to them, economists use
an entirely different approach, willingness-to-pay (Hammit, 2002).
When discussing the adverse human health effects of cigarette smoking, the concept
of morbidity is important. Besides increasing premature deaths, cigarette smoking
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contributes to nonfatal morbidity. Thus, morbidity could have effects on the quality
of life experienced by smokers, independently from premature mortality effects.
For example tobacco influences periodontal health and disease. Smokers experience
more periodontal disease, including greater bone- and attachment- loss, increased
pocket formation and calculus formation. Depending on the criterion, smokers are
2.6-6.0 times more likely to exhibit periodontal destruction than nonsmokers
(Bergstrom & Preber, 1994; Grossi et al., 1994; Haber et al., 1993; Page & Beck,
1997). The mechanisms involved in smoking associated periodontal disease are
not known. Smoking may affect the periodontal vasculature, inflammation, or
immunity, including humoral immunity (Christen, 1992; Haber, 1994; Palmer et al.,
1999). However, periodontal disease, even severe periodontal disease, does not
necessarily lead to premature death.
Whether any morbidity, not leading to premature mortality, might occur at lower
exposures remains uncertain and speculative. In the sense that LSRO knows of
no predictive tests for such non-lethal morbidities, such testing is not feasible.
Only if a smoking-related morbidity occurs out of relation to (independently from)
smoking-related mortality, and if an ingredient exerts an effect on this morbidity
through one of the three pathways summarized in section 5.1, could a smokingrelated morbidity become a health concern associated with the presence of an
additive.
5.3.7 Diseases and Exposures not Covered in This Report
LSRO has not attempted to cover all health effects of cigarette smoking in a
comprehensive way. For example, scientists have observed some beneficial effects,
such as improvements in mortality from Alzheimer’s disease (Lee, 1994). In
addition, the relative prevalence (or relative incidence) of some effects or some
exposure pathways is so low that even the most comprehensive and extensive of
studies could not detect the changes an ingredient might cause. For example,
environmental tobacco smoke (ETS) remains a controversial subject, but the
maximum relative prevalence of mortality of diseases noted in only a few studies
is near 1.3 (Tweedie & Mengersen, 1992; Vutuc, 1984).
5.3.8 Relationship of Risk Analysis to Safety Assessment
The process that LSRO initiates with this report is based on the available evidence
and involves identifying levels of ingredients added to cigarettes such that expert
scientists do not expect unfavorable incremental health effects. This process has
parallels with the safety assessment process used by the U.S. Food and Drug
Administration (FDA) to set levels for additives and pesticides in the food supply
(Raiten, 1999). While based on scientific principles, FDA’s safety assessment
process is administrative and regulatory in nature. Therefore, this report describes
how the processes differ.
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The U.S. Environmental Protection Agency (EPA) also has a new policy, one
modified from FDA’s policy presented as a process of science-based, regulatory
risk estimation. Mechanically, operationally, and scientifically, the policies of these
two agencies differ. For example, under regulatory risk assessment, EPA generally
assembles data from available sources to estimate potencies and exposures for all
environmental carcinogenic substances, including agricultural pesticides, which enter
the U.S. food supply. However, FDA essentially excludes carcinogenic substances
from consideration as additives to the U.S. food supply.
For non-carcinogenic substances, both agencies seek a no-observed-adverse-effectlevel (NOAEL) based on experimental animal data, essentially a regulatory
determination of dose, or of experimental exposure, which the agency can later
convert into some estimate of human exposure. Both agencies divide these
NOAELs by additional factors, called safety factors at FDA, or uncertainty factors
at EPA, to decrease human exposure still more.
Under a regulatory risk assessment (Byrd & Cothern, 2000):
(1) some person or organization, usually a governmental agency, assesses the
risks of exposures of a substance, process or use of a product, which should
be specified as the probabilities of future losses under specified conditions,
(2) the agency usually publishes the assessment for public comment, often
through a process that mimics informal rulemaking, takes comments on the
assessment, and issues a final assessment, and
(3) the agency usually will engage in some form of communications program
about the agency’s assessment and the agency’s perceptions of the risk.
Note that for cigarette smoking, the primary loss would be lives or life span under
specified conditions. EPA tries to keep risk management, the process of regulating
the sources of risk specified in the assessment, well separated from the process of
assessing the risk. While the implications of the risk assessment for a pesticide
tolerance, whether from a carcinogenic potency or from a maximum allowable amount
of a non-carcinogen, may seem obvious to all, issuing and enforcing a regulation
will come later. However, under a safety assessment (Byrd & Cothern, 2000):
(1) a manufacturer submits required data about a candidate, non-carcinogenic
substance under specified conditions in a petition to a governmental agency,
either to FDA as a proposed additive to the food supply or to EPA as a proposed
agricultural pesticide,
(2) the agency reviews the data and sets a tolerance, as subject to public
comment, to allow or not to allow the substance in the food supply under
certain conditions, and
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(3) the agency publishes its decision in the Federal Register and the Code of
Federal Regulations.
Both EPA’s and FDA’s paradigms have undergone analysis, and differences have
been noted (Carrington & Bolger, 2000). Thus, the essential difference between
safety assessment and risk assessment is that the functional objective of a safety
assessment is an administrative action, whereas the functional objective of a risk
assessment should be a statement of a population risk.
EPA has confused the separation of these two processes by redefining a safety
assessment-like process, as applied to non-carcinogenic substances in the
environment, as a risk assessment. However, EPA does not generate a probability
statement as part of its non-cancer assessment procedure. Instead, EPA
demarcates a point of exposure, which after division by an uncertainty factor, the
agency asserts will have no risk whatsoever at lower exposures.
Often, the same individual at FDA carries out both steps 1 and 2 of a safety
assessment. No division of risk assessment and risk management exists. EPA
strives to separate risk assessment and risk management. Both agencies rely
heavily on animal test data in making decisions about tolerances for noncarcinogenic substances allowed in the food supply. For non-pesticides, EPA often
limits itself to data available in the scientific literature. Often, the safety or uncertainty
factors are described as two independent tenfold factors, one for interspecies
variation and the other for intraspecies variation. However, the tenfold safety
factors of safety assessment cannot function independently of each other. FDA
really has a hundredfold safety factor for everything – species differences, human
variation, “uncertainty,” and everything else.
Regardless of the reasoning used by FDA’s regulators to achieve the first use of
the safety assessment process, on balance, the policy has worked for food additives.
The U.S. and other Western nations now have nearly fifty years of experience
with the process. Wilson (1998) has made an accounting of its performance. One
significant failure occurred. When Canadian regulators allowed the use of cobalt
salts in beer, they miscalculated the prodigious amounts of beer that brewery workers
consumed, and several workers died of heart failure presumably due to cobalt
toxicity. Otherwise, the track record is clear over perhaps 6,000 substances allowed
as food additives worldwide. So, the failure rate of additives submitted to the
worldwide safety assessment process is perhaps 2 X 10-4. This rate is per chemical
substance or a risk of administrative failure. It is not a risk in the sense of loss of
human lives or reduction of human life span under specified conditions.
FDA may, on average, over-regulate food additives. Scientists have no way to
tell. The failure estimate cited above has a large associated uncertainty, one difficult
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to estimate. The number of regulated substances does not change the estimate or
its uncertainty by much. For example, if only 3,000 substances have undergone
evaluation as food additives, the failure rate changes to 1 X 10-4. Instead, the
uncertainty resides in a lack of knowledge about the numerator of the fraction.
Further, fifty years of regulatory experience provide data about a changing process,
in the sense that FDA keeps making improvements and expanding the data
manufacturers have to submit. The net effect has been lowering of NOAEL
exposures and decreases in the uncertainties of these NOAELs.
The analytical procedures that LSRO contemplates for ingredients added to
cigarettes, and the tests to support these procedures, are not analogous to either
safety assessments or regulatory risk assessments. Instead, the scientific basis
for the feasibility of testing resides in three recommendations.
(1) Scientists can evaluate data from tests that compare highly similar, virtually
identical test cigarettes, which differ only in the presence of specific amounts
of an ingredient or ingredients.
(2) LSRO will not confine itself to a review based primarily on animal toxicology
data. Instead, LSRO will proceed with an evaluation of tests balanced among
various approaches (physical, chemical, cell culture, animal model, clinical,
and epidemiological), setting priorities and using cost of information approaches
to adjust resources to the tests most likely to provide informative data. LSRO
also will seek post-marketing surveillance information.
(3) Some analogies exist between a NOAEL and a point of no detectable
change in an exposure-response relationship. However, the two concepts
differ. Although subject to some statistical clarification, LSRO essentially relied
on the concept of no detectable change in deciding that testing ingredients
added to cigarettes was feasible. Under specified experimental conditions
and detection limits, scientists can define a point of no detectable change such
that a difference cannot be detected between smoke from an experimental
cigarette and smoke from a reference cigarette.
5.4 SUMMARY
Chapter 5 primarily summarizes the adverse health effects of cigarette smoking.
If an ingredient changed the cigarette smoke matrix in such a way that it changed
the known adverse health effects of cigarette smoking, as documented in
epidemiological studies, LSRO would like a test program that at least signaled
such a possibility for further investigation. In addition, a test program should signal
an ingredient that changed human smoking behavior, such that known adverse
health effects of cigarette smoking might change. In LSRO’s view, such a test
program is feasible.
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Adverse human health outcomes are biological measures of the cigarette smoke
exposure. To this end, one purpose of this chapter was to establish an approximate
point in the human exposure-response relationship in readily observable units. Most
smokers consume approximately one pack of cigarettes per day, seldom more than
two packs per day. Using this exposure rate from the date of initiation for a
lifetime smoker and Cohen’s estimate, the typical male lifetime smoker loses a
significant portion of his lifespan, approximately seven years.
The annualized chance of a lifetime cigarette smoker dying prematurely is
approximately one-in-one-thousand, and the chance of that lifetime smoker dying
from lung cancer is approximately 13% of the overall excess risk of death, or 0.13
x 10–4. Over the typical one pack a day smoker’s lifetime, the cumulative lifetime
risk of death from cigarette smoking-related causes becomes approximately 7 x
10-2 (nearly one-in-ten), the cumulative chance of dying of lung cancer becomes 9
x 10-3. Pathologists observe few deaths from lung cancer among nonsmokers.
Rate-based estimates, like a probability of 10-3 of premature death for male smokers
of more than one pack per day per year, dramatize the fates of deceased smokers
but do not convey all of the risks of smoking. Overall, lifetime smoking degrades
lung performance in all members of the smoking population, whether their death is
premature or not (Davis & Novotny, 1989). Pathologists observe that the lungs of
smokers are colored black with tar and lack many cilia, regardless of their causes
of death (Auerbach et al., 1979). Further, the driving forces in human health
responses to cigarette smoking are dose and time, not exposure rates or packyears. Both dose and time are complex factors (Rozman & Doull, 2000).
Overall, the remarkable characteristics of cigarette-related mortality are (a) smoking
does not generally increase all causes, or even most causes, of mortality seen in
the nonsmoking population, and (b) smoking increases the incidence of mortality
from diseases already seen in the nonsmoking population. Deaths associated with
cigarette smoking are not associated with causes not seen in nonsmokers. Chapter
7 addresses the possibility that epidemiological studies might practically reveal
useful information about the effects of ingredients on the human health effects of
cigarettes. However, this chapter shows that the most important outcome to study
for direct effects on smokers’ health is premature mortality, not the causes of
premature mortality. For epidemiological studies, direct observation of premature
mortality will produce the highest quality of data in the shortest period of time with
the smallest populations under observation, compared to any associated causes of
cigarette smoking-related mortality and morbidity.
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6
TESTING: ISSUES AND METHODS
OUTLINE
6.1
INTENT & PURPOSE
6.2
TESTING ISSUES
6.2.1 Gold Standards
6.2.2 Predictivity
6.2.3 External Validity
6.2.4 False Negative and False Positive Results
6.2.5 Combination of Tests
6.2.6 Signal-to-Noise
6.2.7 Internal Validity
6.2.7.1 Experimental details and specifications
6.2.7.2 Sampling
6.2.7.3 Sample size and power to discriminate
6.2.7.4 Adequate and appropriate controls
6.2.7.5 Gradient of effect and kinetics
6.2.7.6 Relevant experimental conditions
6.2.7.7 Measurable biological endpoints
6.2.7.8 Minimization of bias and differential error and
confounding
6.2.7.9 Randomization and control
6.2.7.10 Negative effects
6.2.8 Interpretation of Data
6.2.8.1 Association, correlation, regression, and concordance
6.2.8.2 Classical (qualitative) criteria for causation
6.2.8.3 Bayesian criteria for causality
6.3
TESTING METHODS
6.3.1 Programs of Toxicity Testing
6.3.2 Tests of Smoke Composition and Deposition
6.3.3 Tests of Biological Activity and Toxicity
6.3.4 Overall Programs and Methods for Testing
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6.4
SOME TEST DATA: MENTHOL AS AN EXAMPLE
6.4.1 Background Information About Menthol
6.4.2 Effect of Smoking on Menthol
6.4.3 Effect of Menthol on Smoke Chemistry and Physics
6.4.4 Biological Activities of Menthol
6.4.4.1 Cell cultures and tissues
6.4.4.2 Animal models
6.4.4.3 Absorption, deposition, metabolism and elimination
of smoke constituents
6.4.4.4 Biomarkers of exposure
6.4.4.5 Smoking behavior
6.4.4.6 Other human experimental studies
6.4.4.7 Other human observational studies
6.4.5 Summary of Test Data About Menthol
6.5
SUMMARY
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TESTING: ISSUES AND METHODS
6.1 INTENT & PURPOSE
Scientists can test an ingredient added to cigarettes in many ways relevant to establishing whether the substance might change the morbidity and premature mortality associated with smoking. Added ingredients could change the relative risks
of cigarette smoking through different mechanisms. Added ingredients could change
the exposure to other substances in cigarette smoke, intensify the biological activity
of cigarette smoke, or create potentially new biological effects. Thus, scientists
need to investigate the potential adverse health effects of added ingredients with a
testing program that comprehensively explores many possible mechanisms.
Manufacturers currently have available the technology to prepare approximately
identical test cigarettes for scientific study, only lacking one or more ingredient(s).
However, such test cigarettes have been used in only a limited number of published
experiments. The capacity to prepare paired test cigarettes is key to the experimental design and overall strategy adopted by LSRO. Such pairs exactly fit with a
relative risk approach to added ingredients. By comparing data from a cigarette
with an ingredient to data from a nearly identical cigarette without the ingredient,
scientists can understand relative risks and can identify whether a detectable change
has occurred, given that the test is appropriate and that scientific testing principles
have been applied.
An aim of this Phase One report is to provide a basis, developed from a review of
the literature, that a feasible process can evaluate whether, given certain conditions, ingredients added to cigarettes change the relative risks of smoking. Test
data will support this process. In this regard, the rigor of the testing program is
important. Therefore, the tests should be precise and accurate. Moreover, test
performance should be standardized based on reference materials.
Section 6.2 describes conditions relevant to the use of test data in risk assessments
and decisions based on those assessments, some of the issues that scientists generally encounter in determining the quality and accuracy of data. These issues include: establishing criteria for rendering a test (or a test result) scientifically valid,
limitations of test methods, and suggestions for improving a testing process. In
addition, a definition of scientific testing and a description of the parameters of
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testing are necessary adjuncts to a discussion of the feasibility of testing. Section
6.2 also bridges to the previous chapter, which concerned direct observation of
adverse human health effects of cigarette smoking, and to the subsequent chapter,
where areas of research that might merit further testing are highlighted. Additional
research needs, described in Chapter 7, clearly will be subject to the principles laid
out in section 6.2.
Section 6.3 reviews and addresses the limitations of tests and testing programs
traditionally used to determine the toxicities of specific substances, for example, as
added to the food supply. Section 6.3 inquires whether LSRO can usefully adapt
these programs to the review of ingredients added to cigarettes.
LSRO briefly examined several examples of added ingredients in preparing this
report, deliberately choosing substances used in greater amounts, including sucrose
and cocoa, to facilitate the discussion of feasibility. LSRO focused on menthol as
an example of an added ingredient to explore the feasibility of testing for changes
in smoke and biological activity relevant to the toxic effects posed by smoking
cigarettes because menthol transfers with smoke and because some epidemiological
data are available. Published studies compare smoke chemistry or potential health
effects of menthol containing cigarettes to similar cigarettes without menthol.
Section 6.4 summarizes some salient points of this exploration.
6.2 TESTING ISSUES
6.2.1 Gold Standards
A “gold standard” is a test, or a material used in a test, that scientists consider
reproducible, accurate, and reliable. Data from gold standard tests often form
databases to evaluate other tests (Faraone & Tsuang, 1994). Gold standard data
may be compared to other data about an added ingredient. Subsequently, new
tests also undergo comparisons for their ability to replicate data obtained with the
gold standard test or material.
In testing for adverse human health effects of smoking cigarettes (and in this
report, for the adverse human health effects of ingredients added to cigarettes),
the epidemiology of human health effects of smoking is the gold standard. The
existing knowledge base about the adverse health effects of smoking comes almost
entirely from human epidemiological studies. LSRO needs tests that predict human
adverse health effects to insure that the test results will provide useful information
about an added ingredient. To have usefulness, a test must predict an outcome of
a human epidemiological study, or an outcome from the aggregated evidence of
many epidemiological studies of the health effects of cigarette smoking.
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Some tests are conducted in attempts to predict the presence of a disease (Kraemer,
1992). Investigators select two groups of subjects, some believed to possess the
disease, some thought not to possess it. The validity of a test is defined by its
relative ability to identify the patients with the disease and to avoid detecting the
patients without the disease. No one pretends that the diagnosticians have infallible
skills. However, test evaluation usually proceeds as if no uncertainty attaches to
classification into the disease and control groups.
The application of a test and its evaluation in the above situation, involves defined
reference populations. It should be distinguished from a situation frequently addressed in this report, attempting evaluation of a test without knowledge of any
prior, defined reference populations. The distinction is crucial. In the first situation,
the test predicts a known reference. In the second situation, the only characteristic
defining the two groups is the result of the two tests, e.g., a ninety-day inhalation
toxicity test in rats.
In many situations in this report, LSRO had to evaluate test data, or the potential
performance of a test, in the absence of reference populations. For example, LSRO
usually lacked information about the prevalence or incidence of diseases between
populations that smoked cigarettes with added ingredients compared to similar populations that smoked cigarettes without the same ingredients. In this circumstance,
test data often become self-referential.
6.2.2 Predictivity
Human studies directly generate human health data, as opposed to test methods
involving animal models, which require interpretation when applied as indicators of
potential human health effects and which do not necessarily yield definitive
information about humans. One potential limitation of human studies, for example,
clinical studies, is that the number of subjects may be small. Small numbers of
subjects make the resulting data more dependent on chance (Bukowski et al.,
2001). The number of subjects determines statistical precision and limits the ability
to test hypotheses. Investigators can achieve greater sensitivity in each individual
subject (See Appendix H). However, many epidemiological studies have manyfold more subjects than traditional animal bioassays.
Human epidemiological studies usually have lengthy durations - at least several
years - because most human diseases of interest have long latencies. Diverse
human populations, different living conditions and environments, and variable
background rates of diseases, all limit quantitative epidemiological observations.
Susceptibility factors, such as predisposing genetic traits, age, ethnicity, gender, or
nutritional status, vary in human populations (Perera, 1997).
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As an example of test predictivity consider the concordance of rodent cancer
bioassays. For new substances, mixtures and processes, epidemiological data do
not exist; therefore, studies that utilize model systems are used as substitutes. In
the absence of human epidemiological studies of human carcinogenic effects of
chemical substances, regulators have used lifetime animal bioassays to predict the
carcinogenicity of chemical substances, particularly new substances and/or mixtures
entering commerce. Generally, these studies employ two rodent species, rat and
mouse. The status accorded to these tests suggests that the scientific community
and society in general regards them as good quality tests. However, even the
performance of good tests needs to be analyzed (Kraemer, 1992). Test evaluation
includes specification of both internal and external validity. The two-year rodent
carcinogenicity assay has received criticism for many reasons, including the high
exposures involved in testing (McConnell, 1989). The test design explicitly substitutes
high exposures to compensate for small numbers of subjects. However, this
substitution often is not expressed when communicating the results of carcinogenicity
bioassays, and whether the substitution is scientifically valid remains unclear.
Higher exposures do not necessarily reflect effects observed at lower exposures,
yet, lower exposures typify human exposures. For example, at high exposures the
normal metabolic pathway may saturate and an overflow pathway may generate a
carcinogenic metabolite, one not seen at lower exposures. Other arguments favor
testing at higher exposures, including use of the maximum tolerated dose (MTD),
often defined as “the highest dose of the test agent during the chronic study that
can be predicted not to alter the animals’ longevity from effects other than
carcinogenicity” (Sontag et al., 1976). The MTD is used to increase the probability
of a statistically significant number of animals with tumors in a small sample (Krewski
et al., 1993). According to Omenn (2001), concerns about the use of the MTD
include whether it is valid to extrapolate results down to the doses that are likely to
be relevant. Additional criticism of lifetime cancer bioassays derives from their
duration. Experiments that could estimate human carcinogenic risks within a shorter
time might prove preferable. Even with lower predictivity, a tradeoff exists (Omenn
& Lave, 1988).
Scientists sometimes regard a qualitative response of tumor induction in one species
as raising a question about the test substance but insufficient evidence of
carcinogenic activity to classify the substance for regulatory purposes. At present,
carcinogenicity tests continue to use two rodent species, rats and mice. Depending
on the regulatory scheme, chemicals that cause tumors in one or both rodent species
are considered potential human carcinogens (Lave et al., 1988). In the U.S. National
Toxicology Program experiments, however, results in rats do not predict results in
mice very well, and vice versa (Lave et al., 1988). The rodent bioassay detects all
known human carcinogens as rodent carcinogens (World Health Organization, 1987),
thus it is a highly sensitive assay. However, the value of a test is measured by its
specificity as well as its sensitivity (Coburn et al., 1965). Specificity is lacking,
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since the concordance of data for rats and mice was 70% of chemicals (Zeiger,
1987).
The term, “concordance,” can have two different meanings. In toxicology,
investigators often compare the responses of different species to the same chemical
substance. When doing so, a quantitative concordance is a similarity between the
exposures that produce any biological response in two species. Quantitative
concordance differs from qualitative concordance, in which chemical substances
produce the same biological responses in two different species. For qualitative
concordance, the exposure inducing a response in one species predicts the exposure
that induces a different response in a second species.
One way to improve on the gold standard approach to testing is to take multiple
tests and evaluate the combined results of the tests (Kraemer, 1992). Although
individual tests of carcinogenic effects may fallibly predict the risk of human diseases, if these tests provide information about different mechanisms of toxicity,
analysts may combine test results and improve the quality of information (Rosenkranz
et al., 1990). However, the inability to predict results of chronic bioassays of one
rodent species, based on results in the other rodent specie, when both bioassays
have met high standards of internal validity, does not inspire confidence in predictions
about human carcinogenic effects, using bioassay results with both rodent specie.
6.2.3 External Validity
In this report, LSRO’s goal in testing is to determine the relative health risks of
ingredients added to cigarettes. Although tests on human subjects will yield the
most scientifically valuable information, many considerations limit the allowable
human tests. Despite close biological and genetic similarities, LSRO remains
skeptical of animal to human extrapolation. The available animal models lack
external validity for the adverse human health effects of cigarette smoking.
Currently, no animal model predicts smoking related premature mortality or morbidity
well enough that we could be secure in saying that an added ingredient would not
increase human risk if it does not increase risk to the animal.
6.2.4 False Negative and False Positive Results
Minimally, tests of health effects should distinguish between individuals with and
without a disease. An assessment of the validity of a test involves the determination
of the relationship between the expected results — the prediction, and the measured
results — the outcome. Test accuracy (or validity) and precision are indices of
test performance. Precision is the extent to which multiple measurements under
specified conditions agree (Dybkaer, 1995). Repeatability (intra-laboratory) and
reproducibility (inter-laboratory) are two types of precision (Greiner & Gardner,
2000b).
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Error may be defined as the difference between a value obtained and a true value
of that which was measured (Dybkaer, 1995). Random error is the difference
between the measured value and a large number of measurements of the same
parameter that was measured (Dybkaer, 1995). Random error compromises
precision (Jekel et al., 2001).
Accuracy defines the agreement between a measurement and its true value
(Dybkaer, 1995). Thus, test results may be accurate and imprecise, or precise and
inaccurate. For a disease, the accuracy of a test is described by its sensitivity,
defined as the probability of a positive test outcome in the presence of the disorder,
and its specificity, the probability of a negative test result from individuals that do
not have the disorder (Kraemer, 1992). The true positive rate of a test is equal to
its sensitivity, and the false positive rate of a test is equal to 1-sensitivity (Baker,
1995). If a test indicates, for example, presence of disease when disease is absent,
this is referred to as a false positive result, type 1 error or alpha (α) error. If a test
indicates disease absence when a diseased state is occurring, this is a false negative
result, type II error, or beta (β) error (Jekel et al., 2001). A highly sensitive test is
important for identifying ill individuals. A highly specific test is especially useful,
when the cost of missing individuals without the disease is high (Faraone & Tsuang,
1994).
An ideal test will exhibit both high sensitivity and high specificity (Kraemer, 1992).
However, a test that is only highly sensitive or highly specific is not necessarily a
legitimate test. Such terms sometimes fail to show a frame of reference. The
predictive value of a test shows the performance of the test (Smith & Slenning,
2000). The positive predictive value of a test describes the probability of that an
individual with a positive test result will have a disorder. The negative predictive
value of a test refers to the probability of not having a disorder, given a negative
test result.
As an example of false negative and false positive test results, consider the use of
the Ames test, a classic assay of genotoxicity of chemical substances, specifically,
of bacterial mutagenicity to predict carcinogenic effects (Ames et al., 1973).
Certain mutations in Salmonella typhimurium render the bacterium unable to
synthesize the essential amino acid, histidine, and the mutated bacteria cannot
grow in the absence of histidine. Some substances directly, or after metabolic
activation by enzymes from mammalian liver homogenates, can additionally mutate
the bacteria, which revert to histidine independence.
One theory of cancer is somatic mutation (See Chapter 3). If so, substances
which cause mutations in bacteria and in mammals should prove positive in the
Ames test and also carcinogenic in lifetime bioassays. Thus, any catalogue of
substances tested for carcinogenic effects and substances tested for Ames positive
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results should reveal a portion of the tested substances negative in the Ames test
but positive in the lifetime rodent bioassay (false negative with respect to the Ames
test) or positive in the Ames test, but negative in carcinogenicity tests (false positive
with respect to the Ames test). Multiple factors could contribute to the lack of
concordance between assays such as the shortcomings of each assay and the
distinct criteria needed to give rise to the two measured endpoints (Elespuru, 1996).
For example, a substance with activity in bacteria but not in mammals would classify
as false positive in this example. For additional information, see results of Ashby
and Tennant (1988). Chapter 3 describes tests that have been conducted on cigarette
smoke and on the urines of smokers, using bacteria from the Ames test.
6.2.5 Combinations of Tests
If a group of tests is to outperform the individual tests, analysts must choose
combinations of tests to outperform individual tests not simply based on the sensitivity
and specificity, but on the qualities of these values. Combinations of tests will
often yield less specific and less sensitive results than individual tests, because the
results of different tests may disagree (Kraemer, 1992). Before assuming that a
group of tests will perform better than an individual test, the investigator should
have knowledge about test efficiency, and the extent to which tests agree. The
information value of an individual test depends on the results obtained with other
tests in a group. The new combined group result attempts to predict some gold
standard, but its information value depends on many factors, including the true
positive test rate, the false positive test rate, and the prevalence the gold standard
(disease) in the population (U.S. Food and Drug Administration, 1994a).
Test results with Ames test strains, both using liver enzymes and not using liver
enzymes, and using strains with sufficient loci representing sites like those involved
in mutational processes which cause carcinogenic alterations, should detect almost
all substances that cause cancer through somatic mutation. Thus, if all carcinogenic
events are somatic mutations caused by exposure to chemical substances in the
environment, scientists would expect the Ames test to detect all carcinogens also
detected by the whole animal bioassay. However, the Ames test will not detect
processes that derange the replication apparatus, such as substances that cause
genomic instability or aneuploidy (Barrett & Shelby, 1986). In addition, not all
carcinogenic events may involve somatic mutations, even somatic mutations through
processes of DNA adduction (Ashby et al., 1989; Elcombe et al., 2002; Moolgavkar
& Knudson, Jr., 1981).
Recently, the U.S. Food and Drug Administration (FDA) made progress in
computationally predicting the results of Ames tests from structural characteristics
of chemicals, by analyzing the results with various chemical structures and individual
test strains, instead of using the standard Ames test result, achieved by aggregating
the results of several different test strains (Matthews et al., 2000). FDA’s approach
is to aggregate the Ames test results after predicting results for individual
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Salmonella strains. Thus, the structure-activity relationship with one Salmonella
strain is not like the structure-activity relationship of another strain.
Similarly, exposure to substances that selectively kill subpopulations of mammalian
cells will increase cell turnover, as damaged organs replace lost cells. In this
circumstance, cell replacement can fix normal background mutations, some of
which give rise to carcinogenic alterations. Instead of direct mutation, as detected
by the Ames test, cell killing could induce cigarette-smoke associated lung cancer
(Moolgavkar et al., 1989; Moolgavkar et al., 1993). However, testing for both
effects, direct mutation through adduct formation and cell killing, would detect
both potential mechanisms of carcinogenicity.
6.2.6 Signal-to-noise
Important goals of any test are to detect, discriminate and describe a signal. The
signal is a property that one intends to measure. Noise may be defined as fluctuations
associated with the signal. Noise usually arises from multiple sources. In biological
testing, it may arise from the sample itself, the equipment used to measure the
signal, or factors in the external environment (Sherman-Gold, 1993). The signalto-noise ratio of a test is, therefore, crucial to the quality of information of the test.
This ratio delineates the limitations of the test and provides a framework for
interpretation of results. The signal to noise ratio reflects the ability of an assay to
detect a signal. A test with a high signal to noise ratio implies minimal distortion
and an ability to detect small changes in the signal. Conversely, a low signal-tonoise ratio suggests a compromised ability to discern subtle changes in the signal
from large effects of noise. Such measurements will be less reliable. An ideal test
will have a high signal to noise ratio.
Given the omnipresence of noise in testing, no test achieves a perfect assessment
of its signal. Each test needs to undergo an assessment of the reliability of its data.
One approach to such an assessment is to consider the signal analogous to a mean,
and the noise analogous to the standard deviation of the mean. Within this framework, the signal-to-noise ratio and the mean to standard deviation ratio become
equivalent. This relationship may be expressed as:
_
S/N = x /sd
_
where S is the signal, N is the noise, x is the mean and sd is standard deviation of
the mean. The assessor needs to compare data from the two groups and determine
whether the groups differ in a statistically significant way. For example, does the
concentration of a biomarker in individuals who smoke cigarettes with an added
ingredient differ from the concentration of the same biomarker in individuals who
smoke an otherwise identical cigarette without the ingredient? The student’s t-test
allows the assessor to estimate whether the two groups of individuals differ
significantly with respect to the biomarker.
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The numerator of the equation reflects the change in the signal, that is, the difference
between the average value of the signal obtained under standard or untreated
conditions (e.g., cigarettes with an added ingredient) and the average value of the
signal obtained upon deviation from this condition (e.g., cigarettes without the added
ingredient). The denominator reflects the average fluctuation of the difference in
the average value of the signal. This ratio may be compared to tabled values to
determine if the observed difference is too large to be plausibly explained by chance
fluctuations.
Experimental design is an important topic involving signal-to-noise consideration.
In the presence of noise, assessors need to establish a standard for the level of
noise that significantly alters the signal.
6.2.7 Internal Validity
Traditionally, when scientists discuss test validity, they mean internal validity, the
ability of a test to predict itself. The ability to repeat tests independently, either in
the same laboratory (the original laboratory) or in other laboratories (independent
laboratories), and obtain the same results, is the essence of science. The
characteristic of internal validity requires good test design as a first step. The
scientific validity of tests within a program for testing ingredients added to cigarettes
will contribute critically to its acceptance. For example, scientists should find the
tests reproducible and accurate. Test performance also should undergo calibration.
Internal validity of tests is a necessary, but not final, step in development of valid
tests. Tests may have internal validity, but lack external validity (Omenn & Lave,
1988). Gad and Rousseaux (Gad & Rousseaux, 2002) outlined the criteria for
good test design. In this section, LSRO summarizes some commonly discussed
elements of internal validity.
6.2.7.1 Experimental details and specifications
The first requirement for replication of a test is adequate description of test methods
and materials, such that well-trained but independent scientists can conduct the
test. For example, in animal toxicology experiments, the authors must adequately
specify the test species and/or strain. Different animal species and strain have
different metabolic pathways and rates, sensitivities to toxicants, differences in
disposition, lifespan, and background prevalence of diseases (MacGregor et al.,
1995). Strain is an important consideration in testing. For example, the A/J mouse
strain possesses a genetically based susceptibility to lung cancer (Manenti et al.,
1997), whereas the C57BL/6 mouse strain does not (Dontenwill et al., 1973).
Knowledge of this strain characteristic helps in experiment design and interpretation
of results.
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In this report, LSRO has called for investigators to test reference cigarettes in
parallel. When reviewing literature that use “cigarettes” as the substrate, it is not
possible to discern whether unique characteristics detected are correctly attributed
to, for example, the analytical method, or to the brand of cigarette analyzed. In
principle, inclusion of reference cigarettes for calibration purposes will prove
worthwhile, even when comparing a matched control and experimental cigarettes.
Similarly, LSRO has requested that experimenters use FTC smoking machine
conditions as a standard reference method to generate cigarette smoke, when
smoking itself is not the subject of investigation, as when mimicking human smoking
patterns. The rationale for this request is that although no simple smoke generation
conditions replicate human smoking, the FTC conditions are standardized and have
been used for many years in many publications. FTC conditions generate
mainstream and side stream smokes for analysis. Thus, a large body of reference
information exists about smoke chemistry and physics as generated under
standardized conditions. If FTC conditions are included in a paper that uses a
different smoke generation system, readers have a much better chance of
understanding the experimental setup.
6.2.7.2 Sampling
In epidemiology, sampling refers to the selection of individuals from the
population for study participation (Rothman, 1986). Approaches to sampling
individuals or materials for studies include restriction, random sampling, and
stratifying (Kleinbaum et al., 1982). Restriction refers to the decreasing the
eligibility of individuals or materials for inclusion in the study. Random sampling
refers to choosing individuals or test samples for a study so that the probability
of their inclusion is known (Hansen et al., 1953). In stratified sampling, study
participants are organized into subgroups that differ with respect to a covariate
which is a source of variability (Chow & Liu, 1998). The goal of sampling
techniques is to remove the effect of extraneous factors from affecting the data
obtained (Kleinbaum et al., 1982). In probabilistic modeling, elaborate methods
exist to attempt to sample data randomly, including Latin square methods.
These approaches (and the terminology) also apply to chemical and physical
analyses, and to the study materials of any test.
6.2.7.3 Sample size and power to discriminate
Two groups may be compared using the null hypothesis that the two groups are
identical for the characteristic of interest. The power of a statistical test has been
defined as the probability that a test will not accept a null hypothesis if a different
hypothesis is correct (Campbell & Machin, 1993) (See Appendix H). The power
of the test is equal to (1- β), where β is the Type II error. The power of a test may
be increased with a larger sample size when α (Τype I error) is constant (Sokal &
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Rohlf, 1987b). Statistical formulas for calculation of these values are available
(Hall & Going, 1999; Jekel et al., 2001).
An important objective in experimental design is to minimize, to as great an extent
as possible, Type I and Type II errors and to determine a balance between the two
of them that will preclude committing the type of error that is considered to be the
more serious.
6.2.7.4 Adequate and appropriate controls
Experiments should identify and include appropriate controls. Controls refer to
individuals (or materials) who (or which) have similar, if not identical, qualities as
the experimental group, differing only in the experimentally manipulated exposure
(Friis, 1996). Proper controls reduce the effects of confounding factors. For
example, human epidemiological studies should include studies of never smokers
as a negative control for health effects observed in current and previous smokers.
In determining the relative risk of ingredients added to cigarettes, the obvious control
for a cigarette with an ingredient is the identical cigarette with a different amount
of the same ingredient.
6.2.7.5 Gradient of effect and kinetics
Useful tests often allow investigators to measure a variable signal, such that the
investigator can measure a change in signal over time. Because LSRO contemplates the use of “no detectable change” to set excursion limits on the amounts of
ingredients in cigarettes, for tests which LSRO wants to investigate, inclusion of
exposure-response relationships and pyrolysis products in the test data become
important.
6.2.7.6 Relevant experimental conditions
Conditions under which experiments are conducted influence applicability of results.
For example, to determine precursor-product relationships between cigarette
ingredients and other ingredients, experiments should be conducted under conditions
relevant to cigarette smoking.
6.2.7.7 Measurable biological endpoints
Histopathology is a traditional endpoint for toxicology studies (MacGregor et al.,
1995). Approaches are being developed to measure biological endpoints other
than histology. Such approaches include the study of damage-inducible (stress)
genes, fluorescence in situ hybridization, transgenic animal models, single cell gel
electrophoresis, and accelerator mass spectroscopy studies (MacGregor et al.,
1995). Since such endpoints may be measurable in both humans and laboratory
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animals, these approaches could yield information about the applicability of animal
toxicology data to human health effects.
6.2.7.8 Minimization of bias and differential error
Bias, the systematic interference that results in the misrepresentation of a measured effect, may compromise the internal validity of experiments (Greiner &
Gardner, 2000a; Last, 1988; Nurminen, 1983). Biases may be divided into three
subtypes: selection, information and confounding bias. Selection bias refers to the
misrepresentation of the estimated influence of an exposure on a specific health
effect (Last, 1988). The source of selection bias is the method employed in choosing individuals from the population to participate in a study (Choi & Noseworthy,
1992). Information or observer bias takes place during the collection of data and
results in misassignment to categories or incorrect measurement (Feinleib, 1987).
Confounding represents the confusion of the effects of two variables, such that the
effect of the variable of interest is partly indistinguishable from the effects of the
confounding variable (Rothman, 1975). A confounding variable in an epidemiological study is related to the exposure of interest, and is a disease risk factor. It is not
a component of the process between exposure and disease development (Rothman,
1986). Confounding bias arises around data when investigators omit methods for
control such as stratification (Choi & Noseworthy, 1992). Incorrect estimation of
the effect of exposure occurs from mixing exposure and other factors independently associated with an outcome (Choi & Noseworthy, 1992). Good experimental design and interpretation require careful attention to, and minimization of potential sources of bias. [For a more detailed review of each type of bias, see Choi and
Noseworthy (1992)].
6.2.7.9 Randomization and control
Randomization refers to a specified probability of individuals in a population to
undergo inclusion in an experiment and assignment to a control or treatment group
(Glantz, 1992). Random number tables or random number generators may achieve
these assignments. Many investigators consider randomization an important aspect
of study design. Some describe it as the sole assurance of unbiased selection and
obtaining a representative sample of the population (Glantz, 1992). Many kinds of
randomization exist. Simple random studies refer to those in which each member
of a sampling unit has a similar probability of being included in a study (Forthofer
& Lee, 1995). Simple random studies do not guarantee equal representation of
members of different groups in the sample. A stratified random sample and/or
random block experimental design attempts to correct for sampling that results in
unequal representation of members of specific groups. After dividing members of
a population into mutually exclusive groups, a within group random sampling is
done (Elston & Johnson, 1987b). However, many Bayesian statisticians believe
that control, not randomization, is the essential element of scientific experiment
design (Howson & Urbach, 1993).
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Unlike deliberate control (see above), randomization may control for variables that
might influence experimental results but remain unknown to the investigator. Some
authorities, however, discount randomization in favor of controlling factors known
to influence test outcomes. Blinding is a method of controlling for effects of
experimenter or study participant knowledge of treatment (or control) status. When
both the experimenter and the participant are unaware of the treatment, the
experiment is double-blinded (Elston & Johnson, 1987b).
6.2.7.10 Negative Results
A bias against the publication of scientific articles in which investigators report
negative results or results that suggest that a particular exposure will yield minimal
or no benefit has been noted (Gad & Rousseaux, 2002). This tendency could
contribute to an inaccurate expectation of some process (Gad & Rousseaux, 2002).
According to Omenn (2001), for results of epidemiological studies where there is
no statistically significant difference between groups, it is important to note the
difference between groups that would have been necessary in order to have a
statistically significant difference (Omenn, 2001). This type of approach will help to
provide a framework for understanding the results of the experiments, and underscores the need for good experiment design.
6.2.8 Interpretation of Data
Whatever the experimental model employed to test ingredients added to cigarettes,
some common approaches are employed in the interpretation of results. The importance of data interpretation here relates to setting an excursion limit on the
amount of an ingredient per cigarette, which in turn is crucial to providing assurances about the lack of change in risk from smoking a cigarette with the added
ingredient.
6.2.8.1 Association, correlation, regression and concordance
Several statistical terms describe the relationships between parameters. When
two variables are interdependent, they are associated. Tests of association also
are known as tests of independence (Sokal & Rohlf, 1987b). Variables may have
distinct types of relationships. One variable may lead to a change in another,
changes in both variables may arise from another variable, or changes in the two
variables may come from several other variables in common (Sokal & Rohlf, 1987a).
Correlation is a measure of the extent to which two variables vary together. (See
Appendix B). Variables correlate positively if they have a direct relationship (an
increase in one variable relates an increase in the other) and correlate negatively,
if they have an inverse relationship (an increase in one variable relates to a decrease
in the other). Both association and correlation have formal statistical definitions.
Regression refers to the determination of the relationship between variables by
expressing one variable as functions of the other variables (Sokal & Rohlf, 1987b).
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Concordance is a less formal measure of the agreement or similarity between two
or more sets of data. (See Appendix B).
6.2.8.2 Classical (qualitative) criteria for causation
In scientific studies, one objective is to learn whether exposures “cause” the
observed effects, meaning that elimination of the exposure will eliminate the
observed effects (Byrd & Cothern, 2000). In epidemiology, confounding factors
make discovery of causality particularly difficult (Elston & Johnson, 1987a). The
1964 Report of the Advisory Committee to the Surgeon General outlined the criteria
necessary to show causality, including consistency, strength, specificity, temporal
relationship and coherence of the association (U.S. Department of Health Education
and Welfare, 1964). Hill later expanded these criteria to include demonstration of
a biological gradient, biological plausibility, experimental control, and reasoning by
analogy (Hill, 1971).
For example, a temporal relationship, having the exposure precede the effect, is
the most important and most accepted component of these causality criteria
(Kozlowski et al., 2001a). Consistency of association between event and outcome
is another criterion for causality. Here, investigators would probe whether the
ingredient produces an effect in only a few studies, in most studies, or in all studies
and whether that effect is observed with multiple approaches. Specificity of
association provides support for a relationship between an exposure to a specific
substance and specific health effect, as opposed to linkage between many
substances, especially without apparent chemical linkage, and panoply of effects,
especially without some biological linkage. Coherence of explanation describes
whether the biological explanation adduced to explain the association between the
putative cause and effect is supported by other data and prevailing theories (Susser,
1991). As test data support more of these criteria, they lend more support to causality.
6.2.8.3 Bayesian criteria for causality
Many scientists and mathematicians find the above “criteria based” approach to
causation unsatisfactory. Except for the temporality criterion, each of the other
criteria has known exceptions. More comprehensive and systematic approaches
are used to elicit causality from data. Cox (2002) outlined methods to address
existing loopholes and proposed an alternative tripartite process to detect causality,
which includes evidential reasoning, hypothesis-testing and law-based explanation.
Evidential reasoning refers to a comparison of the consistency of the body of
evidence in support of causation with the consistency of evidence of non-causation.
Hypothesis-testing is the evaluation of whether data supports the larger theory
behind the espoused causal theory. Law-based explanation uses known scientific
mechanisms to explain the hypothesized causal relationship. Law-based explanation
may suggest a causal relationship in isolation (Cox, Jr., 2002). However, statistics
approaches also are useful for evidential reasoning and theory testing (Nurminen,
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1997). A gradient of evidence supports causation. As the evidence along the
gradient increases, evidence for a causal relationship increases (Cox, Jr., 2002).
Shared components of the Bayesian and traditional epidemiological approaches
are refutation of potential bias and multiple levels of experimentation with the
objective to reject alternate explanations of causality.
6.3 TESTING METHODS
6.3.1 Programs of Toxicity Testing
The regulation of added ingredients to cigarettes varies. The United Kingdom
program for regulation of new additives to cigarettes includes toxicity testing. The
Government of British Columbia, requires disclosure of ingredients added to tobacco (Government of British Columbia, 1998). In addition, there are a number of
toxicology testing programs in the United States that are overseen by the government and states. These testing and regulatory programs are described below.
The National Toxicology Program (NTP) is an arm of the U.S. Department of
Health and Human Services that coordinates toxicology testing conducted by the
National Institute of Environmental Health Sciences, the National Institute for Occupational Safety of the Centers for Disease Control and the National Center for
Toxicological Research of the Food and Drug Administration. NTP has refined
and standardized protocols and statistical analysis of lifetime bioassays. NTP protocols apply to inhalation studies (National Toxicology Program, 2002a).
The U.S. Food and Drug Administration (FDA) provides guidance for toxicological
testing of food ingredients for ingestion (U.S. Food and Drug Administration et al.,
2001). In addition to providing guidelines for specific types of testing, the FDA
provides considerations for statistical and other common problems encountered in
reviewing such data. FDA’s approach relies extensively on animal models. Although methods for testing foods are not directly transferable to testing ingredients
added to cigarettes, the two situations overlap sufficiently, when the test substance
is shown to transfer in smoke and when the animal model is appropriate for inhalation. Guidelines for inhalation toxicity testing may also be applicable to studies of
added ingredients in cigarettes. For example, De George and coworkers (1997)
recommended general guidelines for toxicology studies for respiratory drug development, which provide scientific considerations relevant for the testing of added
ingredients in cigarettes.
Testing of ingredients added to cigarettes also may be subject to state laws.
Currently, State of Massachusetts (2001) regulations establish procedures for
manufacturers to test cigarette smoke from their brands for specified constituents
and report them. Massachusetts modified the FTC smoke generation protocol by
increasing the required puff volume, puff frequency and number of blocked
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ventilation holes. The State of Massachusetts (2001) also requires that
manufacturers analyze urinary metabolites in samples from sixty smokers per brand
tested. Subjects must have smoked for at least five years, currently smoke at least
fifteen cigarettes per day, and smoke the brand tested or a brand with equivalent
levels of tar and nicotine, for at least three months. Massachusetts specifies the
analytic methods required for measuring human urinary 4-(methylnitrosamino)-1(3-pyridyl)-1-butanol, its glucuronide, the urinary benzene metabolite, trans, transmuconic acid, and additional relevant background information.
The United Kingdom Department of Health established guidelines for approval
and permitted use of new additives in tobacco products in the UK (UK Department
of Health, 1997; UK Department of Health, 1998; UK Department of Health,
2002). The approval process included a review of toxicological evidence of the
ingredient, its pyrolysis products, and the condensate of a reference cigarette
containing the new additive. The Department may require biological studies to
identify the principal metabolites and to test for genotoxicity and changes in clinical,
biochemical, anthropometric and histopathological parameters after inhalation
exposure in an animal model. Required tests are determined on a case-by-case
basis, depending on the composition, activity and quantity of the added ingredient.
The government of British Columbia has implemented the Tobacco Testing and
Disclosure regulation which mandates that Canadian manufacturers of tobacco
submit to the Minister of Health Planning a list of all ingredients (including added
ingredients) of cigarettes of each brand of cigarette “sold, offered for sale,
distributed, advertised or promoted for sale”. Manufacturers are also required to
provide annual reports of the levels of specified smoke constituents in each brand
of cigarette as well as test the efficiency of the filter and pH for cigarettes that
have more than a 1.25% market share of cigarettes sold in British Columbia
(Government of British Columbia, 2001).
6.3.2 Tests of Smoke Composition and Deposition
The addition of an ingredient to a cigarette might change the chemical and/or
physical nature of its smoke. Thus, analyses of smoke chemistry and physics will
be important. For example, the size distribution of particles affects the location
and magnitude of smoke deposition in the respiratory tract (Baker, 1999). Changes
in deposition might alter adverse health outcomes of cigarette smoking.
Burning an ingredient in isolation by heating it in the presence of oxygen and
identifying the chemicals produced using sophisticated gas chromatographic mass
spectrometry or related instrumentation can identify pyrolysis products. Cocoa
powder, a more complex mixture, produced more than one hundred pyrolysis
products (Chung & Aldridge, 1992). However, the identification of pyrolysis products
after pyrolysis of a pure ingredient does not assure that the ingredient will produce
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the substances, when incorporated into the more complex matrix of a cigarette
prior to smoking.
The quantitative identification of added ingredients and their pyrolysis products in the
cigarette smoke matrix is markedly more difficult than measuring the pyrolysis
products of an isolated substance. For example, pyrolysis of an added ingredient
containing organic compounds results in cleavage of the molecular compound into
one, two, or three carbon units. Theoretically, these ingredients should yield carbon
dioxide, formaldehyde, acetaldehyde, acrolein, 1,3-butadiene, and similar smoke
components.
As indicated in Chapter 3, tobacco yields thousands of compounds when smoked,
including carbon dioxide, formaldehyde, acetaldehyde, acrolein, and 1,3-butadiene.
If an ingredient adds only a small increment of a substance to the smoke, the
resulting low signal-to-noise ratio would be analytically difficult, if not impossible,
to detect. Thus, specification of reasonable limits on detection limits, using modern
isotopic labeling and sensitive instrumentation, will be important. Jenkins and
coworkers (1977) provided recommendations for standard methods to produce
14C labeled cigarettes for test purposes. LSRO also will engage in some
specification in the Phase Two report of this series.
Data about the pyrolytic fate of the added ingredient during cigarette smoking
conditions will be uniformly desirable for all ingredients to understand whether the
ingredient changes the amount or type of pyrolysis products in smoke. Strategies
for testing added ingredients may differ depending on whether the ingredient is
volatilized and transferred directly into smoke or undergoes complete combustion,
resulting only in products of pyrolysis and pyrosynthesis.
The conditions for both isolated pyrolysis experiments and machine smoking of
cigarettes can differ from those inside a burning cigarette, as smoked by humans.
Analytical tests of cigarette smoke provide no definitive information about the
biological relevancy of the chemical components of the smoke and only limited
information about the potential toxicity of the smoke components. Additional data
derived from alternative tests will contribute to an understanding the relative risk
of the inhalation exposure of an ingredient and its pyrolysis products.
6.3.3 Tests of Biological Activity and Toxicity
Tests of biological activity have been conducted on cultured cells, tissue samples,
isolated organs, model animals, and human subjects. Investigators have studied
the acute effects of smoking on human biomarkers, such as altered expression of
LFA-1 and HLA-DR II molecules by human alveolar macrophage and lymphocytes
obtained from bronchoalveolar lavage fluids (Mancini et al., 1993). Whether these
acute changes relate to long-term chronic disease associated with smoking remains
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Figure 6.1 Biomarker categories along the continuum from exposure to disease. Reprinted with
permission from (Rundle & Schwartz, 2003). Copyright 2003 American Association for Cancer
Research.
unknown. At present, no test or group of tests can predict the human response to
smoking.
Whole animal models can identify the absorption, distribution, metabolism and excretion of ingredients added to cigarettes within the smoke matrix (Farrell et al.,
1980). When kinetic data also are available, physiological modeling of the data to
the species of origin will permit meaningful extrapolation to humans, by substitution
of human physiological and anatomical variables in the model (Andersen, 1995).
Moreover, such models can elicit the effects of an ingredient, if any, on the dosimetry of nicotine or other smoke constituents.
For testing the toxicity of food ingredients, the U.S. FDA recommends a battery of
short-term tests to evaluate induction of both gene mutations and chromosomal
aberrations (U.S. Food and Drug Administration et al., 2001). The standard threetest battery used to test genotoxicity of a substance includes a test for gene mutation
in bacteria, an in vitro cytogenetic test (chromosomal damage in mammalian cells
or mouse lymphoma thymidine kinase gene mutation assay), and an in vivo test for
chromosomal damage using hematopoietic rodent cells (MacGregor et al., 2000).
Investigators often use short-term genotoxicity tests to indicate whether a substance
causes damage to DNA or reduces repair of DNA damage. Chromosomal damage
has the potential to induce cancer or lead to an inheritable mutation. Point mutations
involving substitution, addition, or deletion of one or a few DNA base pairs have
been detected using bacterial tests (U.S. Food and Drug Administration et al.,
2001). Many bacterial strains have been used to test for mutagenicity of substances
(International Conference On Harmonisation Steering Committee, 1995). Selecting
several strains and tests helps reduce the risk of false negative results for substances
with genotoxic potential. However, a positive assay result in cell culture does not
necessarily indicate that the substance poses a genotoxic/carcinogenic risk to
humans.
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For in vitro evaluation of food ingredients to detect structural chromosomal
aberrations, the FDA recommends a functional test, the mouse lymphoma thymidine
kinase gene mutation assay, instead of a cytogenetic test of chromosomal damage
to mammalian cells. The mouse lymphoma assay measures heritable genetic
damage in living cells and can identify test substances that induce either gene
mutations or heritable chromosomal events associated with carcinogenesis (U.S.
Food and Drug Administration et al., 2001). Heritable chromosomal damage detected
by this assay is not necessarily lethal. The International Conference On
Harmonisation (1995; 1997a) has reviewed cytogenetic analysis of chromosomal
aberrations of hematopoietic rodent cells in bone marrow and peripheral blood
samples for the assessment of genotoxicity.
According to the Society of Toxicology (1999), in the absence of human data,
research with experimental animals is the most reliable means of detecting important toxic properties of chemical substances and for estimating risks to human and
environmental health. Animal models can be useful in evaluating differences in
dosimetry, local toxicity (biochemical, functional or morphological changes), systemic toxicity (clinical observation, clinical pathology, necropsy, histopathology) and
premature mortality from cigarettes with added ingredients. For the development
of drugs intended for human inhalation, general guidelines for animal toxicology
studies include consideration of the subject population and the route, level and duration of exposure (DeGeorge et al., 1997; U.S. Food and Drug Administration et al.,
2002). The general principles outlined in these documents are relevant to testing
the effects of inhaled smoke with animal models.
In testing the carcinogenicity of drugs, the FDA has chosen the experimental approach of coupling one primary long-term carcinogenicity rodent study with one or
more other supplemental studies designed to provide additional relevant information that is not easily yielded by the long-term carcinogenicity rodent study. FDA
bases inclusion of additional studies on their relevance to the potential human mechanism of carcinogenesis (Klaassen, 2001). Tissue assessment applies morphological, histological and functional criteria to determine whether the substance has
caused changes at the cellular level (International Conference On Harmonisation
Steering Committee, 1997b). The National Toxicology Program (2002a) described
five categories of evidence for reporting results of carcinogenic activity: clear evidence, some evidence, equivocal evidence, no evidence and inadequate study.
Overall evaluation of the carcinogenic potential of a substance would include consideration of the kinetics of the substance in the animal model, tumor incidence and
latency, relative carcinogenic and toxic potencies, dose-response characteristics,
and an evaluation of relevance to human carcinogenicity.
In a review, Coggins (2002) emphasized that long periods of exposure to high concentrations of cigarette smoke did not induce the cancers evident in epidemiological studies of human smokers during inhalation studies with animal models (rats,
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mice, hamsters, dogs, or nonhuman primates). Differences in laboratory animal
responses between cigarettes with added ingredients and similar cigarettes without
added ingredients would interest LSRO. Since animal models can lack predictivity
for the endpoints of interest, of cigarette-related human diseases, LSRO will
restrict the interpretation of such results to the species tested.
The National Cancer Institute conducted three series of experiments to investigate
differences in the tumorigenic activity of cigarette smoke condensate collected
from machine smoked cigarettes (Gori, 1977). Reference cigarettes included the
1R1 University of Kentucky cigarette and standard experimental blends (33%
flue-cured, 27% reconstituted sheet, 20% Burley, 11% Turkish, 1% Maryland
tobaccos, 5% invert sugar, 3% glycerol) that resembled the 1970 market blend but
varied by crop year of tobacco used. Either 12.5 mg or 25 mg of dry smoke
condensate in 0.1 mL of solution was applied daily to mouse skin, i.e., skin painting
bioassay, for 18 months or until death. In addition, some mice received only vehicle
control (acetone), negative control (hair clipping) and positive control
(benzo[a]pyrene in acetone) treatments. Tumorigenic activities of the condensates
of the standard experimental blend (SEB III) of cigarette (1) with humectant,
without sugar (2) with sugar, without humectant, (3) without sugar and humectant
(4) with powdered cocoa and without sugar and humectant (5) with magnesium
nitrate (6) with zinc oxide and (7) with magnesium nitrate and zinc oxide were
determined. There was statistically significant less tumor growth when mouse
skin was painted with 12.5 mg of condensate of SEB III cigarettes plus magnesium
nitrate (at least a 5% confidence level) as compared to SEB III with humectant
and without sugar; SEB III without sugar and humectant; SEB III with zinc oxide
or SEB III with powdered cocoa, without sugar and humectant (12.5 mg and 25
mg condensate). When mouse skin was painted with 25 mg of condensate of SEB
III with powdered cocoa, without sugar and without humectant, there were
significant differences in number of tumors as compared to SEB III without sugar,
without humectant and SEB III with magnesium nitrate and zinc oxide. Cigarette
concentrate (25 mg in 0.10 ml) from smoke of cigarettes that contained powdered
cocoa but were without sugar and humectant were significantly more tumorigenic
than smoke from cigarettes that lacked both powdered cocoa and humectant or
contained magnesium nitrate and zinc oxide (Gori, 1977). Statistically significant
differences in tumorigenesis were found between groups of mice painted with
condensate of SEB III without sugar, without humectant and SEB III with sugar
and without humectant. In addition, there was a statistically significant difference
in tumorigenesis between groups of mice painted with condensate of from cigarettes
with zinc oxide and 1R1 cigarettes.
Roemer and Hackenberg (1990) also investigated whether addition of cocoa alters
biological effects of cigarette smoke condensate. All experimental cigarettes were
American blend filter cigarettes made with 36.5 % Flue-cure tobacco, 23.7% Burley
tobacco, 12.4% Oriental tobacco and 27.4% reconstituted tobacco. Whereas the
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reconstituted tobacco used in the NCI experiment (Gori, 1977) consisted of 43%
flue-cured stems, 18 % flue-cure scrap, 28% Burley stems, and 12 % Burley
scrap, the reconstituted tobacco in this experiment was composed of 26% flue
cured stems, 9% flue cured scrap, 57% Burley stems, and 8% Burley scrap. To
the tobacco, 0, 1, or 3% cocoa powder was added. Cigarettes were smoked using
a smoking machine. Smoke condensate from each type of cigarette was collected
separately and three concentrations of cigarette smoke condensate from each
type of cigarette were prepared. Mice were randomly divided into treatment groups.
After an 8 week dose adaptation period, each mouse received five applications
per week of either acetone (control) or a total of 60, 90, or 125 mg dry cigarette
smoke condensate prepared from one of the 3 types of cigarettes to an area of the
back that was clipped twice weekly. Mice were monitored for general health
status, behavior, tumor or other lesion development and mortality for 75 weeks.
No differences were noted in the general health and behavior of mice treated with
acetone and condensates from cigarettes with varying amounts of cocoa nor
between condensates from cigarettes with varying concentration of cocoa. Based
on these results, Roemer and Hackenberg (1990), concluded that there is no
significant difference in the biological activity of condensates from cigarettes with
the concentration of cocoa tested in this study and condensate from cigarettes
without cocoa.
Multigenerational rodent studies have been used to test the reproductive toxicity
and teratogenicity of a substance by examining its effect on reproductive systems
of males and females, postnatal maturation of offspring and the reproductive
capacity of offspring (U.S. Food and Drug Administration et al., 2001).
Multigeneration studies measure parameters of reproductive and developmental
health, including estrous cycles, conception, parturition, neonatal growth and
mortality. Study designs include direct exposure of the parents, in-utero exposure
followed by direct exposure of offspring, in-utero only exposure of the pups born
to those offspring, and concurrent controls. Treatment consists of exposing parental
male and female rodents to the test substance during the period of mating
(conception), gestation and through weaning of offspring. Randomly selected
rodents from the first litters of offspring are continuously exposed to the test
substance from weaning through their adult mating period. Of this group, randomly
selected pregnant females continue to be exposed until the weaning of their pups.
Some of the pregnant females is euthanized one day prior to the expected date of
parturition and fetal pups are examined for fetotoxic effects of the test substance.
Primary manifestations of developmental toxicity in the rodent fetus are: death
during gestation, structural anomaly, altered growth and functional deficiency. The
dose, timing of exposure and duration of exposure contribute to the developmental
toxicity of a substance.
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A preliminary study with pregnant animals may be required to determine whether
the substance’s toxicity varies according to reproductive status and to select three
levels of test doses. A sufficiently high dose is necessary to elicit reduced body
weight or rate of weight gain in the parental rodents, but not cause more than 10%
parental mortality. The lowest dose should not produce any observable adverse
effects in parental rodents.
Cigarette smoke is associated with early menopause in women. Hoyer and
coworkers (2001) speculate that this may be due to the destruction of oocytes by
contaminants in smoke, which are known ovotoxicants. Once the oocyte is
damaged, the result is ovarian failure because the oocyte cannot be regenerated.
Hoyer and coworkers (2001) developed a generalized rodent model to study
ovotoxicity, which may be of use in testing the reproductive effects of added
ingredients to cigarettes.
Cigarette smoking increases the risk of delivering low birth-weight offspring. In a
recent review, Carmines et al. (2003) summarized data on effects of exposure to
cigarette smoke. Studies suggest that babies whose mothers smoked during
pregnancy weigh less and are smaller for gestational age than babies whose mothers
did not smoke during pregnancy (U.S. Department of Health Education and Welfare,
1964; Working Group Party of the Royal College of Physicans, 1992). While
some studies report no association between maternal smoking habit and
malformation of offspring (Malloy et al., 1989), other studies suggest an association
between cigarette smoking and oral clefts (Beaty et al., 1997; Werler et al., 1990)
and the possibility of a weak association between maternal smoking and congenital
reduction limb defects in children (Aro, 1983; Czeizel et al., 1994; Kallen, 1997).
Numerous groups report that female rats or mice that are exposed to cigarette
smoke give birth to pups of lower birthweight than those not exposed to cigarette
smoke (Carmines et al., 2003; Essenberg et al., 1940; Reznik & Marquard, 1980;
Seller & Bnait, 1995) while others report no effect of maternal smoking on
birthweight of offpsring (Bertolini et al., 1982; Peterson et al., 1981; Reckzeh et
al., 1975; Wagner et al., 1972). One rabbit study revealed a decrease in birthweight
due to exposure to cigarette smoke (Schoeneck, 1941). Rodent studies also report
developmental delays (Seller & Bnait, 1995) and malformations, while other studies
report no malformations (Bertolini et al., 1982; Peterson et al., 1981; Reckzeh et
al., 1975). It is generally held that cigarette smoking does not lead to congenital
malformations in offspring (Schardein, 1985). Further characterization of the
relationship between cigarette smoking and birth outcomes are needed (Carmines
et al., 2003).
Neurotoxins directly and indirectly elicit diverse biochemical, structural and functional
abnormalities (U.S. Food and Drug Administration et al., 2001). For example,
seven chronic smokers with toxic rhinitis had changes in their nasal nerve mucosa
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compared to control subjects without toxic rhinitis, who did not smoke tobacco
products (Groneberg et al., 2003). Specifically, smokers with toxic rhinitis had
increased expression scores for neuropeptide tyrosine and vasoactive intestinal
peptide in mucosal nerve fibers. U.S. FDA suggests a tiered approach to testing
substances for neurotoxicity, which screens substances for potential neurotoxic
effects, followed by more in-depth testing of the screen positive substances to
characterize effects and dose-response relationships (U.S. Food and Drug
Administration et al., 2001). Screening tests should encompass a variety of
pathological changes and functional disorders of the nervous system. Components
of the neurotoxicological screen can be integrated into other toxicological testing.
Accumulating evidence implicates cigarette smoking in immunosupression,
overactivation of the immune system and promotion of autoimmune reactions
(Gardner, 2000). Sopori and Kozak (1998) concluded that chronic exposure to
cigarette smoke was associated with T cell unresponsiveness in human smokers.
A number of studies demonstrate enhanced leukocytosis (Corre et al., 1971;
Silverman et al., 1975; Tollerud et al., 1989), increased percentages of total T
lymphocytes (Ginns et al., 1982) and decreased natural killer cell activity (Ferson
et al., 1979; Hersey et al., 1983; Hughes et al., 1985; Tollerud et al., 1989) in
cigarette smokers. Data on effects of cigarette smoke on immunoglobulin levels
show inconsistent results (Gardner, 2000). Decreased levels of IgA have been
reported in rats after inhalation exposure to cigarette smoke (Miller et al., 1996)
and in smokers (Barton et al., 1990; Sato et al., 1993; Sopori et al., 1989). Cigarette
smoking could therefore lead to increased susceptibility to respiratory infections
(Gardner, 2000).
There are conflicting data about mechanisms of immunotoxicity. Whether cigarette
smoke inhibits or stimulates mucociliary clearance is not known (Gardner, 2000).
Reviews of data suggest that the effects of factors such as age, sex, and smoking
intensity and duration are not consistent (Johnson et al., 1990) and the severity and
breadth of the effects of smoking cigarettes on the immune system have not been
fully described (Johnson et al., 1990). Tiered approaches to screen chemicals for
immunotoxic effects have been outlined (Karol, 1998; National Research Council,
1992). A major limitation to detecting changes in immune system function is that a
healthy individual typically has a large functional reserve, therefore, only a major
impairment may reveal an effect on immune function. Additional information about
the effects of cigarette smoking on the immune system would be beneficial.
Only human studies can generate relevant data about variations in smoking behavior
in response to added ingredients. Without considering other factors, such as
acceptability, an ingredient added to a cigarette in an amount that changed the
number of cigarettes smoked in a detectable way would fall within the scope effects
considered undesirable in this report.
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Short-term clinical studies with designs limited to the assessment of other biological
effects may be useful on a case-by-case basis for specific ingredients, depending
on the organ system affected. For example, a useful animal model of the human
cardiovascular or respiratory system may not be available. The assumption
underlying the use of short-term clinical studies is that the changes observed, if
any, will relate to changes observed after chronic exposure to smoke from cigarettes
with added ingredients.
Detection of biomarkers of effect could have great clinical significance, allowing
for early detection of predisposing lesions, especially if measuring the biomarker
were non-invasive (Srinivas et al., 2001). Once discovered in humans, biomarkers
would open research agendas with animal models. Researchers often have
suggested potential biomarkers, for example, lipid peroxidation or markers of platelet
activation as early events in the pathways of cardiovascular and lung disease.
Clearing the Smoke contains a list of potential biomarkers of harmful effects of
cigarette smoking including several strengths and limitations of each (Institute of
Medicine, 2001).
Lacking suitable animal models, only observational studies of chronic smoking can
reveal human health effects associated with ingredients added to cigarettes.
Epidemiologic studies of smokers identified the diseases associated with smoking
and enabled quantification of the health risk of exposure to cigarette smoke (Thun
et al., 2002). The benefits and limitations of retrospective and prospective
epidemiological studies for assessing the risks and toxic effects of substances
have been discussed from a regulatory perspective (Byrd & Cothern, 2000; U.S.
Food and Drug Administration et al., 2001). General guidelines exist for the design
and reporting of epidemiological studies, including selection of study populations,
control subjects, assessment of exposure, control of confounding variables and
statistical analyses (Verhalen et al., 1981). Some of the limitations of such
approaches are discussed below.
Observational studies of chronic smoking must include adequate information about
exposure. Leffondre and coworkers (2002) reviewed outcomes associated with
smoking among ethnic and regional populations. Measures of smoking history
varied markedly. Some investigators reported a simple categorization of smoking
status (never, smoker, former smoker). Others provided detailed information about
smoking intensity (i.e., cigarettes per day) and duration. Even studies that reported
smoking history in similar units analyzed the data differently. They suggest that
reported smoking intensity and duration be treated as separate variables. Simmons
(2002) emphasized the importance of obtaining yields directly from smokers by
collecting data about the length and amount of each cigarette typically smoked and
by including an index of body mass index. Lee (2001) also reported considerations
for improving the design and analysis of epidemiological studies of smokers.
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Epidemiological studies are capable of providing sufficient information to evaluate
the added risk of an ingredient added to a brand of cigarette. Such studies will
have limitations, as even to detect large differences, they will impose immense
logistical demands, require prolonged exposure periods, and need large study
populations. Harris (1993) cautioned that human epidemiological studies are not
practical for short-term assessment of small differences in the toxic effects of
various cigarette prototypes.
Data from previously conducted studies, particularly multiple studies investigating
a similar outcome can be reanalyzed in combination using meta-analysis. Metaanalysis is a method of analyzing results from different studies without the need to
access raw data from experiments (Putzrath & Ginevan, 1991). Such analyses
are conducted with the assumption that combining data from multiple experiments
will provide more accurate information about exposure-response relationships than
the analysis of individual experiments. Meta-analysis may improve statistical power,
identify differences in effects of studies and research needs (Blair et al., 1995).
The use of appropriate statistical procedures in meta-analysis protects against
incorrect analysis of results (Putzrath & Ginevan, 1991). Among others, Thacker
(1988), Sacks and coworkers (1987), and Blair and coworkers (1995) have published
recommendations for conducting meta-analysis. In a regulatory context, metaanalysis can provide useful data integration (U.S. Food and Drug Administration et
al., 2001). For example, Lee (2001) conducted a meta-analysis of 54 epidemiological
studies, each reporting over 100 lung cancer cases with the objective of examining
effects of type of cigarette smoked. Lung cancer frequency was lower in smokers
of filtered than plain cigarettes. This review usefully illustrates the limitations on
detecting differences in effects on smokers of different kinds of cigarettes.
6.3.4 Overall Programs and Methods for Testing
Except for direct observation (epidemiological studies), no tests correlate with
human health effects of smoking cigarettes. Therefore, the contribution of any
program of toxicity testing in cell culture or animal models of cigarettes with added
ingredients would arise from the detection of potential novel effects, added to the
existing adverse health effects associated with cigarette smoking. Limitations
exist to the ability to observe the human health consequence of adding ingredients
to cigarettes, which need to be considered when developing review criteria for
testing the health effects of added ingredients to cigarettes. (See Appendix H).
Cigarette smoke is a complex, highly variable, chemical mixture of gaseous and
particulate agents formed by the combustion of cigarette ingredients. Therefore,
detecting effects of exposure at low concentrations of mixtures, examining
interactions among components in smoke, and identifying components of smoke
that cause disease is challenging (Samet, 1995).
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6.4 SOME TEST DATA: MENTHOL AS AN EXAMPLE
6.4.1 Background Information About Menthol
Eccles (1994; 2000) published authoritative reviews of menthol. The Joint FAO/
WHO Expert Committee on Food Additives (1976) published a Toxicological Evaluation of Menthol and promulgated an acceptable daily intake for humans of 0 to 0.2
mg/kg body weight, obtained from a no-adverse-effect-level of 200 mg/kg body
weight/day (derived from an unpublished 1961 German 5.5 week rat feeding study
and a safety factor of 1000). The Cosmetic Ingredient Review Expert Panel reviewed the safety of peppermint for use in cosmetics and included a discussion of
menthol, as a component (Nair, 2001). Other reviews of menthol toxicity have
been published (TNO Bibra International Ltd., 1990).
The National Toxicology Program summarized lethal doses for different species
and routes of exposure to dl-menthol, including rat oral LD50 = 2,900 mg/kg and
mouse LD50 = 3,100 mg/kg (National Toxicology Program, 2002b). Bernson and
Pettersson, (1983) studied the toxicity of menthol in several short-term assays and
found 50% inhibitory concentrations for the cellular and subcellular systems ranged
between 0.32 mM and 0.76 mM. Perfetti and coworkers (1993) summarized doses
of menthol corresponding to irritation or lack of irritation of skin or eyes of rabbits
and guinea pigs. Mentholated products, including cigarettes, are associated with
sensitivity reactions in humans (Martindale, 1996; McGowan, 1966; TNO BIBRA
International Ltd., 1990). Menthol at concentrations of 2%-5% can have an irritant
or local anesthetic effect (Eccles, 1994; Lu et al., 1992). Among 877 patients with
contact dermatitis or unclassified eczema in Poland, 1% tested positive at 48 h or
96 h after application of a skin patch containing 5% menthol (Rudzki & Kleniewska,
1970).
The biological action of menthol in producing a cooling sensation is mediated through
members of the TRP family of receptors, specifically TRPM8 (Peier et al., 2002).
Menthol and capsaicin interact with the same receptors, although capsaicin produces a sensation of heat. TRPM8 receptors occur prominently on nerve fibers of
dorsal root ganglia in the spinal cord (Zuker, 2002).
Manufacturers add menthol (C10H20O, m.w. 156) to flavor cigarettes, to distinguish
brands, to reduce throat irritation, and to impart aromas (Fisher, 1999). Both natural and synthetic menthol are used to flavor smoking tobacco (Gutcho, 1972). Menthol is widely distributed in herbs (fennel, coriander). It is obtained commercially
from oils on the leaves of plants in the mint family, Mentha avernsis or synthesized
by hydrogenation of thymol (Budavari, 1989). Standard regulatory methods for
colorimetric and gas chromatographic analyses of menthol in cigarette filler have
been published (AOAC, 2000). Menthol is highly lipophilic (Wokes, 1932).
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Menthol is metabolized in the mammalian liver by conjugation to a glucuronide
(Eccles, 1994).
Menthol is a “Generally Recognized As Safe” (GRAS) flavoring additive for food.
It is added to baked goods, frozen dairy products (e.g., ice cream), candies, puddings, alcoholic and non-alcoholic beverages, and chewing gum. The U.S. FDA
permits the addition of menthol to over-the counter, non-prescription drug products,
however, due to inadequate data to establish safety and effectiveness, the FDA no
longer permits manufacturers to add menthol as an ingredient in over-the-counter
drugs used for smoking deterrence (U.S. Food and Drug Administration, 1994b).
Efforts to determine the amount of menthol that would evoke a 50% reduction in
respiratory rate by Swiss-Webster mice differed by less than four-fold depending
on the method used to estimate exposure (Alarie, 1986). The reduction in respiratory rate was calculated by subtracting the minimum respiratory rate during 30
minutes of exposure from the rate at baseline prior to exposure. In all cases,
recovery to baseline respiratory rate was incomplete for 10 minutes following exposure. Mean percent reduction in respiratory rate for the seven levels (n = 4/
group) ranged from 16% to 52%. However, only one group of mice reduced their
respiratory rate by as much as 50%.
Menthol was first added to cigarettes in the 1920s (Covington & Burling, 1981;
Gaworski et al., 1997). Substantial effort in the form of patented technologies did
not occur until the 1960s (Perfetti et al., 1993). Presently, menthol is added to inner
foil wrappers, to filters, or to tobacco during the latter stages of the manufacturing
process. Because menthol is volatile, it does not remain where applied. For example, when 2.7 mg menthol was applied directly to tobacco, only 70% remained
on the tobacco after four weeks of storage, and when the same amount was applied to the cellulose acetate filter tow of a cigarette, 40% migrated to the tobacco
by four weeks of storage (Riehl et al., 1972).
For cigarettes containing between 2 and 3 mg of menthol, approximately 20%, 30%
and 40% migrated from the tobacco rod to the cellulose acetate filter by the first,
second and eighth month, respectively, of storage at room temperature with individual cigarette packs contained in cartons (Brozinski et al., 1972). Alternative
menthol-like flavorants were developed and tested to reduce the amount of menthol lost during manufacturing and storage. Examples include 1-menthyl
chlorocarbonate, menthyl acetoacetate, and benzaldehyde dimenthyl acetal (Gutcho,
1972).
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6.4.2 Effect of Smoking on Menthol
Several studies provide examples of heating menthol, in isolation and measuring
the product yield. Van Duuren and coworkers (1968), reported results of thermal
degradation experiments of approximately 1 g of dl-menthol heated in a tube for 5
minutes. Menthol did not pyrolyse at 200-500 °C, and more than 98% remained at
500-700 °C. The authors concluded that brief pyrolysis of menthol does not yield
any appreciable amount of degradation products. Schmeltz and Schlotzhauer (1968)
reported lower recoveries of unreacted menthol with higher temperatures of pyrolysis.
They recovered nine times more unreacted menthol at 600°C than 860ºC. Menthol
produced six times more phenol at the higher temperature than the lower
temperature. The predominant pyrolysis products at 860°C were benzene, toluene,
styrene, naphthalene, and ethyl benzene.
With a boiling point of approximately 100 ºC, menthol is highly volatile. Theoretically,
added menthol would be swept up from tobacco downstream from the fire cone
and transferred directly into smoke rather than being subjected to 600 to 950 ºC
temperatures in the combustion zone of a burning cigarette (Eble et al., 1987).
Therefore, little menthol would be available for pyrolysis at the higher temperatures
present in a burning cigarette. Eble and coworkers (1987) determined that the
maximum transfer of menthol from unburned tobacco occurred 5 mm from the
fire cone and that transfer declined linearly up to 30 mm from the fire cone. These
data show that menthol transfers in cigarette smoke. The results of Van Duuren
and coworkers (1968) also are consistent with the transfer of menthol in smoke.
Using four commercial cigarette brands containing 1.6 to 2.2 mg menthol, Mitchell
and coworkers (1963) reported that 19% to 26 % of menthol in each cigarette
transferred into mainstream smoke, as measured in the total particulate matter
fraction by gas-liquid chromatography.
Riehl and coworkers (1972) applied 2.7 mg menthol to cigarettes during
manufacturing and stored them up to seven weeks. After 28 days, some of the
cigarettes were smoked under FTC conditions and mainstream smoke total
particulate matter was collected and analyzed for menthol. Approximately 0.43
mg menthol (16% of total application) from the smoked cigarette (FTC protocol)
transferred to mainstream smoke particulate matter. However, the rate of transfer
differed by location of initial application. For menthol applied to tobacco, transfer
to mainstream smoke increased in a uniform manner from approximately 40 µg/
puff for puffs 1-3 to approximately 58 µg/puff for puffs 7-8. For cigarettes in
which the menthol was applied to the filter, the transfer of menthol into mainstream
smoke was lower, approximately 35 µg/puff for puffs 1-3, but higher, approximately
90 µg/puff for puffs 7-8. Riehl and coworkers (1972) hypothesized that the
temperature of the filter and the concentration and surface area of the aerosol, all
factors that increase with increasing puff number, influence the transfer of menthol
into mainstream smoke.
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Jenkins and coworkers (1970) used 1.1 µCi of biosynthesized [14C]menthol, added
to the tobacco of individual cigarettes, to measure the distribution of menthol in
smoke and butt. They added additional, nonradioactive menthol to adjust to a
constant 3 mg per cigarette. After a four-week equilibration period, machines
smoked the cigarettes. For [14C]menthol added to cigarettes, 44% was detected
in sidestream smoke, 29% in mainstream smoke and 27% remained in the butt.
They found most (96.5%) of the menthol in mainstream smoke in the total particulate
matter. Consistent with the results reported by Riehl and coworkers (1972), the
per puff activity of [14C]menthol in total particulate matter of mainstream smoke
increased linearly as puff count increased from puffs 2 through 8 (Jenkins, Jr. et
al., 1970). Nearly all (98.9%) of the total mainstream activity of 14C smoke product
was identified as [14C]menthol. Of the remaining 14C activity in mainstream smoke,
0.1% was in the form of carbon dioxide, 0.2% in menthane, 0.4% in menthone (an
impurity in the source of menthol) and 0.3% in unidentified constituents (Jenkins,
Jr. et al., 1970).
6.4.3 Effect of Menthol on Smoke Chemistry and Physics
Menthol changes smoke chemistry. Application of menthol at 0.12% to 1.31% wt/
wt to cigarettes had no effect on yields of tar and nicotine in the total particulate
matter of mainstream smoke (Perfetti & Gordin, 1985). However, the addition of
1.8% menthol in a mixture with cocoa shells, licorice extract and corn syrup decreased nicotine yield compared to a control cigarette containing the same tobacco
blend (35% Bright, 23% Burley, 15% Oriental, 27% reconstituted) (Rustemeier et
al., 2002). The added mixture also increased tar significantly by at least 20% and
total particulate matter by at least 16%. Whether these changes resulted from
menthol, another ingredient, or alterations in the amount of tobacco is unclear.
6.4.4 Biological Activities of Menthol in Cigarette Smoke
6.4.4.1 Cell cultures and tissues
Cigarette smoke has genotoxic effects. For example, cigarette smoke condensate
and smoke bubbled through buffered saline solution increases sister chromatid
exchange in Chinese hamster ovary cells (1989). Several investigators have failed
to find alterations in the genotoxicity of cigarette smoke induced by menthol. In a
preliminary report, no significant differences occurred in Ames, sister chromatid
exchange, or cytotoxicity of mainstream smoke condensate from mentholated and
nonmentholated cigarettes (Bombick et al., 2001). The mutagenic response of
Salmonella strains following exposure to total particulate matter collected from
cigarette smoke did not differ between results from cigarettes with menthol (1.8%),
added as a mixture in combination with cocoa shells, licorice extract and corn
syrup, compared to results from a control cigarette containing the same tobacco
blend (35% Bright, 23% Burley, 15% Oriental, 27% reconstituted) but without
added ingredients (Roemer et al., 2002). Similarly, the cytotoxic response of mouse
embryonic cells following exposure to gas phase and total particulate matter
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collected from cigarettes did not differ between results from cigarettes with menthol
(1.8%), added as a mixture compared to results from a control cigarette without
added ingredients (Roemer et al., 2002).
Using human epithelial tissue within 24 h of excision and rabbit and rat crosssectional tracheal rings within 1 h of extraction, Rakieten and coworkers (1952)
observed microscopically that l-menthol (0.4 mg/cc) had little effect on epithelial
ciliary activity. Effects on activity of respiratory cilia did not differ between
exposures to mentholated and nonmentholated cigarette smoke collected in LockeRinger’s solution.
6.4.4.2 Animal models
Gaworski and coworkers (1997) conducted an animal study to determine whether
menthol in cigarettes would affect respiratory tissues. Rats exposed to mentholated
cigarette smoke did not differ in weight gained at any smoke concentration (target
smoke particulate matter of 200, 600 and 1200 mg/m3) compared to rats exposed
to non-mentholated cigarette smoke. In contrast, an exposure-related increase of
26% and 50% occurred in the incidence of clear nasal discharge for the nonmentholated group exposed to target smoke total particulate matter concentrations
of 600 and 1200 mg/m3. This effect was not observed in rats exposed to
mentholated cigarette smoke.
Vanscheeuwijck and coworkers (2002) generated mainstream smoke, using ISO
protocols, from cigarettes with menthol (1.8%), added as a mixture in combination
with cocoa shells, licorice extract and corn syrup, and control cigarettes containing
the same tobacco blend (35% Bright, 23% Burley, 15% Oriental, 27% reconstituted)
but without added ingredients. They diluted the smoke with fresh air at a rate of
1:150 to 1:170 depending of the yield of total particulate matter. Sprague-Dawley
rats were exposed (nose only) to either smoke from the menthol mixture (four
groups; two each male and female) or control smoke (two groups; one each male
and female) at a concentration of 150 µg/L for 6 h/d, 7 d/wk for 90 days. No
differences were evident for respiratory parameters, blood carboxyhemoglobin or
urinary metabolites of nicotine between the menthol mixture and control groups.
No significant differences occurred in ophthalmologic or hematologic observations,
except for one group of female rats exposed to the menthol mixture that had lower
neutrophil counts compared to the female control rats. Absolute and relative weights
of the thymus were significantly greater for both male groups and one female
group exposed to the menthol mixture compared to control groups. Rats exposed
to the menthol mixture did not have an increased incidence of histopathology in
nasal, laryngeal, tracheal, bronchial or lung tissues compared to control rats.
Vanscheeuwijck and coworkers (2002) did find that male rats in the menthol mixture
group had significantly increased blood concentrations of total cholesterol and serum
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urea. Other studies reported decreased serum cholesterol among non-mentholated
cigarette smoke-exposed female rats relative to air controls (Heck et al., 2002)
and rabbits injected with nicotine-free cigarette smoke extract (Yamaguchi et al.,
2000). Similarly in humans, children with long-term passive cigarette smoke
exposure have lower high density lipoprotein cholesterol concentrations suggesting
the possibility that cigarette smoke exposure may negatively affect cholesterol
(Moskowitz et al., 1999). Menthol inhibits lecithin-cholesterol acyltransferase
activity (Cooney et al., 1984) and 3-hydroxy-3-methylglutaryl coenzyme A reductase
activity (Clegg et al., 1982).
6.4.4.3 Absorption, deposition, metabolism and elimination of
smoke constituents
Under experimental conditions, menthol is known to increase dermal drug permeation
(Beutner et al., 1943; Giannakou et al., 1998; Heng, 1987; Kommuru et al., 1999;
Lu et al., 1992; Morimoto et al., 2002; Shojaei et al., 1999; U.S. Environmental
Protection Agency, 2002; Zanker et al., 1978). It has been proposed that menthol
may increase the risk of adverse health effects from smoking by increasing uptake
of cigarette tobacco smoke substances (Kobayashi et al., 1994; Miller et al., 1994).
MacDougall and others (2003) investigated the effect of menthol on nicotine
metabolism. Incubation of liver microsomal fractions with menthol was found to
inhibit nicotine oxidation to cotinine. This effect of menthol on nicotine metabolism
could lead to an increase in risk of adverse health effects for menthol cigarette
smokers (MacDougall et al., 2003). Metabolism of the cigarette flavoring ingredient,
coumarin, by coumarin 7-hydroxylase, was also inhibited in the presence of menthol.
Additional research on the effects of menthol in cigarette smoke on the deposition,
clearance and absorption of other smoke constituents is needed.
6.4.4.4 Biomarkers of exposure
Gaworski and coworkers (1997) measured several biomarkers of exposure in rats
exposed to menthol in cigarette smoke. Groups of male and female rats were
exposed (nose only) to filtered air (control) or smoke from American style filter
cigarettes, with or without 5000 ppm of synthetic l-menthol, at target mainstream
smoke total particulate matter concentrations of 200, 600, or 1200 mg/m3. The
frequency of exposure was 30 minutes, twice per day, five days per week for 13
weeks, followed by a six-week non-exposure recovery period. Serum nicotine
and cotinine concentrations did not differ between rats exposed to the smoke from
non-mentholated cigarettes and mentholated cigarettes except for a lower cotinine
concentration in rats exposed to mentholated smoke at the 200 mg/m3 smoke
concentration. In their animal model study, Gaworski and coworkers (1997) used
smoke concentrations that correspond to v/v dilutions of less than 5% of the
mainstream smoke. In contrast, cigarettes smoked by humans produce a
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concentrated smoke. The FTC tar yields with total volumes of 10 puffs at 35 ml
each yield 40,000-60,000 mg/m3 for mainstream smoke. The menthol concentration
in typical American blend cigarettes can be more than three times higher than in
this study.
Lugton and coworkers (1978) measured conjugated l-menthol in urine samples
from five smokers who consumed 18-23 mentholated cigarettes over an eighthour period. Conjugated l-menthol was not detected in urine samples collected
prior to the smoking period. Maximal excretion of conjugated l-menthol occurred
between the fourth hour of smoking and the fourth hour post-exposure for four of
five subjects.
Miller and coworkers (1994) injected commercially available cigarettes (1992 FTC
yield: 1.1 mg nicotine, 16 mg tar, 14 mg carbon monoxide) with 0 mg, 4 mg, or 8 mg
of menthol. They provided these experimental cigarettes to smokers in random
order, for three controlled-exposure sessions, conducted one week apart. Subjects
were African American male smokers (n = 12) participating in a substance abuse
program. Six subjects identified themselves as smoking mainly mentholated
cigarettes and the other six subjects identified themselves as smoking mainly regular
cigarettes. Following an overnight abstinence from cigarettes, confirmed by a
70% decline in expired carbon monoxide level, subjects drew puffs at 30-second
intervals from an experimental cigarette until they inhaled a total of 600 mL of
smoke. Breath samples were collected, the smoking protocol was repeated, and
final breath samples were collected. On average, subjects had to draw 18 puffs to
inhale 1,200 mL of smoke. The change in expired carbon monoxide from before to
after the 1,200 mL total inhalation volume, increased linearly as menthol dose
increased (p < 0.01). Subjects who preferred mentholated cigarettes at baseline
had a larger increase in expired carbon monoxide, (7.6 ppm) vs. subjects that
described themselves as having a preference for regular cigarettes (5.6 ppm).
Ahijevych and coworkers (2002) examined the association of menthol with some
exposure parameters in women. In contrast to the report by Miller and coworkers
(1994), subjects who preferred nonmentholated cigarettes at baseline had a greater
increase in expired carbon monoxide than subjects smoking mentholated cigarettes.
Baseline plasma nicotine and cotinine concentrations did not differ significantly
between smokers of mentholated and nonmentholated cigarettes. However, a
more recent study by Ahijevych & Parsley (1999) contradicted these findings and
indicated that women smoking menthol cigarettes had significantly higher blood
cotinine concentrations than women smoking nonmentholated cigarettes. Results
were not adjusted or controlled for nicotine intake. The more recent report also
differed in women smoking mentholated cigarettes who had significantly larger
puff volumes than women smoking nonmentholated cigarettes. Clark and coworkers
(1996) found higher blood cotinine concentrations among menthol cigarette smokers
(n = 76) than nonmenthol cigarette smokers (n = 85) after adjusting for ethnicity,
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cigarettes smoked per day, and the mean amount of each cigarette smoked. Menthol
smokers may smoke cigarettes with higher nicotine yields or smoke cigarettes to
obtain greater absolute yields of nicotine.
6.4.4.5 Smoking behavior
African-American smokers prefer mentholated cigarettes. Among 1424 adult
smokers in four states, 89% of African Americans reported smoking mentholated
cigarettes compared to 30% of Caucasian smokers (Wagenknecht et al., 1990).
Benowitz and coworkers (1999) reported striking racial differences among smokers
in San Francisco, where 76% of African Americans (n = 51) smoked mentholated
cigarettes compared to 9% of non-Latino white smokers (n = 51). Similarly, Clark
and coworkers (1996) reported that 83% of African American smokers used
mentholated cigarettes compared with 23% of the non-Hispanic white smokers.
Menthol in cigarettes influences smoking behavior. Nil and Battig (1989) recruited
15 smokers (11 women) in Sweden to measure several parameters of smoking
behavior. Each subject smoked seven different cigarettes: the subject’s own
preferred brand and six others, representing three types of cigarette (mentholated,
dark tobacco, blond tobacco) and two levels of yield (high, low). Smoking sessions
were conducted one week apart for seven weeks. When subjects smoked in their
own manner, changes in heart rate from pre to post smoking were as high or
higher for mentholated cigarettes than for other cigarettes. In contrast, post puff
inspiration time was as low or lower for mentholated cigarettes than for other
cigarettes. Likewise, as subjects smoked in their own manner, they drew fewer
puffs per cigarette from mentholated, high yield cigarettes than from all other
cigarettes except dark tobacco, high yield cigarettes. Correspondingly, the subjects
had the longest puff intervals when they smoked mentholated, high yield cigarettes
than when they smoked all other cigarettes except dark tobacco, high yield cigarettes.
McCarthy and coworkers (1995) conducted a crossover design study with 29 male
smokers who were in treatment for substance abuse. At baseline, smokers who
preferred mentholated cigarettes (n = 11) had a higher average heart rate than
smokers who preferred nonmentholated cigarettes (n = 18), 80 vs. 69 beats per
minute, respectively. Two smoking sessions were conducted one week apart,
using one type of commercially available cigarette at each session, mentholated or
nonmentholated. Both types of cigarettes had 1991 FTC yields of 1.2 mg nicotine
and 17 mg tar, but differed slightly in carbon dioxide yield (mentholated: 17 mg,
nonmentholated: 15 mg). The mean puff volume of subjects smoking mentholated
cigarettes was 7.5 mL less than the mean puff volume of subjects smoking
nonmentholated cigarettes. Smokers of mentholated cigarettes took an average
of four fewer puffs per cigarette. Therefore, the total puff volume smoked per
mentholated cigarette was 39% less than that smoked per nonmentholated cigarette
(p = 0.001).
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A later report by the same research group described a similar crossover design
with 20 male smokers (Jarvik et al., 1994). Each type of commercially available
cigarette tested had a reported yield of 1.2 mg nicotine, 15 mg carbon monoxide,
and 16 mg tar. Subjects drew smaller mean puff volumes and less total puffs from
mentholated cigarettes. However, no significant differences for cigarette types
were reported for peak puff flow, puff duration, puff interval, lung retention time or
yield of total particulate matter.
6.4.4.6 Other human experimental studies
Pritchard and coworkers (1999) measured heart rate and encephalographic (EEG)
activity in 22 male smokers, 12 of whom were regular smokers of menthol cigarette
(8 African Americans, 4 Caucasians) and 10 of whom were regular smokers of
non-menthol cigarettes (Caucasian). Each subject smoked two denicotinized test
cigarettes (yield: 0.06 mg nicotine, 8 mg tar, ~8.5 mg carbon monoxide), one of
which contained 4.1 mg menthol. The pre-to post-smoking increase in heart rate
was greater for regular menthol cigarette smokers than for regular non-menthol
cigarette smokers. The dominant alpha frequency was more than 0.7 Hz slower
for regular menthol cigarette smokers than for regular non-menthol cigarette
smokers. Regular non-menthol cigarette smokers showed a marked difference in
the effect of smoking menthol versus non-menthol test cigarettes on eyes-closed
beta1 power EEG activity. In contrast, menthol smokers showed a consistent
slight increase in beta1 activity after smoking menthol and nonmenthol cigarettes.
At locus Pz, menthol cigarette smoking led to a lowering of beta2 power. Beta2
power was unchanged by non-menthol cigarette smoking. The groups were not
racially balanced.
6.4.4.7 Other human observational studies
In a case report, a 58 year-old woman, who had smoked cigarettes for 30 years,
developed digestive and nervous disorders (vomiting, irritability, unsteadiness,
insomnia, speech disorders, tremors, confusion) and slowed heart rate three months
after switching to mentholated cigarettes. After the switch, she had rapidly increased
consumption to 80 cigarettes per day. Her symptoms disappeared seventeen days
after switching back to a non-mentholated brand of cigarette. Her physician
challenged her with one gram of menthol, three times daily. Symptoms returned
on the third day and increased in severity by day seven, at which time the apparent
oral challenge was withdrawn and symptoms abated (Luke, 1962).
TNO BIBRA International (1990) compiled and reviewed five case reports of
smokers of mentholated cigarettes who experienced hives, dermatitis or skin rash
that cleared after restricting exposure to menthol. At least two of these smokers
also used other mentholated products. Clinical conditions reappeared when pa-
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tients were challenged with a mentholated product (dermal, oral or smoking cigarettes).
Based on analysis of the literature, Garten and Falkner (U.S. Congress, 2002)
propose that mentholation of cigarettes decreases smokers’ ability to detect early
symptoms of respiratory disease, such as shortness of breath and chronic cough.
Menthol has decongestant (Eccles & Jones, 1983), antitussive (Laude et al., 1994;
Morice et al., 1994), expectorant (Boyd & Sheppard, 1969), antibacterial (Moleyar
& Narasimham, 1992; Pattnaik et al., 1997) and anti-allergic activity (Arakawa et
al., 1992) which may obscure warning symptoms of disease. The delay in detection
of symptoms by menthol cigarette smokers may contribute to a delay in seeking
medical care and worsening of health outcomes (U.S. Congress, 2002).
In a case control study, Hebert and Kabat (1989) examined the relationship between
currently smoking mentholated cigarettes and risk of esophageal cancer, using
data collected between 1969 and 1984 in nine U.S. cities, part of the data collected
in the American Health Foundation study. Control smokers were matched by
hospital, gender and age. The study suggested that women, but not men who
smoked mentholated cigarettes, had an elevated prevalence of esophageal cancer.
In another case-control study, Kabat and Hebert (1991) examined the relationship
between smoking mentholated cigarettes and prevalence of lung cancer using data
collected between 1985 and 1990 at eight hospitals in four U.S. cities. They matched
control smokers by hospital, age, gender, race/ethnicity and date of interview. The
results did not support an association between the use of mentholated cigarettes
and lung cancer in either sex. In a third study, these authors found that men who
ever smoked mentholated cigarettes exhibited a positive association with cancer
of the pharynx after adjusting for age, education, filter use, and ethnicity (odds
ratio 1.7, 95% CI: 0.8-3.4) (Kabat & Hebert, 1994).
Similarly, Friedman and coworkers (1998) assessed the incidence of non-lung
cancers among smokers of mentholated and nonmentholated cigarettes in a cohort
study of members of the Northern California Kaiser Permanente Medical Care
program from 1979 to 1985 who had smoked for 20 or more years. The ageadjusted rate ratio of cancer cases for use of mentholated compared to
nonmentholated cigarettes was 1.06 or less for all cancers (mouth, pharynx, larynx,
esophagus, pancreas, cervix, urothelium) except renal adenocarcinoma in men (1.28,
95% CI: 0.39, 4.15), 4 cases in 1,575 male mentholated cigarette smokers and 9
cases in 4,182 male nonmentholated cigarette smokers. The incidence of non-lung
cancers did not increase among long-term smokers who preferred mentholated
cigarettes compared to smokers who preferred nonmentholated cigarettes.
Kabat and Hebert (1991) examined the relationship between smoking mentholated
cigarettes and risk of lung cancer using data from 2,368 smokers collected between
1985 and 1990. Control smokers were matched by hospital, age, gender, ethnicity
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and date of interview. The adjusted odds ratio for risk of lung cancer was not
significantly increased for either short-term (1 to 14 years) or long-term menthol
use (15 or more years).
Sidney and coworkers (1995) examined the risk of lung cancer and use of
mentholated cigarettes among smoking members of the Northern California Kaiser
Permanente Medical Care program from 1979 to 1985. The duration of smoking
mentholated cigarettes was directly related to lung cancer in men (p = 0.02), but
not women. Among men who were long-term smokers, mentholated cigarette use
was associated with a statistically significant, 45% increase in the incidence of
lung cancer relative to those who smoked nonmentholated cigarettes. In contrast,
smoking mentholated cigarettes did not significantly alter the risk of lung cancer in
women who were long-term smokers.
Carpenter and coworkers (1999) examined the association between smoking
mentholated cigarettes and lung cancer risk by comparing cases of lung cancer in
smokers from 35 hospitals with population control smokers in Los Angeles County.
Exclusive menthol smokers were not at increased risk of lung cancer when
compared to exclusive non-menthol cigarette smokers. The rates for women were
not statistically distinguishable from those for men, but were consistent with the
findings of Sidney and coworkers (1995) discussed earlier. There was a significant
(p=0.04) linear trend for decreased lung cancer rate among women as pack-years
of mentholated cigarette smoking increased compared to women exclusively
smoking nonmentholated cigarettes.
Lee (2001) reanalyzed epidemiological data from three U.S. studies (Carpenter et
al., 1999; Kabat & Hebert, 1991; Sidney et al., 1995) and concluded that the data
were inconsistent for a difference in the risk of lung cancer for those who regularly
smoked mentholated cigarettes compared to those who smoked nonmentholated
cigarettes. His meta-analysis showed a decreased relative prevalence in women,
0.70 (95% CI: 0.52-0.95), but not men, 1.23 (0.88-1.72).
Brooks and others (2003) assessed the role of smoking mentholated cigarettes on
the risk of developing lung cancer by analyzing data from the Slone Epidemiological
Center Case Control Surveillance Study. They examined the smoking histories of
643 cases (cigarette smokers for at least 20 years with no history of any form of
cancer before diagnosis of lung cancer within a year of current hospitalization) and
4,110 controls (individuals hospitalized for conditions not regarded as attributable to
cigarette smoking) and used logistic regression to determine the relative risk of lung
cancer as it relates to the number of years that an individual smoked. Their results
indicate no increased risk of developing lung cancer from smoking menthol cigarettes as compared to smoking cigarettes without menthol and no apparent influence of race (Black or White) or sex on risk of developing lung cancer due to
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smoking menthol cigarettes. The interpretation of these results is complicated by
the investigators’ assumption that subjects for whom brand-smoking histories were
incomplete smoked non-menthol cigarettes prior to 1956.
6.4.5 Summary of test data about menthol
Menthol is commonly added to cigarettes. Menthol typically comprises
approximately 2% of the weight of a cigarette. Menthol does not pyrolyse
appreciably during smoking, but transfers into the smoke. Intact menthol is the
primary substance transferred in mainstream smoke. Menthol can change smoke
chemistry. Menthol in cigarette smoke is absorbed into the blood stream and
metabolized. Smokers of mentholated cigarettes have higher blood cotinine
concentrations, increased puff volumes, shortened post puff inspiration times, and
decreased puff numbers per cigarette. The exposure-response relationships for
these effects are poorly known.
6.5 SUMMARY
Except for observational studies of smokers, the tests and testing programs
traditionally used in toxicology can most usefully be adapted for the review of
ingredients added to cigarettes to understand metabolic effects and detect novel
toxic effects. Application of traditional testing programs to ingredients that do not
transfer in smoke seems irrational and wasteful. Even for substances that transfer
in smoke, dosimetry estimates and scientific judgment will come into consideration
in making decisions about subsequent testing for health effects. For example,
some ingredients added to cigarettes are complex natural products. Some proportion
of the mass of such products likely will transfer in smoke. Determining the proportion
of mass sufficient to merit subsequent inhalation toxicology testing will best be left
to case-by-case decision-making. Identification of the transferred products and
prior knowledge will strongly influence decisions about subsequent testing. LSRO
will review this subject in the Phase Two report of this project.
It is a different matter to determine the maximum amount of an ingredient added to
a cigarette that does not change stated parameters of, for example, smoke chemistry, or to determine the maximum amount that does not change the parameter by
more than a predetermined amount. The judgment that neither a substance nor its
pyrolysis products transfers in smoke in biologically meaningful amounts can mostly
be made without reference to cell culture and animal model information.
The principle that multiple, independent tests do not necessarily provide better quality information than a single test, will prove crucial to the construction of LSRO’s
approach to feasibility. It justifies the notion that a few well-chosen tests will
potentially provide more useful data about the relative risk of an ingredient added to
cigarettes than an extensive list of tests.
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A case study of menthol proved particularly helpful to LSRO in assessing the feasibility of testing ingredients added to cigarettes, although the review did not offer
the in-depth perspective of a more extensive and comprehensive examination suitable for regulatory purposes. In addition, the review did not aim to recommend a
limit on the maximum quantity of menthol added to a cigarette. However, the
review did help reveal that testing of ingredients is feasible.
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RESEARCH NEEDS
OUTLINE
7.1
INTENT & PURPOSE
7.2
THE CHEMISTRY AND PHYSICS OF SMOKE
7.2.1 Smoke Chemistry
7.2.1.1 Analytical methods
7.2.1.1.1 Pyrolysis
7.2.1.1.2 Isotopic labeling of substances in cigarettes
7.2.1.2 Standardization of analytical methods
7.2.1.2.1 Reference tobaccos
7.2.1.2.2 Identification of smoke toxicants
7.2.2 Smoke Physics
7.2.3 Reduction of Variables
7.3
SMOKE EXPOSURE
7.3.1 Smoking Behavior
7.3.1.1 Smoking initiation and maintenance
7.3.1.2 Smoking cessation
7.3.1.3 Cigarette brand choice
7.3.2 Smoking Inhalation and Depositon
7.3.2.1 Smoking inhalation
7.3.2.2 Smoke deposition
7.3.2.3 Biomarkers
7.4
ADVERSE EFFECTS OF SMOKE
7.4.1 Premature Mortality
7.4.2 Causes of Mortality
7.4.2.1 Cardiovascular diseases
7.4.2.2 Chronic Obstructive Pulmonary Disease (COPD)
7.4.2.3 Cancer
7.4.2.3.1 Lung cancer
7.4.2.3.2 Other cancers
7.4.3 Immune System Effects
7.4.4 Free Radicals
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7.4.5 Risk Factors
7.4.5.1 Genetics
7.4.5.2 Race/Ethnicity
7.4.5.3 Gender
7.4.5.4 Age
7.4.5.5 Culture
7.4.5.6 Diet and nutrition
7.4.6 Individual Variation in Health Effects and Behavior
7.5
TESTING ISSUES AND METHODS
7.5.1 Animal Models
7.5.2 Clinical Studies (behavior)
7.5.3 Analysis of Data
7.6
SUMMARY
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RESEARCH NEEDS
7.1 INTENT & PURPOSE
To assess the feasibility of determining the relative risk of smoking cigarettes with
added ingredients as compared to smoking cigarettes without added ingredients
LSRO extensively reviewed the relevant scientific literature. Specifically, LSRO
staff evaluated data concerning cigarette smoke, human exposures to cigarette
smoke, the adverse health effects of smoking, and issues about the methods used
to study cigarette smoke. In addition, LSRO studied menthol, an example of an
added ingredient. The overall review revealed areas where additional data would
help an assessment of the feasibility of testing whether added ingredients increase
the relative risk of adverse health effects of smokers. In this chapter, LSRO
identifies areas of research that warrant further scientific investigation.
7.2 THE CHEMISTRY AND PHYSICS OF SMOKE
7.2.1 Smoke Chemistry
LSRO adopted a criterion of “no detectable change,” as an initial basis for
elimination of potential relative risks of adverse health effects associated with
ingredients added to cigarettes. Molecules of an ingredient in a cigarette will
encounter diverse circumstances on ignition of a cigarette; they may transfer intact
with smoke, convert into one or more other substances, or undergo a combination
of chemical and physical processes. As such, the burning of added ingredients in
cigarettes could generate chemicals absent from the smoke of cigarettes that lack
or contain lesser amounts of the added ingredient. Other potential effects of the
inclusion of added ingredients include increases or decreases in the amounts of
one or more chemical components of the smoke matrix and multiple, diverse effects
on smoke substances, such as alteration in lung absorption. Thus, the inclusion of
added ingredients in cigarettes could alter the exposure of smokers to smoke
chemicals and change the morbidity and mortality associated with smoking cigarettes.
LSRO has suggested an initial step in the assessment of the relative risks of adverse
health effects imparted by ingredients added to cigarettes of comparing, under
standardized conditions, the chemical composition of smoke from reference
cigarettes to smoke from cigarettes approximately identical to the reference
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cigarette, except for the presence or absence of an ingredient or a defined mixture
of added ingredients. Testing a range of concentrations of the added ingredient(s),
including the concentration of the ingredient used in commercial cigarettes and
lower and higher concentrations of the added ingredient will be desirable. Any
significant change detected in smoke quality or quantity arising from inclusion of
an added ingredient, or mixture of ingredients, might imply a change in risk of
adverse health effects of smoking cigarettes. The meaning of change is a matter
for scientific evaluation and interpretation. However, the information derived from
relative chemical analyses of smoke will have vital importance in setting upper
limits on the amounts of added ingredients per cigarette.
The question of whether an added ingredient transfers into smoke and how much
of the ingredient transfers, also will contribute important insight about the identity
of smoke substances and the exposure of smokers to these substances. An
understanding of the process is of importance since knowledge about what transfers
in smoke may be applied to understanding relevant exposure levels of smoke
substances. Boiling point, vapor pressure, and volatility of smoke substances are
useful for predicting what will transfer in smoke, however, we lack complete
understanding of the factors that determine what transfers in smoke.
7.2.1.1 Analytical Methods
7.2.1.1.1 Pyrolysis
Cigarette smoke is a complex mixture of approximately 4,800 different compounds
(Green & Rodgman, 1996). Diverse approaches have been employed in an attempt
to elucidate the relationship between cigarette ingredients and substances in smoke.
Hobbs (1957) predicted a low concentration of oxygen within the burning zone of
a cigarette, which was later confirmed by Newsome and Keith (1965). Based on
this information, pyrolysis studies of pure added ingredients in cigarettes and tobacco
fractions were proposed to study the relationship between specific ingredients in
cigarettes and smoke substances. Other studies, including those of Wynder and
Hoffmann, include pyrolysis studies of added ingredients in inert atmospheres
(Wynder & Hoffmann, 1964; Wynder & Hoffmann, 1967). Criticism of this method
included the use of an oxygen-free atmosphere for pyrolysis, which does not exist
under normal smoking conditions, as well as the dependence of oxygen concentration
on the amount of diluting air and paper porosity (Rodgman et al., 2000).
Pyrolysis of leaf extract has also been examined (Schlotzhauer et al., 1976).
Although these studies may identify potential contributions of some chemicals to
compounds in smoke, pyrolysis is not the only process that occurs in a burning
cigarette (Schmeltz et al., 1978). Changing smoke generating conditions affects
the yields of smoke substances. Bell and coworkers (1966) suggested that the
pyrolysis of certain carbohydrates in air generates higher amounts of phenols than
are generated in a nitrogen atmosphere. Brunnemann and Hoffmann (1982)
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proposed that the fate of chemicals should be determined in the context in which
cigarette smoking occurs.
Pyrolysis studies of ingredients added to cigarettes provide information about the
relationship between added ingredients and cigarette smoke substances. The
identification and measurement of substances in smoke is important for making
decisions about which smoke substances are important for testing.
Some added ingredients may not transfer into smoke, but could disturb the smoke
matrix and affect the dose of smoke substances to which the smoker is exposed as
well as the toxicity of the smoke. The presence of an indicator component that
accurately reflects dosimetry and toxicity of other substances in the mixture that
are applicable to the health effect of interest is a useful tool (Fleming et al., 1994).
The ability to detect changes in the smoke matrix would be extremely useful for
assessing the feasibility of studying the relative risk of adverse health effect of
smoking cigarettes with added ingredients as opposed to smoking cigarettes without
the added ingredient. If scientists could identify an indicator component for changes
in the smoke matrix, we would better understand whether the smoke matrix changes
when a specific added ingredient is present. If any particular substance is a better
signal of changes in the chemical composition in the smoke than any other, LSRO
is not aware of it. Other than convenience and signal-to-noise ratio, a scientific
rationale to select chemical markers for routine analysis is not apparent. One
approach to prioritizing is to select chemical substances that have high concentrations
in smoke e.g. acetaldehyde and 1,3-butadiene in the gas phase. Additional research
might provide better candidate substances to signal changes in the chemical
composition of cigarette smoke. Information about the best chemicals to analyze
as an index of change in the smoke matrix would be informative for determining
feasibility.
7.2.1.1.2 Isotopic labeling of substances in cigarettes
As noted previously, LSRO could not find evidence that pyrolysis of isolated added
ingredients will provide information about interactions of added ingredients and
their pyrolysis products in cigarette smoke. Growing tobacco plants in a [14C]
carbon dioxide atmosphere and performing chemical analyses of the smoke yielded
from cigarettes containing the radioactive label, provides an alternate approach to
pyrolysis (Schmeltz et al., 1978). This process reflects, more accurately, the
contributions of specific added ingredients to the formation of smoke chemicals
and is useful for identifying degradation products at low levels (Jenkins, Jr. et al.,
1970; Schmeltz & Schlotzhauer, 1968). Syringe-spiking cigarettes with [14C] carbonlabeled cigarette ingredients, spraying with radioactive compound, and sheetcastingblending are other approaches to incorporating radioactive label into cigarettes
(Jenkins, Jr. et al., 1970; Jenkins, Jr. et al., 1977). Challenges in conducting
radioisotope analysis of smoke constituents include assuring that the chemicals
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demonstrate the requisite activity and are labeled in multiple positions (Schmeltz et
al., 1978). The use of stable isotopes is another approach. Insufficent baseline
data exist to document the performance of mass spectrometer detection combined
with chromatographic processing of smoke. It may be that introduction of a stable
isotopically-labeled marker substance into a cigarette will simultaneously provide
adequate data to evaluate the transfer of an added ingredient and changes in smoke
composition during cigarette combustion. Knowledge of the fate of cigarette
ingredients in an intact cigarette provides more pertinent information about the
chemical mixture that comprises smoke and new smoke substances that may form
during the burning of a cigarette, relevant to the feasibility of testing for the relative
health risk of added ingredients.
7.2.1.2 Standardization of analytical methods
The Federal Trade Commission (FTC) standards for smoking machines were
designed in 1967 to compare mainstream smoke yields of nicotine and tar (Federal
Trade Commission, 1967a; Federal Trade Commission, 1967b), and later carbon
monoxide (Federal Trade Commission, 1979) from different cigarettes smoked
under identical standardized conditions. Subsequent to the development of these
standards, other standards for smoke generating machines have been introduced.
In 1991, the International Organization for Standardization (ISO) harmonized then
existing standards, which Canada and the United Kingdom adopted (International
Organization for Standardization, 2000; International Organization for
Standardization, 2002). In the United States and Japan, the FTC standard still is
used, while some European countries use CORESTA standards (CORESTA, 1994).
For purposes of testing ingredients added to cigarettes, standardization will be
useful for controlling conditions and limiting variations in measurements. One
approach to generating comparable data is to use the FTC standard of smoking
cigarettes, because extensive data on cigarette smoke chemistry generated using
the FTC methods are presently available. The use of such an established technique
to produce smoke is of value since the yields of some chemicals in cigarette smoke
have not been measured with validated techniques (Rodgman & Green, 2002).
Standardization of techniques could also help standardize terminology. For example,
the FTC and CORESTA methods define tar differently (CORESTA, 1991c;
Pillsbury et al., 1969). Standardization of techniques and terminology could provide
more comparable results, which will assist the interpretation of data, thus assisting
decisions about feasibility.
Assessment of existing methods to detect and quantify chemical substances in
smoke might allow the determination of their usefulness at the concentrations found
in smoke. Thus, the process of validation of testing methods currently in use and
determining new approaches applying sophisticated methodologies and
instrumentation would advance the study of smoke chemistry. There are, however,
limitations on the applicability of smoke chemistry studies. Because the specific
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substances responsible for human morbidity and mortality remain unknown, sideby-side chemical analysis of cigarettes with and without an ingredient cannot yield
information directly relevant to human morbidity and mortality. Instead, such studies
show that a kind of cigarette generates smoke similar to the cigarettes previously
consumed by cohorts in published epidemiology studies.
7.2.1.2.1 Reference tobaccos
Reference cigarettes are essential controls for testing ingredients with and without
ingredients. Tar and nicotine yields of older reference cigarettes are not reflective
of today’s cigarettes (Baker, 1999; World Health Organization, 1986). The use of
standardized reference tobaccos would generate data on yields of chemicals in
cigarettes currently being smoked. Consistent and definitive documentation of the
use of reference cigarettes in experiments would provide useful information for an
assessment of feasibility (Baker, 1999; World Health Organization, 1986).
Information about the source of the tobacco crop used in the cigarette, the quantity
of reconstituted tobacco, as well as its processing, housing, packaging and transport
would provide researchers with useful detail. The specification of the concentration
and technical grade of the added ingredient used is a useful approach (CORESTA,
1991b; World Health Organization, 1986).
7.2.1.2.2 Identification of smoke substances
Various chemical substances in smoke are thought to be toxicants, however, no
standardized method exists for the identification of the majority of these chemicals
(Rodgman & Green, 2002). In addition, other groups have not verified the presence
and concentrations of some putative toxicants. Rodgman and Green (2002)
proposed that some substances may be present as artifacts. Rodgman and Green
(2002) described a process to develop a more complete information base, by
confirming the presence of the chemical in smoke, recording toxic properties, and
evaluating of the process used to characterize amount of the substance found in
smoke. However, LSRO knows of no test of toxic properties that is predictive of
the human health effects observed in epidemiological studies.
7.2.2 Smoke Physics
Cigarette smoke has been defined as an “aerosol of aqueous droplets which contain
dissolved and suspended substances dispersed in a mixture of vapors and gases,
including air” (Davies, 1988). The microenvironment, and processes such as
nucleation and particle growth, influence smoke formation (UK Department of
Health, 1998). Cigarette smoke aerosols exhibit cloud-like behavior, which may
result in different patterns of movement than are predicted for the individual particles
that make up the cloud (Chen & Yeh, 1990). Such differences in patterns of
movement will affect lung deposition. The physics of smoke affects deposition of
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cigarette smoke aerosols in the trachea and lung bifurcation regions (Chen & Yeh,
1990).
As smoke ages, smoke particle size increases and particle concentration decreases
(Morimoto et al., 2002). Knowledge of the substances contacting smokers’ lungs
will provide insight into exposure. The relationship between chemical variability
and the size of a smoke particle has not been fully described (McRae, 1990).
Whether the presence of added ingredient affects these processes is not known.
Light scattering studies (or other measurements relevant to particle sizes) of smoke
(Ingebrethsen, 1984) generated from cigarettes with and without added ingredients
would contribute to knowledge about aerosol dynamics and to decisions about
feasibility.
7.2.3 Reduction of Variables
The process of human cigarette smoking is complex. One research approach to
the chemistry and physics of cigarette smoke would be to simplify the process by
removing many variables from study. Investigators could pack different tobaccos
at different densities in impermeable tubes, such as stainless steel tubes. They
could send defined gases through the tubes at different rates and temperatures,
such that the gases sustain combustion. Scientists could study the chemical and
physical characteristics of the smoke, either at specific time points or collectively
through measurements of the collected total smoke. Similarly, the spectrum of
light absorption might provide instantaneous estimates of changes in chemical
composition. The application of such techniques might be useful for improving
understanding about the process of smoke formation. An approach to studying
pyrosynthesis is to supply a potentially reactive substance in the gas mixture to a
tobacco column undergoing combustion. Reaction products incorporating this
substance would essentially trap pyrosynthesis in progress.
Given a better understanding of how variables, such as oxygen tension, temperature,
flow rate, packing density, and kind of tobacco affect the chemical and physical
composition of smoke; the scientific community would be better positioned to
understand the more complex phenomenon of human smoking. Knowledge about
this would provide additional understanding about the relationship between chemists’
test cigarette smoke and relate it to human exposure in order to make predictions
about health effects.
7.3 SMOKE EXPOSURE
Cigarette smoke exposure refers to the external relationship between tobacco
smoke and the body (Shields, 2002). Changes in exposures to chemical substances
in smoke change adverse health effects, most obvious in smoking cessation, where
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smoking intensity (cigarettes consumed per unit time) and smoking duration (years
of smoking), both change. However, more subtle differences reflecting changes
in smoke composition remain elusive.
7.3.1 Smoking Behavior
7.3.1.1 Smoking initiation and maintenance
Many biological factors have been implicated as contributory to the initiation of a
smoking habit. Socio-cultural factors, including attitudes of individuals and their
peers toward smoking and smoking behaviors of individuals in the immediate
environment, may also play a role in smoking initiation (Rennard & Daughton,
2002). Fowles (2001) suggested that some added ingredients, such as licorice,
sugars and artificial sweeteners, and glycerol and methylglycerol, which alleviate
throat irritation caused by cigarette smoke may increase the appeal of cigarettes.
Menthol produces a cooling effect, which also may alleviate the burning sensation
experienced during smoking (Miller et al., 1994). Whether the presence of an
added ingredient could influence the decision to initiate smoking remains unknown
and elusive. Studying smoking initiation could contribute to an understanding of
feasibility of assessing the relative risk of added ingredients.
According to Levin and Rose (1995), some smokers perceive an improvement in
brain function and an energy surge from smoking a cigarette. The expectation of
such effects of smoking may influence the development of a smoking habit in nonhabitual smokers. The role of anticipation of effects, and whether added ingredients
contribute to the anticipation of smoking, are unknown. LSRO would like to know
whether anticipation changes smoking behavior in a measurable way, such as
consumption of cigarettes or biomarker levels.
Surveys of youth from diverse racial and ethnic backgrounds, who initiate cigarette
smoking, would be beneficial for dissecting the process of initiation and maintenance
of smoking habit (Ahijevych, 1999). If ingredients added to cigarettes are shown
to be a factor in the initiation and maintenance of smoking, LSRO will want to
revisit this topic.
The role of genetics in smoking initiation is not fully described (Minnesota
Department of Health, 2003). The rate of nicotine metabolism may affect the
initiation and maintenance of cigarette smoking (Ahijevych, 1999). The roles of
polymorphisms in dopamine b-hydroxylase, monoamine oxidase A and B, and other
enzymes in the development and maintenance of a smoking habit are discussed
later in this chapter.
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7.3.1.2 Smoking cessation
Little is known about the physiological, cognitive, behavioral, and psychological
effects of withdrawal from smoking cigarettes with additives. It will be important
to determine whether the presence of added ingredients affects the ability of
individuals to cease smoking. According to Sommese et al. (1995), many studies
report psychological, and/or physiological responses to nicotine withdrawal, including
a decrease in carboxyhemoglobin, digit recall, serial addition/subtraction, and job
satisfaction; and an increase in blood pressure, depression, absenteeism, caloric
intake, craving, aggressiveness, confusion, and impulsivity. Whether any added
ingredient alters responses to cigarette withdrawal and successfully quitting smoking
remains unknown.
7.3.1.3 Cigarette brand choice
In a review of compensation and smoking, Scherer (1999) noted that smokers can
compensate for smoke taste, cigarette smoke irritation, the draw resistance of the
cigarette, and yields of nicotine and tar. Studies of the effects of nicotine yield on
cigarette smoking report conflicting results. If the nicotine yield does not change
cigarette smoking, LSRO might revise its decisions about feasibility. Thus, changes
in nicotine yields of smoke from cigarettes with and without added ingredients will
prove useful to know. In addition, the role of the presence of added ingredients on
the decision to smoke a specific brand of cigarette, or to switch cigarette brand
has not been fully explored, although LSRO has no insight into better research
methods to pursue these topics. However, changes in behavior could increase the
exposures of smokers to toxicants.
7.3.2 Smoke Inhalation and Deposition
7.3.2.1 Smoke inhalation
The risk of adverse health effects of inhalation exposure to unpyrolyzed and pyrolyzed
added ingredients would be useful to know. LSRO endorses approaching this
problem by conducting a case-by-case by study of added ingredients. Davies
(1988) describes a relationship between the amount of smoke inhaled and the
intakes of carbon dioxide and particles, which affect the physiological and
psychological responses to smoking. Human body temperature may affect the
surrounding ambient air, which influences the inhaled fraction of airborne particles
(Ogden & Birkett, 1975). Reliable analytical methods for the determination of the
bioavailability of components of the smoke and levels of metabolites in tissues
would contribute to an understanding of the relationship between whole smoke
and inhalation. A better baseline of data about smoke inhalation would contribute
more information to decisions about the feasibility.
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7.3.2.2 Smoke deposition
Davies (1988) defines the deposited fraction of an aerosol as the fraction that,
during steady breathing, does not exit the respiratory tract on exhalation. The
deposited doses of smoke components determine the risk of adverse health effects
of smoking cigarettes. Factors that affect the deposition of inhaled aerosols include
the physical and electrical characteristics of aerosol particles, the structure of the
respiratory system, and the way that the smoker breathes (Martonen et al., 2000).
Particles are deposited via three primary mechanisms: inertial impaction,
sedimentation and diffusion (McKemy et al., 2002). Multiple theoretical models
have been developed in an attempt to predict the deposition pattern of inhaled
cigarette smoke. However, these models have not been validated. Factors such
as age, anatomical anomalies, the physics of airflow, hygroscopicity, and aerosol
charge, have not be addressed satisfactorily (Phalen & Oldham, 2001).
Identification of the effective dose and its relationship to individual features, such
as body size, have not been fully explained (Phalen & Oldham, 2001). These data
gaps are relevant to a decision about feasibility, since better information might
provide some insight into the susceptibility of specific groups of individuals in the
population to diseases associated with cigarette smoking. Studies of factors
contributing to locations of deposition in human airways and explanations of how
exposure leads to long-term tissue pathology would be useful. Better information
about how to study deposition could also affect a decision about feasibility of
assessing relative risk of adverse health of smoking cigarettes with and without
added ingredients.
The mechanisms of smoke uptake into lung tissue have not been characterized.
Although nicotine may be absorbed by more than one mechanism, the predominant
mechanism of absorption is not known, nor is the influence of added ingredients on
nicotine absorption known. Gray and Kozlowski (2003) propose banning ingredients
which increase nicotine bioavailability. Information about the molecular structure
and function of nicotinic receptors could provide insights about potential interactions
between added ingredients and nicotine receptors (Society for Research on Nicotine
and Tobacco, 2002).
Heeschen and coworkers (2001) showed that nicotine promotes growth of
atherosclerotic plaques, decreases apoptosis of endothelial cells, and enhances
development of capillary networks. The overall effects of tobacco smoke may
differ from the effect of nicotine in isolation. Detailed characterization of the
physiological actions of nicotine and the effects that nicotine may elicit on behavior
and cognition would provide useful information.
Proposed mechanisms of action of one added ingredient, menthol, include altering
the effect of known carcinogens and an effect on smoking behavior (McCarthy et
al., 1995). Jarvik and coworkers reported that smoking mentholated cigarettes
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involved smaller puff volumes and decreased numbers of puffs as compared to
smoking non-mentholated cigarettes (Jarvik et al., 1994). In contrast, McCarthy
and others (McCarthy et al., 1995) found increased number of puffs and higher
puff volumes when smoking mentholated cigarettes as opposed to smoking nonmenthol cigarettes. Smokers of mentholated cigarettes have higher blood levels of
carboxyhemoglobin and greater expired carbon monoxide levels than smokers of
non-mentholated cigarettes, although retention times are similar for all smokers
(Jarvik et al., 1994). The mechanism by menthol achieves increased levels of
carboxyhemoglobin has not been fully described (Jarvik et al., 1994).
Thus, whether menthol affects the deposition, absorption and clearance of smoke
components remains to be determined. Research questions to be addressed include
whether menthol in the presence of heat increases “penetration” of smoke
constituents, exposure to toxic components of cigarette or other alterations of
biological activity. Neuropharmacological tests of how added ingredients might
impact the pharmacology of addiction might be applied (Fowles, 2001).
7.3.2.3 Biomarkers
A biomarker or biological marker has been described as “a measure that is used as
an indicator of normal biologic processes, pathogenic processes or pharmacological
responses to therapeutic intervention” (De Gruttola et al., 2001). The three
categories of biomarkers include biomarkers of exposure, biomarkers of biologically
effective dose, and biomarkers of potential harm (Institute of Medicine, 2001).
General principles for use of biomarkers have been outlined (Shields, 2002).
Important processes include biomarker validation and evaluation for sensitivity
and specificity at realistic exposure concentrations and at lower amounts. In addition,
testing of the biomarker in different populations and noting the variability of data
on multiple testing of an individual, between individuals in the same laboratory and
between different laboratories also is important. Identification of alternative sources
of biomarker can clarify the relationship between exposure and biomarkers.
Other research needs are the identification of early indicators of disease and the
validation of biomarkers of tobacco exposure (Shields, 2002). Mao and coworkers
(Mao et al., 1997) reported loss of heterozygosity mutations at the 3p14, 9p21 and
17p13 loci in the lung tissue of smokers that are asymptomatic for lung cancer.
These genetic loci code for FHIT (fragile histidine triad gene), p16/ CDKN2 and
p53 tumor suppressor genes, respectively. The investigators propose the
identification of clonal genetic markers to separate out individuals most likely to
develop lung cancer (Mao et al., 1997). A biomarker that fails to detect substances
in cigarette smokers during nicotine replacement therapy, will validate smoke
exposure (McKemy et al., 2002).
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DNA adducts may indicate biologically effective doses of smoke in lung tissue
(Spitz et al., 1999). According to Wiencke and others (Wiencke et al., 1999),
DNA adducts are affected by the intensity of exposure and time elapsed since
exposure to tobacco smoke. Detoxification and the disappearance of adducts can
result from DNA repair or cell turnover. DNA adduct formation, however, is not
sufficient for commencement of carcinogenesis or mutagenesis (Kramer, 1998).
Adducts to proteins, such as hemoglobin, may also be useful biomarkers of
biologically effective dose. Kramer (1998) describes the advantages of using protein
adducts, which include their long half-life (120 days) and dose dependence of
binding.
7.4 ADVERSE HEALTH EFFECTS OF SMOKE
7.4.1 Premature Mortality
Epidemiological studies have identified cardiovascular diseases, cancer and chronic
obstructive pulmonary disorder (COPD) as the primary causes of death associated
with cigarette smoking. The mechanisms by which chemical substances in smoke
contribute to the development and progression of these diseases are not known.
Furthermore, the identity of the smoke chemicals most toxic to humans are not
known. Additional research to elucidate the mechanisms of pathogenesis of these
diseases will aid the development of predictive models.
Statistically, greater accuracy resides in enumerating all of the excess deaths
associated with some exposure, instead of some portion of these deaths associated
with a cause of death. In addition, better quality data are available from the
interpretation that a person has died, than from the cause of death, which requires
an inference based on symptoms and may require an autopsy. For these reasons,
the emphasis placed on specific causes of death over premature mortality in
epidemiological studies of cigarette smoking is difficult to understand. An important
research priority is to ensure that supporting mortality data are made available in
published articles. If tabulations of, for example, lung cancer data are the only
available information, LSRO will review them, but these data will be inferior to the
mortality data from which investigators derived the lung cancer data.
LSRO will continue to seek insights into the health impact of non-tobacco ingredients
added to cigarettes. Thus, comparative studies of health statistics of smoking
from countries where the cigarettes consumed have, or do not have additives will
remain a high priority research topic. LSRO learned much from comparisons of
health outcomes from smoking filtered and unfiltered cigarettes (Lee, 2001).
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7.4.2 Causes of Mortality
7.4.2.1 Cardiovascular disease
Genetic and environmental factors influence cardiovascular disease. Elucidation
of the molecular and cellular events underlying various cardiovascular diseases
would lead to a better understanding of the role of smoke chemicals, and possibly
added ingredients, in development of these diseases. Cigarette smoke increases
the rate of progression of arteriosclerosis (Waters et al., 1996). Various mechanisms
have been proposed for cigarette smoke associated induction of stroke, including
increased platelet aggregability and reduced blood flow by arterial vasoconstriction
(Rogers et al., 1985).
7.4.2.2 Chronic obstructive pulmonary disease (COPD)
COPD is characterized by airflow limitation that is progressive, difficult to reverse,
and associated with atypical lung inflammation (Turato et al., 2001). Perhaps
fifteen percent of cigarette smokers develop “clinically significant” obstruction of
their airways (Fletcher & Peto, 1977; Sethi & Rochester, 2000). Genetic
susceptibility, environmental and host factors are thought to contribute to the
development of COPD (Sethi & Rochester, 2000). Deficiency of alpha-1-antitrypsin
(α-1AT) is a known risk factor for COPD (Eriksson, 1965). Identification of other
risk factors of COPD is in the early stages (Higham et al., 2000; Patuzzo et al.,
2000). Taylor and coworkers (1986) proposed that an imbalance of oxidants and
antioxidants contributes to the development of COPD. The process includes
antiprotease inactivation (Janoff, 1985; Laurell & Eriksson, 1963), inflammation
(Bosken et al., 1992), infection, and activation of transcription factors (Collins,
1993; Schreck et al., 1991). How the inflammation associated with COPD begins
and maintains, is not known (Shapiro, 2002). The roles of bacteria in the
inflammation and the mechanisms by which smoke chemicals give rise to COPD
have not been fully described and remain a research need. Whether added
ingredients contribute to smokers’ outcomes in relation to COPD is not known.
7.4.2.3 Cancer
7.4.2.3.1 Lung cancer
Various groups have reported a trend of increased prevalence of adenocarcinoma
with respect to squamous cell carcinoma in smokers (Travis et al., 1995; Larson,
1960). Additional research is needed to determine whether this apparent shift in
incidence is due to advances in technology used for disease diagnosis, changes in
the classification of the diseases, or a shift of carcinogenesis. Observed changes
in relative incidence of cancers could result from an increase in depth of inhalation
of smoke by smokers of low yield cigarettes (Levi et al., 1997; Thun et al., 1997).
The use of added ingredients has changed during the same time frame. So, a
change in relative incidence might relate to an added ingredient, instead of a low
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yield structure. If on evaluating the data, this trend reflects disease incidence, the
potential effects of added ingredients on the shift from squamous cell and small
cell carcinoma to adenocarcinoma and bronchioalveolar carcinoma will need to be
determined.
7.4.2.3.2 Other cancers
Smokers are more likely to have neoplastic diseases (Read, 1984; Shaw & Milton,
1981). Cigarette smoking is associated with cancers of the mouth, esophagus,
kidney, pharynx, larynx, bladder and pancreas (World Health Organization, 1986).
Additional research is needed in order to determine whether cigarette smoking
underlies other diseases, such as breast cancer and leukemia (University of
California Office of the President, 2000). Whether there are other carcinogenic
effects that have not yet been described is unknown.
7.4.3 Immune System Effects
Studies suggest that deleterious effects of cigarette smoking, which ultimately
manifest as multiple pathologies, may relate, in part, to the suppression of the
immune system by chemical substances in cigarette smoke (Holt & Keast, 1977).
Cigarette smokers reportedly are more likely to contract influenza and mount a
weaker antibody response to the virus (Aronson et al., 1982; Finklea et al., 1969;
Kark et al., 1982). Levels of many immunoglobulins are suppressed in smokers
(Andersen et al., 1982; Gerrard et al., 1980).
Specifically, nicotine has been identified as a candidate suppressor of the immune
system (Geng et al., 1996). Immune suppression may be achieved via multiple
mechanisms, including inhibition of T-cell function (Geng et al., 1996) or increasing
adrenocorticotropic hormone (ACTH) and catecholamines (Sopori & Kozak, 1998).
These results require independent validation. Additional research on the mechanisms
underlying suppression of the immune system might be useful (Sopori & Kozak,
1998). Further understanding of a mechanism by which cigarette smoke suppresses
the immune system would provide a potential bioassay to evaluate whether an
added ingredient causes immunosuppression.
7.4.4 Free Radicals
Halliwell and Gutteridge (1999) defined free radicals as “chemical species capable
of independent existence that contain one or more unpaired electrons.” A minimum
of four classes of free radicals occur in cigarette smoke, as detected with electron
spin resonance (Pryor et al., 1983b). Free radicals in tar may be transported in
respiratory tract lining fluids (Hatch, 1992; Mudway & Kelly, 1998; Pryor, 1992b;
Cross et al., 1994), which contain antioxidants, providing an initial defense system
(Cross et al., 1997). Classes of antioxidants found in respiratory tract lining fluids
include low molecular mass antioxidants, metal binding proteins, antioxidant enzymes,
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Evaluation of Cigarette Ingredients: Feasibility
sacrificial reactive proteins, and unsaturated lipids (Davis, 1998). Free radicals
may interact with lung tissue (Church & Pryor, 1985; Pryor et al., 1983b; Pryor et
al., 1983a; Pryor, 1997; Stone et al., 1994) and cause DNA damage (Church &
Pryor, 1985; Pryor et al., 1983a; Stone et al., 1994; Leanderson & Tagesson,
1990; Lehr et al., 1994; Pryor, 1987; Eiserich et al., 1995). An imbalance of
oxidants and antioxidants may contribute to the development of COPD (Taylor et
al., 1986), and free radicals have been implicated in the development of
cardiovascular diseases (Maxwell & Greig, 2001). Information about the nature
and the mechanisms by which free radicals contribute to the development of various
diseases associated with cigarette smoking would be desirable. Whether added
ingredients in cigarettes contribute to the production and activity of free radicals
has not yet been described.
7.4.5 Risk Factors
The State of California has listed as a research initiative the study of tobacco
smoking initiation and mechanisms of development of adverse health effects–how
factors such as age, ethnicity, race or gender modify these effects (University of
California Office of the President, 2000).
7.4.5.1 Genetics
Various groups report that genes influence smoking behaviors (Carmelli et al., 1992;
Edwards et al., 1995; True et al., 1997). Genes potentially affect smoking initiation,
addiction, metabolism, host repair, and disease defense mechanisms (Thun et al.,
2002). This topic has recently been reviewed (Batra et al., 2003). Polymorphisms
in cytochrome P450 enzymes, the major enzymes involved in the metabolism of
nicotine to cotinine, affect nicotine metabolism and thus, may ultimately change
smoking behaviors (Pianezza et al., 1998). Null alleles of the CYP2A6 enzyme,
the primary enzyme involved in nicotine metabolism, may confer protection from
development of smoking habit. Smokers with CYP2A6*2 and CYP2A6*3 alleles,
which have reduced activity, are less likely to be tobacco-dependent, smoke fewer
cigarettes smoked per day (Messina et al., 1997; Nakajima et al., 1996; Pianezza
et al., 1998), and are better able to quit smoking (Batra et al., 2003; Tyndale &
Sellers, 2001).
Persons with some CYP2D6 polymorphisms may be categorized as poor
metabolizers (Alvan et al., 1990; Daly et al., 1996), extensive metabolizers, or
ultrarapid metabolizers of nicotine (Lovlie et al., 1996). This gene may play a role
in smoking behaviors after development of a smoking habit (Boustead et al., 1997),
as opposed to the initiation of a smoking habit. The role of the CYP2D6 allele and
risk of adverse health effects of cigarette smoking remains to be determined
(Bouchardy et al., 1996; London et al., 1997; Tefre et al., 1994).
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Other genes involved in nicotine metabolism including monoamine oxidases A and
B (Costa-Mallen et al., 2000; Fowler et al., 1996a; Fowler et al., 1996b) and
dopamine B-hydroxylase (McKinney et al., 2000) may also play a role in smoking
behavior. Studies examining the roles of tyrosine hydroxylase (Lerman et al.,
1997) and catechol-O-methyltransferase do not show a relationship with smoking
behavior (McKinney et al., 2000).
The dopaminergic reward pathways of the brain are stimulated by nicotine (Clarke,
1990; Tefre et al., 1994). Dopamine D1, D2 and D4 receptors genes have been
implicated in smoking behavior. Some studies suggest that the Dopamine D2
receptor gene (DRD2) DRD2A1 allele is associated with fewer dopamine receptors
(Thompson et al., 1997) and increased likelihood of being a smoker (Noble et al.,
1994), while others have not found such an association (Bierut et al., 2000; Singleton
et al., 1998). The role of dopamine receptors in the development of a dependence
on nicotine has not been resolved.
The SLC6A3 gene codes for the dopamine transporter proteins. Lerman and
others (1999) showed that individuals that express the 9 tandem repeat allele
(SLCA3-9) were older at initiation of a smoking habit, had an increased likelihood
of quitting smoking, and exhibited longer periods of abstinence from smoking. Data
from another study conflicts with this result (Jorm et al., 2000). Sabol and others
(1999) supported the association between this gene and propensity for longer periods
of smoking abstinence but did not support a role for this gene in smoking initiation
(Batra et al., 2003). Elucidation of the regulatory mechanisms of brain nicotinic
receptors would be useful for determining mechanisms of nicotine addiction.
Whether inclusion of added ingredients influences the mechanism of nicotine
dependence is an area of research that has not been fully explored.
Since nicotine increases brain serotonin release (Mihailescu et al., 1998; Ribeiro
et al., 1993) it has been proposed that serotonin receptor and transporter genes
may also be involved in smoking behavior. The roles of these as well as acetylcholine,
adrenergic, mu opioid, and cannabinoid receptors on smoking behavior have not
been fully characterized (Arinami et al., 2000). Whether the presence of added
ingredients in cigarettes modifies these pathways has not been determined.
7.4.5.2 Race/Ethnicity
A more complete understanding of factors associated with populations that affect
risk of developing smoking related disease could lend insight into how added
ingredients may impact risk and outcomes of smoking related diseases. According
to Burchard and coworkers, (2003) studies of the roles of race and ethnicity in
disease susceptibility may be complicated by genetic admixture, which is the
occurrence within a population of individuals who have multiple racial or ethnic
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origins. However, race and ethnicity are descriptors that could help to identify
differential susceptibility to disease and disease outcomes between groups.
A large disparity exists in preference for menthol cigarettes between black and
white individuals. Between 70 and 80% of African American smokers smoke
menthol cigarettes, whereas approximately 25% of Caucasian smokers smoke
menthol cigarettes (Cummings et al., 1987; Orleans et al., 1989; Sidney et al.,
1995). Black male smokers have a higher rate of morbidity from cancers associated
with smoking than do white male smokers (Day et al., 1993; Harris et al., 1993;
Satariano & Swanson, 1988; Teter et al., 2002; Stotts et al., 1991). Black men
initiate smoking at later ages than white men (Rogers & Crank, 1988), and black
male smokers reportedly smoke fewer cigarettes per day than white male smokers
(Novotny et al., 1988). Black smokers have higher levels of cotinine than white
smokers (Ahijevych et al., 1996; Ahijevych & Parsley, 1999; Benowitz et al.,
1999). The rate of cotinine clearance in African American smokers (Perez-Stable
et al., 1998) and Chinese-American smokers (Benowitz et al., 2002b) is significantly
lower than the rate of cotinine clearance in Caucasian smokers. Several
investigators found that black persons carry an allele of the CYP1A1 gene associated
with lung adenocarcinoma (Cosma et al., 1993; Crofts et al., 1993; London et al.,
1999; Shields et al., 1993; Taioli et al., 1998). Other differences could result from
differences in smoking behavior or exposure to smoke in the home or work
environment (McCarthy et al., 1992).
The risk of morbidity and mortality from COPD is higher for white smokers than
for black smokers (Coultas et al., 1994). However, the mechanism(s) underlying
this phenomenon have not been explained (Sellers, 1998). Several metabolic
enzymes exhibit differences in gene frequencies across ethnic groups. As reviewed
in Sellers (1998), UDP glucoronyl transferase enzymes, which conjugate and
inactivate carcinogens (Richie, Jr. et al., 1997), glutathione transferase enzymes
which inactivate and remove reactive electrophilic intermediates (Strange & Fryer,
1999), cytochrome P450 enzymes CYP2E1, and CYP2D6 could deactivate
chemical substances in cigarette smoke and influence the risk of smokers for
developing various smoking related diseases (Sellers, 1998). The Tobacco Use
Needs Assessment program of the state of Arizona provides statistics on tobacco
use according to geographic area and population (University of Arizona College of
Public Health, 2002). Knowledge about the contribution of race and ethnicity to
differences in smoking behavior and exposure to differences in rates of morbidity
and mortality from smoking related diseases could enhance feasibility of testing
for the health effects of ingredients added to cigarettes.
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7.4.5.3 Gender
Several lines of research suggest that women have a higher risk of developing lung
cancer than do men who smoke (Brownson et al., 1992; Harris et al., 1993; Lubin
& Blot, 1984; McDuffie et al., 1987; McDuffie et al., 1991; Osann et al., 1993;
Risch et al., 1993; Zang & Wynder, 1992). However, other studies do not report
such an increased risk (Thun et al., 2000). Data from Wei and coworkers (1993;
2000) suggests that women have decreased ability to repair DNA damage. This
group proposes that a reduced ability to correct DNA damage is associated with a
greater risk for lung cancer.
Various physiological, psychological and behavioral factors may contribute to an
individual’s smoking behavior. Creighton and Lewis (1978) and Battig et al. (1982)
show that smoking behavior differs between men and women. Both groups found
higher puff frequencies and smaller puff volumes in women. Men and women
also differ in their responses to the removal of olfactory sensory cues during smoking
(Perkins et al., 2001).
As reviewed by Gritz et al. (1996), gender differences in nicotine sensitivity,
tolerance, dependence and withdrawal may affect smoking cessation. Although
other studies revealed no differences in effectiveness of pharmacological smoking
cessation aids (Fortmann & Killen, 1995; Hurt et al., 1994). Female biological
processes such as menstruation, menopause, and pregnancy may also affect the
ability to cease smoking (Gritz et al., 1996).
According to Grunberg et al. (1991), nicotine metabolism occurs faster in men
than in women. In addition, nicotine has a stronger influence on body weight and
food and water intake in women than in men (Grunberg et al., 1986).
Kim and Hu (1998) reported that the efficiency of deposition of aerosol particles is
generally higher in women, however this has not yet been fully described (Phalen
& Oldham, 2001). More research is needed to understand the effects of gender
on smoking behavior and exposure and adverse health effects of smoking. Gender
differences could exist in the response to ingredients added to cigarettes.
7.4.5.4 Age
Approaches to understanding deposition of aerosol particles included models that
often failed to account for the role of aging on particle deposition efficiencies. As
individuals age, the size of distal airways may increase, tissue elasticity may be
altered, and the body may experience deterioration (American Heart Association,
2001). The effect of added ingredients on deposition may vary depending on the
age of the smoker. In addition, Wei and colleagues (1993; 1998) report that as
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individuals age, the ability for DNA repair decreases, however, later studies by the
same group (Wei et al., 2000) did not support this finding. Consideration of age in
modeling deposition may be valuable.
7.4.5.5 Culture
Some data suggests that culture impacts the way that cigarettes are smoked
(Schwartz et al., 1961; Hammond, 1959). The State of California has developed
research initiatives to investigate the contributions of family and environment on
smoking behavior (University of California Office of the President, 2000). Studies
have demonstrated that children with siblings and friends who smoke are more
likely to adopt the habit of smoking (Alexander et al., 1983).
7.4.5.6 Diet and nutrition
The role of diet and nutrition in the development of diseases associated with smoking
cigarettes has not been completely described. Epidemiological studies have shown
that diets rich in fruits and vegetables associate with a decreased risk of lung
cancer (reviewed in (Ziegler et al., 1996). Khaw and others (2001) identified an
inverse relationship between the plasma concentration of vitamin C and risk of
death from cardiovascular disease. Elinder and coworkers (1994) showed that
polyphenolic flavenoids may confer protection from cardiovascular disease
(Kromhout et al., 1995; Hertog et al., 1993). Other studies, however, do not
support a protective effect of antioxidants (Elinder & Walldius, 1994). Maxwell
and Grieg (2001) propose conducting long-term prospective randomized controlled
trials in order to investigate advantages of increases in antioxidants.
Wynder et al. (1987) suggested that fat intake could account for difference in lung
cancer between United States and Japan. Fat may affect the function of some
lung oxidases and lead to additional conversion of carcinogenic form of compounds
(Wynder et al., 1987). It has been suggested that obesity (Alavanja et al., 1996)
and a diet rich in fat (De Stefani et al., 2002) are risk factors for adenocarcinoma
of the lung. Whether diet and body weight could influence the effect of ingredients
added to cigarettes is not known.
7.4.6 Individual variation in health effects and behavior
There is a wide intra-individual variation in uptake of inhaled substances.
Physiologically based pharmacokinetic simulation models using experimental animal
and human data have shown that physiologic factors, such as alveolar ventilation,
substantially impact the kinetic behavior of potentially carcinogenic inhaled
substances (Lin et al., 2001). In addition, rates of metabolism and clearance of
chemicals from the body are known to vary. Exposure time to nicotine is affected
by the rate of nicotine metabolism and clearance from the body. Factors that
affect an individual’s exposure to compounds and health outcomes due to exposure
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have not been fully described. Ingredients added to cigarettes may influence the
uptake of substances already present.
7.5 TESTING ISSUES AND METHODS
7.5.1 Animal Models
There are multiple tests using animal models that may be applied to the study of
heath effects of smoking cigarettes. However, whether they predict the
development of human diseases, particularly the human diseases associated with
smoking, is an important consideration. Validated animal models of smoking-related
diseases will prove useful for a determination of the effects of added ingredients
on the risk of cigarette smoking related diseases.
Various animal models have been used to study the biological effects of inhalation
exposure to cigarette smoke. In addition to the thousands of other substances in
cigarette smoke, animals are exposed to carbon monoxide. The affinity of carbon
monoxide for hemoglobin is 240 times that of oxygen for hemoglobin (Douglas et
al., 1912). Carbon monoxide uptake in rats is faster than the predicted rate of CO
uptake in man (Silbaugh & Horvath, 1982). To decrease the probability of rodent
carbon monoxide related poisoning, investigators dilute the cigarette smoke to which
the animals are exposed and allow intermittent exposure to cigarette smoke.
7.5.2 Clinical Studies (behavior)
The effects of menthol in cigarettes has undergone some investigation (Caskey et
al., 1993; Porchet et al., 1987; Tsujimoto et al., 1975). However, the effects of
many other added ingredients on smoking behavior are not known. Added ingredients
could cause multiple changes in smoking behavior. They could affect smoking
topography, the number of cigarettes smoked per day, the amount of the cigarette
smoked and choice of cigarette smoked. Investigators could assess many
parameters such as the number of puffs per cigarette, puff duration, puff volume,
peak puff flow, inter-puff interval, total smoking time, and total inhalation volume
to describe how a cigarette is smoked and measure the variation within and among
smokers. Measurement of these parameters with paired, otherwise identical
cigarettes, with and without an added ingredient, are not likely to prove useful in
understanding the effect of added ingredients on smoking behavior, because it is
not readily apparent how to integrate these parameters into a measure of behavior.
Measurement of the number of cigarettes smoked and of a biomarker of smoking
behavior will lead to an integrated measure. Conducting of such behavioral studies
is a high priority for LSRO, in the sense that additional data about such studies
would make decisions about the feasibility of testing easier.
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7.5.3 Analysis of Data
Many epidemiological studies have explored information about the health effects
of cigarette smoking. Additional analysis of these surveys and other types of data
may help to determine how cigarette smoking impacts relative risk of adverse
health effects of cigarette smoking and to determine how age, gender, race, and
ethnicity affect that risk (University of California Office of the President, 2000).
cDNA microarray expression profiling and proteomic techniques may prove useful
characterizing individuals risk of morbidity and mortality from cigarette smoking
related diseases.
7.6 SUMMARY
LSRO and its advisors have identified areas where additional research would
improve decision-making about the feasibility of determining the relative risks of
smoking cigarettes with added ingredients as compared to smoking cigarettes without
added ingredients. Additional research is needed to understand the chemistry of
cigarette smoke. The relationship between substances in the cigarette and smoke
from the cigarette could be used as a basis to assess the effects of the added
ingredient on the smoke matrix. Additional validated, standardized techniques for
smoke chemical detection will aid this process. Information about the physics of
cigarette smoke would serve as a basis for determining whether added ingredients
affect exposure levels of individuals to cigarette smoke. Research is needed to
elucidate the biological and social factors that elicit changes in smoking behavior.
Consistent use and further development of biomarkers of exposure and effect will
help to provide insight into health effects of smoking cigarettes with added ingredients.
Improved understanding of the mechanisms of disease associated with cigarette
smoking would provide insight as to whether added ingredients contribute to these
diseases. How risk factors such as gender, race or ethnicity, culture, gender, age
and diet influence risk of modify health risks will also contribute to such analyses.
Clinical studies of the effects of added ingredients on exposure to smoke, as mediated
by smoking behavior, will also be useful to making decisions about the feasibility of
testing for the relative risk of adverse health outcomes from smoking cigarettes
with added ingredients as compared to smoking cigarettes without added ingredients.
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8
CONCLUSIONS
OUTLINE
8.1
PRIMARY CONCLUSIONS
8.2
SCIENTIFIC RATIONALE
8.2.1 A Matrix of Chemical Substances in Cigarette Smoke
8.2.2 Pyrolysis and Transfer
8.2.3 No Detectable Change
8.2.4 Human Smoking Behavior
8.2.5 Case-By-Case Evaluation
8.3
DETAILS & EXCEPTIONS
8.3.1 Biological Testing
8.3.1.1 Prediction of human health effects
8.3.2.2 Biomarkers of human disease
8.3.1.3 Duration of other cumbersome elements of human
epidemiology
8.3.2 Mixtures
8.4
PRIORITY SETTING
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8
CONCLUSIONS
8.1 PRIMARY CONCLUSIONS
LSRO recommends that manufacturers test ingredients added to cigarettes. Such
testing is both feasible and worthwhile as discussed below. (See Appendix G).
Test data will inform judgments about the risks of cigarettes containing these
ingredients, relative to similar cigarettes without the same ingredients. Some of
the assumptions that support this recommendation merit expression.
Absolute safety, defined as the absence of risk, is unattainable. The term, “safe,”
as used in most product safety testing, comes closest to the criterion of “reasonable
certainty of no harm,” as used at the U.S. Food and Drug Administration (FDA).
When most experts refer to a product as “safe,” they mean something operational,
for example, that a food additive has undergone FDA review and passed.
In this report, LSRO has used a different approach from FDA’s, looking at the
effects of ingredients added to cigarettes for relative risk, defined as the risk of
exposure to a cigarette with the ingredient, relative to the risk of exposure to a
cigarette without the ingredient. Employing relative risk created many advantages
in LSRO’s review. A relative risk approach diverges from traditional product
safety testing. An “unchanged relative risk” does not imply that the underlying
activity, cigarette smoking, is safer or that addition of the ingredient has changed
the risk of the underlying activity.
The feasibility of testing for relative risk depends crucially on testing paired cigarettes
that differ only in the presence or absence of an ingredient or mixture of ingredients.
Traditional safety testing would test the ingredient in isolation. Instead, LSRO
recommends testing an ingredient within cigarettes during the act of smoking.
Should experimental pairs of cigarettes, with and without an added ingredient, prove
impossible to manufacture or unworkable in testing, the approach taken here will
not apply.
The feasibility of testing ingredients added to cigarettes also depends on practical
decision-making. For example, initiation of smoking is a public health concern.
However, direct study of the effects of ingredients added to cigarettes on initiation
of smoking, by presenting non-smokers with cigarettes with and without the additive,
is unethical.
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The conclusions of this Phase One report could apply to new or existing ingredients
currently added to cigarettes, not only to the existing ingredients described in Appendix D. However, LSRO’s emphasis has been to review ingredients already in
use first. (See Section 8.4) LSRO’s Phase Two report will consider the scientific
criteria to be applied to the evaluation of ingredients and will address whether
certain ingredients should obtain expedited review. The case studies and discussions within this report revolve around substances already in use.
Potentially, new ingredients and/or mixtures of ingredients may result in effects not
previously associated with cigarette smoking. Thus, the introduction of novel ingredients may require more robust or extensive testing and monitoring than considered in this report. Importantly, LSRO has operationally excluded from this report
considerations pertaining to issues whereby an additive could potentially decrease
the overall intrinsic toxicity of cigarette smoke.
In this report, LSRO’s conclusions have five underpinnings: the matrix of chemical
substances in cigarette smoke, the pyrolysis and transfer of ingredients during smoking, the concept of no detectable change in overall biological toxicity, the notion of
human smoking behaviors, and a case-by-case evaluation using the sum of base
knowledge and newly generated knowledge. The subsections below review each
underpinning. Each has scientific justification.
8.2 SCIENTIFIC RATIONALE
8.2.1 A Matrix of Chemical Substances in Cigarette Smoke
Scientific data, reviewed in Chapter 3 of this report, establish that cigarette smoke
is a complex mixture of many chemical substances, which changes with time.
Throughout this report, LSRO has referred to the “chemical matrix” or the “smoke
matrix,” as a shorthand way of communicating this concept. Many research studies
have attempted to relate the biological toxicities of individual smoke constituents to
human health outcomes. The relative lack of overall success in this endeavor has
persuaded LSRO to regard human health effects in relation to cigarette smoke as
a whole.
When comparatively testing otherwise identical cigarettes, the added ingredient
should alter the baseline smoke matrix as little as is possible. Animal tests are not
sensitive measures of change. Smoking machines, which create experimental
cigarette smoke, do not necessarily reproduce the volume of inhaled smoke delivered
to the human lung. They do represent a fair technology for examining comparative
toxicities of different cigarettes smoked in a similar manner. The experimental
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Evaluation of Cigarette Ingredients: Feasibility
smoke can be regarded as a physico-chemical surrogate for human-generated
smoke.
8.2.2 Pyrolysis and Transfer
The scientific data in Chapter 3 of this report review the evidence that some
ingredients pyrolyze during cigarette combustion, even to complete elimination.
Similarly, Chapter 4 emphasizes that an ingredient, or pyrolysis products of the
ingredient, can transfer into a smoker’s lungs along with the smoke. The current
state-of-the-science is such that exclusively testing ingredients outside of the smoke
matrix does not provide sufficient information to predict the behavior of the ingredient
within the smoke matrix.
The potentials for pyrolysis and for transfer of an ingredient within a smoke matrix
are crucial elements in LSRO’s approach to the relative risks of different amounts
of the ingredient in a cigarette. Testing unpyrolyzed ingredients in isolation,
particularly by different routes of administration, will not provide information about
potential pyrolysis products. Some ingredients and pyrolysis products of ingredients
will undergo complete degradation during cigarette combustion and will not reach
the smoker’s respiratory tract. Other ingredients, like menthol, will resist pyrolysis,
distilling, condensing and redistributing down the tobacco column, transferring nearly
completely intact.
Scientific methods and technology are readily available to generate data about the
effects of an ingredient on smoke chemistry. Thus, tests to resolve questions
about pyrolysis and ingredient transfer to smoke are feasible.
When an ingredient added to cigarettes, or a pyrolysis product of the ingredient
added to cigarettes, transfers in the smoke, traditional methods of inhalation
toxicology can be brought to bear to obtain dose-effect data and exclude an increase
in risk superimposed on the known chronic adverse health effects of smoking.
Because scientific methods and technology exist to meet these concerns, testing
ingredients added to cigarettes to understand the relative risks of those ingredients
when added to cigarettes is feasible.
To decrease the risk that an ingredient added to cigarettes might increase a known
adverse health effect associated with cigarette smoking, it will be necessary to
understand better the chemistry, exposure, and adverse health effects associated
with cigarette smoking. In addition to excluding an additional health effect, one
associated with the added ingredient, a concern exists that the additive might change
the chemistry of the smoke matrix in such a way that the known, chronic adverse
health effects of smoking might change. A change could come about in either of
two ways: (1) the ingredients added to cigarettes, or pyrolysis products of the
ingredients added to cigarettes, might change the toxicity of the inhaled smoke or
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(2) the ingredients added to cigarettes, or a pyrolysis product of the ingredients
added to cigarettes, might change smoking behavior so as to result in greater initiation
or either of these possibilities also might occur in the absence of transfer of the
added ingredient and its pyrolysis products during inhalation of a plume of hot
smoke.
To minimize the risk of adding new adverse health effects to the existing adverse
health effects associated with cigarette smoking, research data will have to reveal
the effects of an ingredient added to cigarettes on smoke chemistry, because one
of the prominent concerns relates to potential pyrolysis products of the ingredients
added to cigarettes, not only to the added substance per se.
While science-based testing can achieve, or at least approach, many of the above
objectives, application of a set of standardized safety tests, such as those used by
FDA’s food additives program or EPA’s pesticides program, could not. Testing a
purified substance could miss a novel health effect associated with a pyrolysis
product. If the purified, isolated substance is pyrolyzed separately, the problem
remains of demonstrating that these pyrolysis products have some identity with
those formed in the cigarette smoke matrix. Testing in experimental animals is
insensitive, relative to testing with techniques of analytical chemistry and physics.
Existing recommended, standardized programs for testing toxic effects, i.e., food
additive methods, are not directly transferable to testing cigarette additives.
Gastrointestinal and cutaneous pathways differ markedly from the inhalation
pathway. In addition, the effects of a chemical substance may not resemble the
effects of its pyrolysis product(s). Therefore, existing test guidelines may have
relevance, but they will require modification. Any valid, useful test must comply in
principle with the testing principles in Chapter 6 of this report.
Data about the pyrolytic fate of each ingredient added to cigarettes under smoking
conditions will be uniformly desirable for all ingredients. In addition to understanding
whether an added ingredient shifts the chemical composition of other substances
in the smoke matrix, test data must be available to understand the relative risks, if
any, of the effects of inhalation exposure to an unpyrolyzed ingredient.
8.2.3 No Detectable Change
A “no detectable change” approach interprets test data and other health effects
information to specify an upper limit on the amount of an ingredient added to a
cigarette. The upper limit relates to test signals, which depend on sampling and
test methods. The approach reduces the amount of an ingredient per cigarette
below a level where change is detected in any of a group of tests. The tests should
be balanced among the kinds of tests (physical, chemical, biological, and clinical)
that generate information about potential human health effects. At some upper
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limit of the amount of ingredient per cigarette, if tests do not detect a difference
between smoke from a cigarette containing an ingredient and smoke from an
otherwise identical cigarette without the ingredient, no a priori reason will exist to
suspect a differential health effect.
Other approaches exist to designating an upper limit on the amount of an ingredient
to add per cigarette. As an example, a manufacturer can limit the amount of an
ingredient by comparison to a highly toxic substance. If the maximum dose acquired
by smoking a cigarette falls below a biological cut-off or limit for a highly toxic
substance, as established by other means, the manufacturer could regard this
maximum amount as acceptable.
Any inference based on testing of an ingredient for pyrolysis or for transfer will
depend crucially on the hypothesis that changes in test cigarette tobacco will not
alter the response relationship between the amount of ingredient and test signal for
either pyrolysis or transfer. If the test cigarette contained Burley tobacco and the
experimenter changes to Maryland tobacco, test signals will change in relation to
the same amounts of ingredient added. If a change in the tobacco substrate alters
the nature or magnitude of changes in the smoke matrix, the logic of testing to
detect a point of no detectable change will still hold, as long as the change in the
tobacco substrate does not change the ingredient-signal relationship. Similarly,
differential changes in the levels of substance in the smoke, given a change in the
tobacco substrate, would not necessarily invalidate experiments to set the maximum
level of ingredients added to cigarettes per cigarette. The tobacco substrate would
need to shift the relationship between the amount of an ingredient added and the
test signal.
If testing revealed no incremental chemical or biological effect, a new test, previously
not used, might detect a difference. Even if LSRO obtained data from every
known test, a new test might arise, which could detect a difference. Like all areas
of science, LSRO’s inference will relate to the state-of-the-art applicable
technologies existing at some point in time. A test also can generate a false negative
result, a finding of no effect when one exists. Employing a battery of tests, instead
of a single test, will reduce the probability that a false negative result will influence
estimation of the upper limit. Each test has characteristic statistical limitations,
such that retesting with more samples could usually detect a difference, not
previously noted. Testing with reliable methods that have good, although presently
unspecified, signal-to-noise characteristics will minimize the possibility of
underestimating the upper limit for an ingredient not to change smoke chemistry.
In this regard, selection of specific tests, which LSRO will describe in its Phase
Two report, Scientific Criteria, will become important.
Most ingredients are added to cigarettes in relatively small amounts, for example,
in parts per million. The ability to detect a change above background noise is
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potentially meaningful, because exceeding background noise could mean that amount
of an added ingredient altered a biological response. Specific cigarette brands also
contain unique mixtures of added ingredients, and the components of these mixtures
may interact with one another. Therefore, an ingredient in a mixture should be
evaluated in the brand of cigarette to which it will be added and marketed.
For existing ingredients added to cigarettes, LSRO will seek useful test data to
make decisions about two objectives: (1) not increasing any of the well-known
adverse health effects associated with cigarette smoking, and (2) not adding a
new, different adverse health effect. Detection of a difference induced by an
ingredient in smoke is sufficient to allow experimental location of a no detectable
change level. Subsequently, LSRO can estimate the maximum level to add per
cigarette. However, “no detectable change,” or “maximum likely change,” is a
difficult concept. It neither implies the absence of any change, nor does it mean
that the specified measure lacks any implications for relative risk.
In making its decision about feasibility, LSRO considered the joint implications of
analyzing a model smoke and of using indicator substances. Neither approach
completely eliminates the possibility that an added ingredient might change the
concentration of some substance in cigarette smoke and that this smoke substance
might induce an adverse human health effect. Thus, both approaches might be
employed, monitoring both the ingredient, and its pyrolysis products, and indicator
substances usually found in cigarette smoke.
At a level of an ingredient where exposure-response relationships change, current
scientific data do not permit LSRO to predict whether known adverse human
health effects might increase or decrease. Therefore, LSRO will regard any change
as an indication of a potential adverse effect or as detection of the added ingredient.
This approach means that added ingredients also will not necessarily decrease
adverse health effects, particularly adverse health effects of smoking cigarettes; it
does not address them. Chemical components of the smoke can serve as indicators
of changes within the matrix. LSRO will describe some substances, which might
make the best indicators, in its Phase Two report, Scientific Criteria. Thus, LSRO
will regard any detected change in an indicator substance as potentially adverse
development and evaluate it as such, unless the manufacturer can provide convincing
evidence to the contrary.
8.2.4 Human Smoking Behavior
Humans have a limited ability to articulate their personal perception of smoke
exposure. The evidence favors human smoking behaviors intended to achieve an
approximately constant amount of delivered and absorbed nicotine. Smoking
behavior determines the delivery of nicotine to the respiratory tract absorptive
surfaces. CNS-mediated feedback mechanisms exist which might create the
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Evaluation of Cigarette Ingredients: Feasibility
phenomenon of compensation, such that increasing smoke intake compensates for
lower nicotine yield in the smoke.
However, the taste and aroma of cigarette smoke also influence smoking behavior.
Ingredients added to cigarettes and designed to affect taste and aroma might
contribute to the maintenance of smoking behaviors. Little definitive published
data shows that added ingredients might or might not alter the sensory experience
of smoking. Sensory cues, such as cigarette flavor and aroma, potentially support
the smoking behaviors. Smoking pairs a distinctive aroma and taste ambiance
with a pharmacological effect.
The behavior of individual smokers is highly variable. The study of the toxic material
to which smokers are exposed is a relatively complex and difficult scientific subject.
Because of high inter-individual variation, human studies that compare different
smokers will likely encounter difficulties. However, cigarette consumption and
smoke exposure are amenable to scientific investigation. Thus, testing of smoking
behavior is feasible.
8.2.5 Case-By-Case Evaluation
The wide range of chemical and biological properties of existing ingredients will
make the application of over-riding rules impractical and unlikely. The evaluation
of an ingredient will require case-by-case expert judgment. The application of
written review criteria will help to avoid an inconsistent treatment of similar
ingredients, maximize transparency of the process, and support the establishment
of an evidence-based consensus.
Evaluation of each ingredient should begin with a scientific review of the available,
relevant information by qualified experts. Scientists often will need to proceed
with the evaluation of an ingredient on a case-by-case basis. General scientific
principles, as described in Chapter 6, should inform and assist the review of a
substance. LSRO will further detail the application of these principles in the Phase
Two report, Scientific Criteria.
The use of every conceivable test is neither necessary nor desirable. LSRO cannot
locate definitive tests that accurately predict specific health endpoints related to
cigarette smoking. Beyond a small number of key tests, to establish identity of
smoke between a pair of experimental cigarettes, additional tests will yield little
additional predictive information and may generate disinformation. A balanced
program of qualitatively different tests of ingredients added to cigarettes will be
desirable (physical, chemical, subcellular, cellular, intact animal, and human tests).
Signal-to-noise ratio and practicality will be important factors in selecting appropriate
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193
tests. LSRO will describe these factors and tests in more detail in its Phase Two
report, Scientific Criteria.
8.3 DETAILS & EXCEPTIONS
8.3.1 Biological Testing
Testing an added ingredient with traditional animal toxicity tests may be insensitive,
because animal bioassays do not predict human health effects associated with
cigarette smoking. Any potential animal model would have to predict at least one
human health effect associated with cigarette smoking, to be useful in feasible
testing. To date, few, if any, short-term biomarkers accurately detect any of the
human diseases associated with cigarette smoking. Direct observational studies of
chronic adverse human health effects in epidemiological studies take too long and
are too cumbersome to help evaluate most added ingredients.
To evaluate the relative risks associated with an ingredient, LSRO will need data
about biological changes in smoke chemistry as a function of the amount added.
In effect, these tests use a biological measurement to compare smoke from a
cigarette containing an ingredient to the cigarettes smoked in existing epidemiological
studies. They are measures of biological similarity. Traditional product safety
testing in laboratory animals would make little sense in the absence of demonstrable
transfer, changes in smoke chemistry, or changes in behavior relevant to smoke
exposure.
8.3.1.1 Prediction of human health effects
Over the last two decades, cigarette manufacturers produced extensive test data
using traditional animal models and exposures to either (1) diluted, cooled smoke or
(2) particulate matter filtered from whole smoke. Both exposure pathways miss
potentially crucial aspects of exposure. The first approach may miss pyrolytic or
pyrosynthetic changes in the smoke that humans inhale. The second may miss
changes in the gas phase of cigarette smoke.
Animal toxicology tests are relatively insensitive methods to detect changes induced
in cigarette smoke by an added ingredient, relative to the known toxicity of cigarette
smoke. Animal toxicity tests also do not predict the adverse health effects of
cigarette smoking observed in epidemiological studies of humans. Testing an added
ingredient in isolation almost certainly will miss the more meaningful and relevant
effects associated with exposure to a cigarette smoke matrix modified by the
additive. Pulmonary absorption of a pyrolysis product in an inhaled plume of hot
smoke containing many thousands of chemical substances, but changed amounts
or ratios by the presence of the additive, would not be detected in experiments
where only the effects of the additive were studied.
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Evaluation of Cigarette Ingredients: Feasibility
Predictive tests of morbidity associated with human smoking also are not yet
available, although proximal markers of risk of diseases associated with smoking,
such as early biomarkers of effect, have been suggested and are actively being
explored. Identification of relevant surrogates of disease will improve the overall
review process.
8.3.1.2 Biomarkers of human disease
Biomarkers of effects for the human diseases associated with cigarette smoking
are not yet readily available. Biomarkers of exposure, such as cotinine levels, will
prove useful to establish whether human smoking behavior changes in response to
an added ingredient. Should biomarkers of human adverse health effects arise,
toxicologists can study these biomarkers in animal models. Should an animal model
prove useful as a predictor of adverse human health effects, toxicologists can
employ the model in the paradigm of paired, similar cigarettes, with and without an
added ingredient, to detect exposure-response relationships and levels of no
detectable change. Toxicologists can compare substances that induce mutagenic
reversion in smoke produced by human smokers to substances that induce reversion
in their urine and seek animal models that either replicate or allow predictions of
these relationships.
8.3.1.3 Duration and other elements of human epidemiology
Observational studies of human epidemiological effects will take too long and prove
too cumbersome to evaluate most added ingredients. At best, LSRO can hope for
inferential studies comparing populations smoking cigarettes with added ingredient
to populations smoking cigarettes lacking added ingredients. Such studies likely
will omit identification of specific ingredients in the cigarettes smoked. When
possible, results obtained or inferred from testing should undergo validation by post
marketing surveillance, epidemiological studies, and/or clinical trials. However,
using pack-years, as a measure of exposure is potentially inaccurate, while better
than no indicator. Epidemiologists usually obtain exposure information about smoking
from questionnaires or interviews. Retrospective, self-reported data may
inaccurately characterize the intermittent exposures of individual smokers. As
smokers change brands, they will also change exposure to the effects of added
ingredients. Measures of cigarette consumption cannot easily account for variable
smoking.
A biological model that does predict human disease outcomes of different durations
of cigarette smoking would greatly simplify testing of ingredients added to cigarettes.
8.3.2 Mixtures
Besides the presence of mixtures of substances of natural origin in cigarettes and
mixtures of many chemical substances in smoke, manufacturers seldom add a
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Conclusions
195
single ingredient in producing cigarettes. Mixtures of ingredients are added to
cigarettes. For testing purposes, an added ingredient can be a single added chemical
compound or substance, or a specific recipe of compounds or substances, such as
a proprietary flavor formula, or a complex mixture, for example, cocoa. The testing
of ingredients added to cigarettes, one-by-one, is subject to exceptions:
Exception one: If predictive tests of significant cigarette-associated health
effects become available, manufacturers also should apply such tests to all
added ingredients in combination.
Exception two: Manufacturers will have to retest the specific mixture of added
ingredients, in the proportions used in their brand(s) of cigarettes, to obtain an
assurance that the mixture lacks the same effect absent when individual added
ingredients are tested, which might arise from an interaction between two or
more ingredients.
8.4 PRIORITY SETTING
Because manufacturers already use many ingredients, setting priorities on the
evaluation of ingredients makes good sense. Dividing added ingredients into
categories of existing and new also makes good sense, because the kinds of tests
and the urgency of testing clearly differ for substances already in use.
Manufacturers can set priorities for evaluation of ingredients based on the relative
maximum mass (or average mass) added to a brand of cigarette, with the highest
mass per cigarette evaluated first. Priorities for evaluation which factor the mass
of ingredients by measures of toxicity, such as a lethal dose, also make sense,
because doses which affect different measures of toxicity in different test systems
usually covary. A division of added ingredients into categories of pyrolyzed and
unpyrolyzed also makes sense, with the unpyrolyzed receiving more attention.
The need to set priorities explains why an incremental approach to testing is prudent.
A checklist of specific tests to apply to each added ingredient is neither feasible
nor desirable. Given these exceptions, a balanced program of testing ingredients
added to cigarettes will greatly reduce the possibility of any adverse human health
effect, beyond the premature mortality and morbidity already seen with cigarette
smoking.
Epidemiological studies of the premature mortality and morbidity of cigarettes containing menthol demonstrate that scientists can obtain definitive evidence about
ingredients added to cigarettes. Epidemiological studies of the effects of changes
in the design of cigarette structure additionally show that premature mortality and
morbidity can change. (See Chapter 6, Section 6.4.)
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Evaluation of Cigarette Ingredients: Feasibility
Without some test data, managing changes in the exposures from ingredients in
cigarettes will prove difficult. Dose is the critical factor in the possibility of a
change in the relative mortality and morbidity of cigarette smoking. However, dose
is difficult to measure. At present, no tests appear to predict the major human
health effects of chronic cigarette smoking (premature mortality, cancer, cardiovascular effects, and chronic obstructive pulmonary disease). Without predictive
tests for specific endpoints, exposure-response relationships from traditional bioassays will prove useful in understanding relative risks and setting limits on the amounts
of ingredients in cigarettes that are unlikely to change the risk of smoking, relative to
smoking identical cigarettes without the same added ingredients.
A program of testing will put the ingredients added to cigarettes into perspective
with each other in a biologically oriented framework and need not prove the complete absence of relative risk. However, such a program raises questions about
action limits, which LSRO will address in its report on the second phase of this
project, Scientific Criteria. Additional information, particularly information aimed
at understanding which categories of added ingredients pose the highest potential
risks, will serve to target strategies for added ingredients posing the greatest potential health risk
Test data should include quality assurance and peer review, both within and outside
the manufacturing industry. A scientific peer-reviewed report will be accessible to
the public and scientific community. Such reports of added ingredient reviews will
be especially useful as criterion-based references to build on, when new testing
technologies become available and additional relevant data accumulate.
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10
STUDY PARTICIPANTS
AD HOC EXPERT PANEL
Carroll E. Cross, M.D.
Professor, University of California, Davis, School of Medicine, Division of
Pulmonary and Critical Care Medicine
Sacramento, California
Shayne C. Gad, Ph.D., D.A.B.T.
Gad Consulting Services
Cary, North Carolina
Donald E. Gardner, Ph.D., F.A.T.S.
President, Inhalation Toxicology Associates
Raleigh, North Carolina
Louis D. Homer, M.D., Ph.D.
Medical Director, Holladay Park Medical Center, Legacy Research
Portland, Oregon
Rudolph J. Jaeger, Ph.D., D.A.B.T., B.C.F.M.
Principal Scientist, Environmental Medicine, Inc.
Westwood, New Jersey
Robert Orth, Ph.D.
President, Apis Discoveries, LLC.
Cedar Hill, Missouri
* Resha M. Putzrath, Ph.D., D.A.B.T.
Principal, Georgetown Risk Group
Washington, District of Columbia
* Active member through July, 2003.
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Evaluation of Cigarette Ingredients: Feasibility
Emanuel Rubin, M.D.
Professor and Chairman, Department of Pathology, Anatomy, and Cell Biology,
Thomas Jefferson University
Philadelphia, Pennsylvania
James L. Schardein, M.S., F.A.T.S.
Independent consultant
Leesburg, Florida
Thomas J. Slaga, Ph.D.
President and CEO, AMC Cancer Research Center, and Chair, Center for Causation and Prevention
Denver, Colorado
LIFE SCIENCES RESEARCH OFFICE STAFF
Michael Falk, Ph.D.
Director
Daniel M. Byrd, III, Ph.D., D.A.B.T.
Deputy Director
James Cecil Smith Jr., Ph.D.
Senior Scientific Consultant
Nancy D. Emenaker, Ph.D., M. Ed., R.D.
Scientific Consultant
Amy M. Brownawell, Ph.D.
Staff Scientist
Catherine J. Klein, Ph.D.
Staff Scientist
Kara D. Lewis, Ph.D.
Staff Scientist
Paula M. Nixon, Ph.D.
Staff Scientist
Pippa Antonio, B.S.
Associate Staff Scientist
For internal use of Philip Morris personnel only.
Study Participants
Negin P-M. Royaee B.S.
Associate Staff Scientist
Karin French, B.S.
Associate Staff Scientist
Kristine K. Sasala, B.S.
Associate Staff Scientist
Robin S. Feldman, B.S., M.B.A.
Literature Specialist Librarian
Shelia McGregor
Executive Assistant
Tonda Savoy-Middleton
Administrative Assistant
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Evaluation of Cigarette Ingredients: Feasibility
TABLES
CHAPTER 3
3-1 Component levels of mainstream smoke from Kentucky research cigarettes (85 mm) as measured under standard laboratory procedures.
3-2 Numbers of substances in different chemical classes identified in tobacco
leaf and/or cigarette smoke.
3-3 Chemical substances and chemical classes in the vapor phase of mainstream
smoke of cigarettes without filters.
3-4 Chemical substances and chemical classes in the particulate matter of mainstream smoke of cigarettes without filters.
3-5 Percent distribution of radioactivity in mainstream and sidestream cigarette
smokes from 14C-labeled nicotine or n-dotriacontane (based on tobacco consumed).
3-6 Carcinogens in cigarette smoke.
CHAPTER 4
4-1 Regional deposition of tar 24 weeks after switching to a lower tar cigarette
(mean ± SE).
4-2 Some estimates of inhaled tar depositon using different exhaled particle collection methods.
4-3 Human deposition of cigarette smoke components after a two-second, 35 mL
puff and inhalation or mouth-only exposure.
4-4 Percentage of inhaled particles (diameter 0.2 µm) predicted to deposit in human lung regions by three models.
4-5 Comparison of exposure biomarkers in nonsmokers and active smokers.
4-6 Breath concentrations (µg/m3) of selected smoke constituents by smokers (S)
and nonsmokers (NS) [Data given as unweighted geometric averages].
CHAPTER 5
5-1 Summary of smoking-related deaths.
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273
FIGURES
CHAPTER 3
3.1 The components of a typical filtered cigarette.
3.2 Diagram of the major processes generating cigarette smoke during a puff.
3.3 Variation in temperatures (°C) inside the combustion zone of a cigarette following one second of a 2-second puff.
3.4 Cumulative number of chemical substances identified in tobacco and cigarette
smoke by year.
3.5 Sales-weighted tar and nicotine values for U.S. cigarettes.
CHAPTER 4
4.1
4.2
4.3
4.4
4.5
4.6
Typical puff profiles.
Average puff volume changes with puff number.
Schematic representation of the nicotine-acetylcholine receptor.
Relative deposition of particles in a lung model.
Pankows’s depiction of four processes for molecular absorption.
Schematic diagram of cigarette smoke deposition in a cast model of the human tracheobronchial tree.
4.7a Ciliated epithelium at a tracheal bifurcation in an unexposed rat.
4.7b Deciliation at a tracheal bifurcation in a rat exposed to cigarette smoke.
4.8 Average benzene concentrations in breath as function of number of
cigarettes smoked.
4.9 Schematic representation of whole body exposure of rats to cigarette smoke.
CHAPTER 6
6.1 Biomarker categories along the contininum from exposure to disease.
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Evaluation of Cigarette Ingredients: Feasibility
APPENDIX A
LIFE SCIENCES RESEARCH OFFICE
Added Ingredients Review Ad Hoc Expert Panel:
Carroll E. Cross, M.D. is a pulmonary critical care physician at the University of
California at Davis School of Medicine, Division of Pulmonary and Critical Care
Medicine. He graduated from Columbia College of Physicians and Surgeons in
1961, completed his internship at the University of Wisconsin Hospital in 1962, his
residency at Stanford Hospital Center in 1964 and his clinical and research fellowship training at the University of Pittsburgh Medical Center in 1968. He was certified in internal medicine in 1969 and in pulmonary disease in 1971. He has been at
the University of California, Davis since 1968, where he is currently a professor of
medicine and physiology. Dr. Cross has published over 200 papers in such fields as
air pollutants, antioxidant micronutrients, inflammatory-immune system oxidants,
ozone, oxides of nitrogen, cigarette smoke, and related aspects of inhalation toxicology as it relates to respiratory tract diseases. He is a member of several professional organizations including the American Physiological Society, the UK Biochemical Society, the Oxygen Society, the Mount Desert Island Biological Laboratory,
the Western Society of Physicians, and the American Society for Clinical Nutrition.
He serves on the editorial boards of the American Journal of Clinical Nutrition
and Free Radicals in Biology and Medicine, and has served on research review
panels for the Veterans Administration, for the NIH and for the Heart, Lung and
Cancer Society Associations.
Shayne Cox Gad, Ph.D., D.A.B.T., F.A.T.S. is the Principal at Gad Consulting
Services. After majoring in chemistry and biology at Whittier College, he obtained
his doctorate in pharmacology/toxicology from the University of Texas. As a fellow at Bushy Run Research Center, Union Carbide Toxicology Laboratory he developed a system for assessment of toxicity of polymer thermal decomposition
products. Later, he worked at the Shell Development Laboratory establishing a
new inhalation toxicology research facility. As Manager of Mammalian Toxicology
at Allied Corporation, Dr. Gad was responsible for all mammalian toxicity testing
including the operation of the entire Department of Toxicology laboratory and all
external contract testing. Dr. Gad has also worked at G.D. Searle & Co. as Director of Toxicology and Senior Director of Product Safety and Metabolism where he
used an interdisciplinary approach to oversee safety/toxicity research programs,
assisted with the prioritization of research and development efforts, interacted with
foreign firms, regulatory agencies, and developed world wide occupational Airborne Control Objectives. He has also served as the Director of Medical Affairs
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Product Support Services at Becton Dickinson and Director of Toxicology at
Synergen. Dr. Gad has directed the design, conduct, writing, and filing of investigational new drug applications, new drug applications, drug disposition studies and
medical device petitions. Dr. Gad’s presentations and publications total over 300
and include 27 books and 35 book chapters.
Donald E. Gardner, Ph.D., F.A.T.S. is the President of Inhalation Toxicology Associates. He received his M.S. in medical microbiology from Creighton University
and his Ph.D. in environmental health and toxicology from the University of Cincinnati. He has worked for the U.S. Environmental Protection Agency (EPA) and the
Public Health Service. Following retirement from EPA, he joined the staff of
Northrop/ManTech Corporation as Vice President and Chief Scientist. He held
academic appointments at Duke University, North Carolina State University, and
the University of Massachusetts. He served on various panels of the National
Research Council since 1989, for part of this time as Vice-Chairman of the Committee on Toxicology. He is presently on the Editorial Board of Toxic Substance
Journal and Environmental and Nutritional Interactions. At Inhalation Toxicology Associates he provides consulting services to several Federal Agencies, the
World Health Organization, private industry, and law firms. He has published more
than 225 peer-reviewed manuscripts and book chapters and is the founding Editor
of the Journal of Inhalation Toxicology. He is also a co-editor of the Target
Organ Toxicology Series and New Perspectives: Toxicology and the Environment. He is a Fellow of the Academy of Toxicological Science. Among his many
accolades, he has received lifetime outstanding achievement awards from the Society of Toxicology in the areas of both inhalation toxicology and immunotoxicology.
Louis D. Homer, M.D., Ph.D., was the Medical Director of Clinical Investigation
and Biomedical Research at Legacy Research, Holladay Park Medical Center.
He acquired a Ph.D. in physiology and a M.D. from the Medical College of Virginia. He has served as Assistant and Associate Professor at Emory University
concentrating on physiological processes and mathematical models. Afterward he
served as Associate Professor at Brown University and then moved on to become
a Research Medical Officer at the Naval Medical Research Institute concentrating on biometrics, physiology, environmental medicine, metabolic research, and kidney transplant histocompatibility. He served as a consultant to scientists regularly
on topics ranging from physiology, mathematics, statistics, and computer applications. He also has reviewed proposals and has served on site visit teams for NIAID,
NHLI, NSF, and the Naval Medical Research and Development Command. He
has reviewed prospective articles for the Journal of Theoretical Biology, Microvascular Research, American Journal of Physiology and the Journal of
Applied Physiology. His interest in using mathematical models of physiology in
his research has led him to become familiar with a number of computer languages,
numerical algorithms, iterative least-squares estimation, and iterative maximum likelihood estimation.
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Evaluation of Cigarette Ingredients: Feasibility
Rudolph Jaeger, Ph.D., D.A.B.T., B.C.F.M., is Research Professor of Environmental Medicine at the New York University School of Medicine. In addition to his
academic teaching and research activities, he is Principal Scientist at Environmental Medicine, Inc., a consulting firm specializing in consumer product evaluation,
environmental health risk assessment and industrial toxicology. Dr. Jaeger also
serves CH Technologies (USA) Inc. as President and Chief Scientific Officer.
CH Technologies, a scientific instrument manufacturing company, specializes in
low volume inhalation exposure systems for pharmaceutical and infectious disease
research. CH Technologies and affiliates manufacture specialized cigarette smoking machines. Dr. Jaeger earned status as a Diplomate of the American Board of
Toxicology in 1980. The U.S. Environmental Protection Agency accredited him as
an Asbestos Inspector, and he is a Registered Environmental Assessor in the State
of California. He is a Certified Lead Inspector and Risk Assessor in the State of
New Jersey. He is a Diplomate of the American Board of Forensic Medicine and
a Board Certified Forensic Examiner. Dr. Jaeger served on expert panels of the
Toxicology Information Program Committee of the Board of Environmental Toxicology of the National Research Council. His research interests include inhalation
toxicology, plastics and their monomers, combustion products, pulmonary pathophysiology, liver toxicity and pathophysiology, and the effects of lead and heavy
metals on the developing nervous system.
Robert Orth, Ph.D. is a physical chemist at Apis Discoveries, L.L.C. He is also a
consultant to Monsanto Company and adjunct Associate Professor of Physical
Chemistry at the University of Missouri, where he teaches undergraduate courses
in physical chemistry, instrumental analysis and general chemistry. After obtaining
his Ph.D. from Case Western Reserve University he completed a post doctoral
fellowship at the University of Utah researching mass spectrometry and developed
High Pressure Liquid Chromatography/Mass Spectrometry interface using ultrasound. He conducted research in secondary ion mass spectrometry and taught at
both the University of Utah and Montana State University. He held positions of
increasing responsibility, including Research Fellow, during a 16-year career with
the Monsanto Company. He worked on complex problems in environmental chemistry and remediation and in food and agricultural science. His current work at
Apis Discoveries, L.L.C. includes setting up business units for ultratrace analysis,
consulting for submission to the U.S. Food and Drug Administration of direct and
indirect food additives, analysis and remediation of organic pollutants. He has
neariy 100 publications and presentations in analytical and physical chemistry, and
he is the author of many internal Monsanto publications and presentations. He
currently holds two patents.
Resha M. Putzrath, Ph.D., D.A.B.T., was a principal at Georgetown Risk Group
during the preparation of this report. After earning an M.S. and a Ph.D. in biophysics from the School of Medicine and Dentistry at the University of Rochester, she
was a Research Fellow in Physiology at Harvard Medical School and then a
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Appendices
277
Fellow at the Interdisciplinary Programs in Health at Harvard School of Public
Health. Dr. Putzrath previously worked for the U.S. Environmental Protection
Agency; National Academy of Sciences; Environ Corporation; Organization Resources Counselors, Inc.; and Step 5 Corporation. She currently holds two teaching appointments at Johns Hopkins University, in the Whiting School of Engineering and Applied Science and in the Department of Environmental Heath Sciences
at the School of Hygiene and Public Health. She develops methods for improving
the accuracy of toxicological evaluations and risk assessments, focusing on models
for combining data, primarily for complex mixtures of chemicals including dioxins,
pesticides, and particulate matter. Her analyses have included: proposing alternative procedures that reduce uncertainty within regulatory assumptions; delineating
the limitations of toxicity equivalency factors; and evaluating numerous hazardous
waste sites, occupational exposures, and consumer products. Near the conclusion
of this report, Dr. Putzrath, went to work for the Risk Assessment Forum at the
U.S. Environmental Protection Agency, and as a full-time employee, she resigned
from the Added Ingredients Review Ad Hoc Export Panel.
Emanuel Rubin M.D. is the Attending Physician-in-Chief of Pathology and Chairman of the Department of Pathology, Anatomy and Cell Biology at Thomas Jefferson
University Hospital and Medical College, respectively. He obtained a medical degree from Harvard Medical School. After completing a residency at the Children’s
Hospital of Philadelphia, he continued as a Dazian Research Fellow in Pathology
and as Advanced Clinical Fellow of the American Cancer Society, both at Mount
Sinai Hospital. After the fellowship Dr. Rubin spent the next fourteen years at
Mount Sinai Hospital’s Pathology Service with increasing responsibility, culminating as the Pathologist-in-Chief. Dr. Rubin then became the Director of Laboratories at the Hahnemann University Hospital. His many academic appointments
include the Irene Heinz and John LaPorte Given Professor and Chairman of the
Department at Mount Sinai School of Medicine, Professor and Chairman of the
Department of Pathology and Laboratory Medicine at the Hahnemann University
School of Medicine, Adjunct Professor of Biochemistry and Biophysics at the University of Pennsylvania School of Medicine, and several appointments at Jefferson
Medical College, culminating in his current position. Among honors received, the
University of Barcelona named him as Doctor Honoris Causa. He was also given
the F.K. Mostofi Distinguished Service Award from U.S.-Canadian Academy of
Pathology and the NIH MERIT Award, which lasts until 2006. He has held many
editorial positions and has served as a consultant to many organizations. He has
published over 300 articles, including 13 textbooks and one CD-ROM.
James L. Schardein, M.S., F.A.T.S., consults in reproductive and developmental
toxicology. Early in his career, he established one of the first pharmaceutical industry laboratories that investigated the effect of drugs on the developing embryonic
animal model. After 23 years of experience in the pharmaceutical sector, Mr.
Schardein joined a contract laboratory, International Research and Development
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278
Evaluation of Cigarette Ingredients: Feasibility
Corporation, as Director of the Reproductive and Development Toxicology group,
where he later was named Division Vice President. In 1992, he additionally acquired responsibilities as Associate Director of Research for managing Study Directors and aiding the direction of all research programs at the laboratory. In 1995,
he joined WIL Research Laboratories as Director of Research and Senior Vice
President, from which he retired March 3, 2000. He has served as consultant to
many organizations, including the National Institute of Environmental Health Sciences, the U.S. Environmental Protection Agency, Interagency Regulatory Liaison
Group, and the International Life Sciences Institute. He is the author of two books,
Drugs as Teratogens (1976) and Chemically Induced Birth Defects (1985), the
latter now in its 3rd (2000) edition.
Thomas Joseph Slaga, Ph.D. is the president and CEO of the Center for Cancer
Causation and Prevention at the AMC Cancer Research Center. He completed
his doctorate in physiology/biophysics at the University of Arkansas Medical Center and a postdoctoral fellowship at the McArdle Laboratory for Cancer Research
at the University of Wisconsin. He held positions of increasing responsibility as an
Assistant Member of the Fred Hutchinson Cancer Research Center, Staff Member at the East Tennessee Cancer Research Center, and Senior Staff Member of
the Skin Carcinogenesis and Tumor Promotion and Biology Division at the Oak
Ridge National Laboratory (part of the Department of Energy). He simultaneously
held faculty positions at the University of Washington Medical School, the Oak
Ridge Graduate School of Biomedical Sciences, Texas A&M University, and the
University of Texas. Several of Dr. Slaga’s former students now work for cigarette manufactures. Currently, his joint/adjunct positions include Interim Deputy
Director and Member of the Comprehensive Cancer Research Center and Member of the Department of Biochemistry and Molecular Biology at the University of
Colorado Health Sciences Center. He has served on many scientific advisory
committees and editorial boards. He also peer reviews submissions to several
scientific journals. His research interests include mechanisms of chemical carcinogenesis, tumor promotion, and mechanisms of action of dermal antitumor agents.
Dr. Slaga has published more than 500 papers.
Added Ingredients Review Meeting Speakers:
Christopher R.E. Coggins, Ph.D., D.A.B.T., is the Senior Vice President of Science and Technology for Lorillard Tobacco Company. Dr. Coggins has been a
board-certified toxicologist since 1986 and has previously been employed by the
following companies and organizations: R.J. Reynolds, Research and Development;
Battelle-Geneva Research Center; and Animal Diseases Research Association,
University of Edinburgh. Dr. Coggins is a member of several professional societies, such as the American Board of Toxicology, the Society of Toxicology, and the
Society of Toxicologic Pathologists. He continues to serve as a manuscript reviewer for several scientific journals and is currently a member of the editorial
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Appendices
279
board of the journal Inhalation Toxicology, and is on the Board of Directors of the
Tobacco Industry Testing Laboratory. Dr. Coggins has published over 40 articles
in the peer-reviewed scientific literature.
Richard N. Dalby, Ph.D. is an Associate Professor and Vice Chair in the Department of Pharmaceutical Sciences at the University of Maryland School of Pharmacy. He was previously a Research Assistant Professor in the Department of
Pharmacy and Pharmaceutics at Medical College of Virginia / VCU (1989-1992).
Dr. Dalby holds a bachelor of pharmacy degree (1983) from Nottingham University in England, and a Ph.D. in pharmaceutical sciences from the University of
Kentucky (1988). Richard’s aerosol research, which encompasses novel lung and
nasal formulation development, devices design and product testing, is founded on
both his Ph.D. work on sustained release metered dose inhalers, and industrial
experience as an Inhalation Formulation Scientist with Fisons Pharmaceuticals (19881989). He has more than 30 published papers and 70 abstracts related to aerosol
technology, has authored several book chapters, and has spoken at many national
and international meetings. He is a co-inventor on three patents concerned with
novel MDI formulations, a reviewer for several international journals, and acts as
an industrial and FDA consultant. He is a member of the Royal Pharmaceutical
Society of Great Britain and the American Association of Pharmaceutical Scientists. He is the director of an annual Inhalation Aerosol Technology Workshop, and
co-organizer / publication coordinator of a major international symposium - Respiratory Drug Delivery, now in its 16th year.
Mark W. Frampton, M.D., is an Associate Professor of Medicine and Environmental Medicine in addition to being the Director of the Pulmonary Research Programs
at the University of Rochester Medical Center. Dr. Frampton expertise and research interests include the health effects of air pollution. His goals are to determine indicators of individual susceptibility to the adverse effects of pollutants and to
develop markers of exposure. He is a member of the American College of Physicians, American Lung Associations of New York State, and the American Thoracic
Society.
Wolf-Dieter Heller, Ph.D., received his doctorate in mathematics at the Faculty for
Economic Sciences of the University of Karlsruhe in Germany. Along with his
teaching responsibilities at the Faculty for Economic Sciences, he is the Editor of
Bieträge zur Tabkforschung International/Contributions to Tobacco Research
and is the Managing Director of the Research Association Smoking and Health in
Berlin, Germany.
Lester B. Lave, Ph.D., is University Professor and Higgins Professor of Economics at Carnegie Mellon University, with appointments in the Business School, Engineering School, and the Public Policy School. He has a B.A. from Reed College
and a Ph.D from Harvard University. His research has focused on health, safety,
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Evaluation of Cigarette Ingredients: Feasibility
and environmental issues, from the effect of air pollution on health to estimating the
benefits and costs of automobile safety standards, risk analysis of carcinogenic
chemicals, testing the carcinogenicity of chemicals, valuing natural resources and
global climate change. Dr. Lave has served as a consultant to a large number of
federal and state agencies and companies. He was elected to the Institute of Medicine of the National Academy of Sciences, is a past president of the Society for
Risk Analysis, and has served on many committees of the National Academy of
Sciences, AAAS, American Medical Association, and Office of Technology Assessment. Dr. Lave is the director of the CMU university-wide Green Design
Initiative. This program is focused on using pollution prevention and sustainable
development to boost economic development. He is engaged in a project on studying the toxicity testing of high volume chemicals and is a member of the Alliance for
Chemical Awareness Advisory Committee.
Peter N. Lee, M.A., C.Stat., received his degrees at the Wimbledon College and
Exeter College, at Oxford. Mr. Lee has served as a Statistician and Research
Coordinator at the Tobacco Research Council (now Tobacco Manufacturers Association) in London, England. Presently he is an Independent Consultant in Statistics and Adviser in Epidemiology and Toxicology to a number of tobacco, pharmaceutical and chemical companies. Mr. Lee has published over 170 papers and
three books. A number of papers describe statistical methods of analysis of longterm animal studies and their application to numerous experiments. Others concern
epidemiological studies and clinical trials, with papers relating to lung cancer, chronic
bronchitis, heart disease, stroke, Alzheimer’s disease, Parkinson’s disease, inflammatory bowel diseases, urinary incontinence and other diseases. Agents investigated include aluminium, PCBs, asbestos, vitamin A and other aspects of diet, personality, drugs, and even pet birds. Smoking and environmental tobacco smoke
(ETS) exposure are particular interests, with papers relating to lung cancer and a
variety of other smoking-related diseases. Two of his books concern ETS, the
second being a comprehensive review of the epidemiological evidence relating to
mortality in adults. A third is a reference book on international smoking statistics.
Luqi Pei, Ph.D. is Pharmacologist and Toxicologist at the U.S. Food and Drug
Administration. He specializes in the nonclinical safety evaluation of inhaled drug
products indicated for respiratory diseases. Dr. Pei received his degree in veterinary medicine from Gansu University of Agriculture, China, degree of Master of
Science from Colorado State University and degree of Doctor of Philosophy from
Texas A&M University College of Medicine. Dr. Pei was a faculty member at the
Gansu Agriculture University for four years and a visiting scientist at the Colorado
State University for a year prior to his graduate training. He completed his
postdoctoral training at the Lovelace Inhalation Toxicology Institute studying toxicity of vapors. Dr. Pei spent about a year in a pharmaceutical company developing
inhaled drug products.
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281
Stephen I. Rennard, M.D., is a physician and a professor of Pulmonary and Critical
Care Medicine at the University of Nebraska Medical Center. He graduated with
honors from Baylor College of Medicine and completed his residency in internal
medicine at Washington University’s Barnes Hospital. He then completed a research associateship in developmental biology and anomaly at the National Lung
and Blood Institute, National Institutes of Health. Dr. Rennard became the Chief
of the Pulmonary and Critical Care Medicine Section at University of Nebraska
Medical Center. His research interests have focused on the inflammatory and
repair processes of lung tissues. This has included investigations in airway epithelial cell biology, mechanisms of epithelial repair, and peribronchiolar airways disease. He has more than 200 peer reviewed publications and has served on the
committees of many organizations including the Pulmonary Subspecialty Section of
the American Board of Internal Medicine, the Graduate Education Committee, the
Council of Governors for the American College of Chest Physicians, the Society
for Research on Nicotine and Tobacco, the Research Advocacy Committee, the
Long-Term Planning committee for the American Thoracic Society and the
Bronchoalveolar Task Group of the European Respiratory Society.
Thomas B. Starr, Ph.D., is the Principal of TBS Associates in Raleigh, North Carolina. Dr. Starr is also an Adjunct Associate Professor, Department of Environmental Sciences and Engineering at the University of North Carolina at Chapel Hill
School of Public Health. He also serves on the Secretary’s Scientific Advisory
Board on Toxic Air Pollutants for the North Carolina Department of Environment,
Health, and Natural Resources. In addition, he serves on the Environmental Medicine Residency Advisory Committee of the Duke University Division of Occupational and Environmental Medicine Training Program. He has had over 30 years of
professional experience in quantitative assessment of health and environmental
risks from exposure to toxic substances, exposure assessment, experimental design and data analysis, comparative risk analysis, toxic torts, and regulatory policy.
He is a member of the Society of Toxicology, American Statistical Association, and
the Society for Risk Analysis. He has more than 80 publications on human and
environmental health effects of exposure to pollutants and other toxic substances,
environmental regulation, and mechanisms of toxicity.
C. Joseph Sun, Ph.D., Capt. USPHS received his degree in pharmacology from
the University of Mississippi Medical Center. Dr. Sun is Pharmacologist at the
U.S. Food and Drug Administration since 1978, where he has served in various
divisions within the Center for Drug Evaluation and Research (CDER). Currently,
Dr. Sun is the Supervisory Pharmacologist/Team Leader in the Division of Pulmonary Drug Products.
Ashok Teredesai, D.V.M., is the Head of the Department of Pathology at the
Institut für Biologische Forschung (INBIFO) in Cologne, Germany. Before his
current position he had been a staff scientist at the Department of Veterinary Pa-
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Evaluation of Cigarette Ingredients: Feasibility
thology in Giessen, Germany. Dr. Teredesai holds a National Specialist Title as
Fachtierarzt für Pathologie. He is a member of many professional societies including the American, British, and German Societies of Toxicologic Pathology, German
Veterinary Medical Association, and the European Society of Veterinary Pathology. He has been published 16 times on subjects including, but not limited to the
histopathology of the rodent respiratory tract.
Piter M. Terpstra, D.V.M., M.B.A., is the general manager of CRC (Contract
Research Center, a Philip Morris Research Laboratory) in Brussels, Belgium. He
received his DVM from the State University of Ghent, Belgium and his MBA from
the University of Brussels. He wrote a thesis on the use of novel anesthetics and
experimental surgery in the horse. After shortly serving for the European Commission as a trainee, he was employed at Eli Lilly in a research and technical support
group for several years. He joined CRC in 1992 and became general manager in
1995.
Patrick Vanscheeuwijck, Ph.D., is the manager of the Bioresearch Department at
CRC (Contract Research Center, a Philip Morris Research Laboratory) in Brussels, Belgium. He received his MS in plant biochemistry and physiology and Ph.D.
in biochemical pharmacology from the State University of Ghent, Belgium. Postdoctoral training was performed at the University of Arizona, Tucson, Arizona,
Department of Pharmacology and Toxicology in molecular pharmacology and at
the Catholic University of Leuven, Belgium, in molecular biology. He has published
more than 20 peer-reviewed manuscripts as author or co-author.
Hanspeter Witschi, M.D., D.A.B.T., F.A.T.S. is a Professor Emeritus of Veterinary Medicine: Molecular Biosciences at the University of California, Davis where
his core research has centered on respiratory toxicology. Dr. Witschi has been a
board certified toxicologist since 1980, and received his medical training at the
University of Berne and University of Geneva in Switzerland. Dr. Witschi has
served as a research associate at the Toxicology Research Unit, British MRC in
Carshalton, England, the Department of Environmental Health, of the University of
Cincinnati, Ohio and at the Department of Pathology, University of Pittsburgh, Pennsylvania. Other professorships include Associate Professor, Department of Pharmacology, and Faculty of Medicine University of Montreal, Canada. He has also
conducted research at the Oak Ridge National Laboratory, Oak Ridge, Tennessee
Dr. Witschi’s principal research interests include the study of cell turnover in the
lungs of animals exposed to common air pollutants such as ozone or nitrogen dioxide, and defining the effects of low level exposure and the study events leading to
adaptation. Additionally his work has focused on the effects of environmental tobacco smoke on intrauterine growth and lung tumor development.
For internal use of Philip Morris personnel only.
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283
LIFE SCIENCE RESEARCH OFFICE BOARD OF
DIRECTORS
Chair:
William Heird, M.D.
Baylor College of Medicine
Houston, TX
Vice Chair: Salvatore (Sam) J. Enna, Ph.D.
University of Kansas Medical Center
Kansas City, KS
Treasurer: Arnold Kahn, Ph.D.
University of California at San Francisco
San Francisco, CA
Past Chair: Donald Beitz, Ph.D.
Iowa State University
Ames, IA
Directors: Gilbert Leveille, Ph.D.
Cargill Health & Food Technologies
Wayzata, MN
Robert Newburgh, Ph.D.
The Protein Society
Bethesda, MD
Terry Quill, M.S., J.D.
International Society for Regulatory Toxicology and Pharmacology
Washington, DC
Robert Russell, M.D.
Tufts University
Boston, MA
Jacob J. Steinberg, Ph.D.
Albert Einstein College of Medicine & Montefiore Medical Center
(ASIP)
Bronx, NY
Taffy J. Williams, Ph.D.
Photogen Technologies, Inc.
New Hope, PA
Secretary: Michael C. Falk, Ph.D.
Life Sciences Research Office
Bethesda, MD
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Evaluation of Cigarette Ingredients: Feasibility
LSRO STAFF:
Amin Akhlaghi was an Associate Staff Scientist at the Life Sciences Research
Office. He received a B.S. degree in biology from the University of Kentucky and
is currently studying at Georgetown University School of Medicine. During his
second year of medical school he was an Adjunct Graduate Fellow at the Center
for Food and Nutrition Policy formerly at Georgetown University.
Pippa Antonio, B.S. worked at the Life Sciences Research Office in Bethesda,
MD, as an Associate Staff Scientist. Ms. Antonio graduated from the University of
Stirling, Scotland with distinction. Her honors thesis was on The Effects of Increased Weight Bearing on the Ultra Structure of Collagen Fibers. She was previously employed as a Research Writer for The Myelin Project, an international organization, whose mission is to accelerate research on myelin repair. She recently
joined her husband in Virginia Beach, VA.
Daniel M. Byrd III, Ph.D., D.A.B.T. is the Deputy Director of the Life Sciences
Research Office and the recent coauthor of the textbook Introduction to Risk
Analysis: A Systematic Approach to Science-Based Decision Making (2000).
He received both B.A. and Ph.D. degrees from Yale University. He first received
certification from the American Board of Toxicology in 1982. Previously, Dr. Byrd
taught pharmacology and conducted independent research into the mechanisms
and dosimetry of chemotherapeutic drugs at Roswell Park Memorial Institute and
at the University of Oklahoma. At the U.S. Environmental Protection Agency he
subsequently held positions in the Office of Chemical Control, the Office of Pesticide Programs, the Carcinogen Assessment Group, and the Science Advisory Board
(SAB) for which he was awarded with the Silver Medal for Management and
Leadership. He also managed committees for three trade associations and was
the president of Consultants in Toxicology, Risk Assessment and Product Safety
(CTRAPS) a scientific support firm that helps clients acquire, interpret, and use
biomedical information. He is the author of more than 100 regulatory documents
and 40 scientific articles.
Bonita R. Condon, B.S. worked at the Life Sciences Research Office as the Scientific Literature Specialist/Librarian through March 2002. She received her B.S.
in Information Resources Management from the University of Maryland. Prior to
joining LSRO, Ms. Condon was Chief of the Information Resources Management
Branch, at the Health Resources and Services Institute (HRSA), where she conducted studies of existing procedures and oversaw a team of specialists who provided solutions and services. Prior to HRSA, Ms. Condon served as a Senior
Auditor at NIH and conducted studies of internal processes and procedures. In the
middle of the Phase One project she left LSRO to join Aspen Associates as the
Project Manager for the National Center for Complementary and Alternative Medicine Clearinghouse, NIH, where she managed teams of science writers, editors,
For internal use of Philip Morris personnel only.
Appendices
285
health information specialists, librarians, and technical specialists. Presently, Ms.
Condon is a Project Manager for BETAH Associates, Inc. BETAH provides professional and technical services to government and private sector professional services markets.
Dr. Michael Falk, Ph.D. is the Director of the Life Sciences Research Office. He
received his Ph.D. in biochemistry from Cornell University and completed
postdoctoral training at Harvard Medical School. He was employed in various
capacities at the Naval Medical Research Institute, Bethesda, MD supervising as
many as 80 senior level scientists. As Principal Investigator he was a key member
of the Scientific Advisory Board and the Acting Director for the Institute. He was
also the Director of the Wound Repair Program and pioneered a new position as
the Director of Biochemistry and Cell Biology. Also, as the Director, he rescued
the Septic Shock Research Program by cutting inefficiencies and increasing productivity in terms of grant funding and publication production. He managed peer
reviews and subject review panels in infectious diseases, environmental sciences,
military medicine, and other health-related fields. He reviewed the research proposals for National Science Foundation, Medical Research Council of Canada, and
Office of Naval Research. As the Director of the Life Sciences Research Office
(LSRO), Dr. Falk manages the evaluation of biomedical information and scientific
opinion for regulatory and policy makers in both the public and private sectors. He
has written white papers on infant nutrition, food labeling, food safety, and military
dental research and has organized two international conferences. He concurrently
works at MCF Science Consultants providing analysis and consultation on emerging
technologies. Dr. Falk has published over 60 research articles, abstracts, technical
reports, and presentations.
Robin S. Feldman, B.S., M.B.A. is the Literature Specialist in the Life Sciences
Research Office. She is a seasoned information specialist with experience in the
electronic acquisition, analysis, and management of scientific, business, and regulatory information. Ms. Feldman obtained her B.S. from the George Washington
University in Washington, D.C. with a major in zoology and her M.B.A. degree
from the University of Maryland at College Park with a concentration in science
and technology. Previously, she worked as a Biomedical Research Assistant at
Consultants in Toxicology, Risk Assessment and Product Safety, where she obtained and researched scientific literature for private and governmental clients. At
the National Alliance for the Mentally Ill, she designed and implemented a document management and retrieval system for the Biological Psychiatry Branch of the
National Institute of Mental Health and served as Managing Editor of Bipolar
Network News, a newsletter for the Stanley Foundation Bipolar Network. At
Howard Hughes Medical Institute, she oversaw the implementation of the HHMI
Predoctoral Fellowship in Biological Sciences program. While serving as Science
Information Specialist at the Distilled Spirits Council of the United States, she managed the installation of a local area network and participated in the development
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286
Evaluation of Cigarette Ingredients: Feasibility
and maintenance of an electronic research database for the beverage alcohol industry. As a Report Coordinator at Microbiological Associates, Inc. she conducted
statistical analyses and prepared technical reports about toxicology studies using
animal models. She served as Data Management Administrator for the National
Toxicology Program’s sponsored studies. Currently, Ms. Feldman maintains LSRO’s
library, responds to requests for reports, and assists LSRO’s scientists in discovering, obtaining, compiling, and documenting the scientific literature required to prepare reports for sponsors.
Catherine J. Klein, Ph.D., R.D., C.N.S.D., is a Staff Scientist at the Life Sciences
Research Office. She graduated magna cum laude from the Department of Human Nutrition and Food Science at the University of Maryland at College Park
where she also obtained her M.S. and Ph.D. in the Graduate Program in Nutrition.
She completed an internship in the Pre-Professional Practice Program in Dietetics
at the University of Maryland Medical System (UMMS), and most recently the
V.A. Kleinfeld Summer Internship Program at The Food and Drug Law Institute.
At the UMMS she developed system-wide guidelines for nutrition assessment,
documentation, and continuity of care. As Clinical Coordinator of Research in the
Division of Critical Care Medicine at the R. Adams Cowley Shock Trauma Center,
she developed, initiated, and administered research projects focused on nutrition
issues in critical care. She established a multidisciplinary nutrition task force, which
resulted in improvements in clinical practice standards. As Program Coordinator
of the Department of Nutrition and Food Science she produced online modules for
continuing education in nutrition for health care professionals. She is the primary
author on 9 peer-reviewed publications including a book chapter and has lectured
or presented at over 25 professional meetings. Dr. Klein received the Pelczar
Award for Excellence in Graduate Study from the University of Maryland Graduate School and Sigma Xi and the Dr. E.V. McCollum Award from the Maryland
Dietetic Association. Her professional contributions include serving on the Advisory Board of the University of Maryland Dietetics Program and as editor for the
Maryland Dietetics Association.
Kara D. Lewis, Ph.D. joined the Life Sciences Research Office in Bethesda, MD,
as a Staff Scientist. Dr. Lewis completed postdoctoral research at Yale University.
She obtained her Ph.D. in biology with a concentration in neuroscience from Clark
University and graduated summa cum laude with a B.S. degree in biology from
Spelman College. Dr. Lewis has conducted research on taste and smell of the
fruitfly, Drosophila melanogaster, and on the molecular mechanisms of sweet
taste transduction in the blowfly, Phormia regina. She has collegiate teaching experience and three peer-reviewed publications. She is a member of the Association
for Chemoreception Sciences.
Negin P-M. Royaee, B.S. worked as an Associate Staff Scientist during the preparation of the Phase One report. She was previously employed at Biophysical Soci-
For internal use of Philip Morris personnel only.
Appendices
287
ety, FASEB, where she assisted in the production of the Biophysical Journal.
Ms. Royaee obtained her bachelor’s degree from Lake Forest College in Chicago,
IL with a major in biology and minor in art history. Ms. Royace has volunteered for
sick and under-privileged children and was a Weekend Resident Manager at The
Children’s Inn at the National Institutes of Health. Currently Ms. Royaee resides
in Boston, MA.
Paula M. Nixon, Ph.D. joined the Life Sciences Research Office in Bethesda,
MD, as a Staff Scientist. Dr. Nixon completed her postdoctoral research at The
Babraham Institute in Cambridge. She obtained her Ph.D. in molecular biology
from Imperial College London as part of Cancer Research UK. She graduated
with an honors degree in molecular biology from the University of Manchester,
UK. Dr. Nixon has conducted research on the MEK5/ERK5 MAP kinase pathway and the role of the AP-2 transcription factors in the control of gene expression
in breast cancer. In addition she was involved in research to identify the Currarino
syndrome gene and the development of mitochondrial DNA profiling techniques
for forensic science. She has three peer-reviewed publications and is a member of
the Biochemistry Society.
Kristine K. Sasala, B.S. recently joined the Life Sciences Research Office in
Bethesda, MD as an Associate Staff Scientist. Ms. Sasala obtained her bachelor’s
degree in biology from the University of Cincinnati, OH with a focus on cell biology. Prior to joining the LSRO, Ms. Sasala was previously employed at Nerac, Inc.
as an Information Scientist. She has been involved in researching the molecular
biology of osetoporosis, anti-Brucella vaccine development, and pharmacology of
xenobiotics, in addition to graduate work in protistology. Ms. Sasala has two peerreviewed publications and several conference publications. At the LSRO, she assists with the Added Ingredients Review and various other projects.
James Cecil Smith Jr., Ph.D., a Senior Scientific Consultant at Life Sciences Research Office obtained his doctorate in nutritional sciences/biochemistry at the University of Maryland and post-doctorate in the Department of Biological Chemistry,
UCLA. He served two years as Health Service Officer at the National Institutes
of Health. Dr Smith completed 36 years in federal research laboratories with increasing responsibilities in the Veterans Administration Medical system and the
U.S. Department of Agriculture (USDA). Best known for contributions to trace
element nutrition, he is also an authority in the area of vitamin and mineral interactions. Original research he directed identified a link between zinc and vitamin A.
His other research accomplishments include studies that revealed an interaction
between copper and dietary carbohydrates. He directed two large human investigations, funded in part by the National Cancer Institute that established the USDA
Laboratory and Research Center at Beltsville, MD as original leaders in research
to elucidate the role of dietary carotenoids. Dr. Smith has publishd more than 375
articles. He served as an editorial board member, assistant editor, and ad hoc
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Evaluation of Cigarette Ingredients: Feasibility
reviewer for several journals and is a member of several professional societies. His
recognitions include the Klaus Schwarz Award for Excellence and Leadership in
Trace Element Research sponsored by the International Association, Bioinorganic
Scientists and was recently named a Fellow of the American Society for Nutritional Sciences.
LSRO STAFF WRITERS:
Ms. Tina Adler, M.S., obtained her M.S. degree from the School of Journalism at
Columbia University, concentrating on health and science issues. She has worked
as a writer for many organizations including the Washington Post, the U.S. National Institutes of Health, Burlington Free Press, the American Psychological Association, and Science News, covering a wide array of scientific subjects.
Ms. Wendy Meltzer, M.P.H., earned her M.P.H. degree from the School of Public
Health and Health Services at George Washington University. She has worked at
Georgetown University Medical Center’s Women’s Interagency HIV Study and at
the Center for Science in the Public Interest. She also has been the Managing
Editor and Executive Producer at FoodFit.com, a nutrition and healthy living web
site. She is a member of the Delta Omega Public Health Honor Society.
Dr. Susan A. Stern PhD., earned a doctorate in biochemistry at the East Tennessee
State University College of Medicine and completed a postdoctoral fellowship in
clinical immunology at the University of Minnesota School of Medicine. She currently is employed as senior manager at Constella Health Sciences. At the time of
her work on this project, she was the Principal at Stern Consulting where she
provided medical and scientific writing, advice on strategic planning, and various
analysis and consulting services to professional service firms and companies. An
immunologist with nearly 20 years of experience in biotechnology, consulting, and
technology management she has held management positions at several biotechnology companies in the Washington metropolitan area. Dr. Stern is the author of
seven original publications and is a member of several professional organizations
including the American Association for the Advancement of Science, Drug Information Association, and the American Writers Association.
For internal use of Philip Morris personnel only.
APPENDIX B
GLOSSARY
Absorption
The transfer of smoke constituents from the lung air space and/or surface into
lung tissue (Byrd & Cothern, 2000).
Accuracy
The ability of a measurement or method to determine the true value of the
quantity being quantified.
Active transport
The process of molecules or ions crossing a membrane against its concentration gradient. This requires energy, saturation, and could be affected by competitive inhibition.
Added Ingredient
A substance added to an already defined mixture.
Additive
A substance added in small amounts to something else to improve, strengthen
or otherwise alter it.
Aerodynamic diameter
For ideal smoke particles (spheres with densities of 1 gram/cubic cm) diameter
and aerodynamic diameter are equivalent. If the particle’s shape or density
differs from ideal, then the diameter and aerodynamic diameter will
differ. Particles smaller than 0.5 µm diameter behave like gases aerodynamically, and diffusion dominates their motion. So, a physical diameter is used.
Aerosol
A two-phase system consisting of a collection of solid and/or liquid particles
(particulate matter) and the gas in which they are suspended (Hinds, 1999).
After cut
Alcoholic solution containing aromatic and/or flavoring agents (e.g., menthol)
and stabilizing agent (e.g., glycol) sprayed onto tobacco after it is cut into shred.
Air-cured
The drying (curing) process of Maryland and Burley tobaccos in open-air barns
or scaffolding, using ambient air conditions without external heat or humidity.
The rate of curing depends on prevailing temperature, humidity, airflow and
sunlight and may extend over a period of 8 weeks or longer.
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Alveolus
Pl. Alveoli. Vascularized air sac extending from the respiratory bronchioles, at
the interface between the airway and the pulmonary tissue where gas is
exchanged.
Animal model
Animals used for in vivo studies as surrogates for humans. Rodents are most
frequently used for studies involving cigarette smoke.
Ash
The powdery residue remaining after combustion of tobacco.
Bias
A statistical sampling or testing error systematically favoring one outcome over
others.
Biological activity
Processes, usually biochemical, that are required to carry out metabolic functions associated with supporting life.
Biologically effective dose
The amount of tobacco constituent or metabolite that binds to or alters a macromolecule; estimates of BED might be performed in surrogate tissues. (Institute of Medicine, 2001).
Biomarker
A biological response variable that is indicative of exposure, and/or effect (i.e.,
disease initiation; disease progression; remission; death).
Blended cigarette
Cigarettes made from tobacco that has been mixed in proportions from other
tobaccos.
Bright tobacco
Flue-cured tobacco.
Bronchiole
Extension of the bronchus; terminal bronchioles are ciliated, respiratory bronchioles extending from terminal bronchioles are not ciliated and contain alveoli.
Bronchus
One of two main branches (bronchi) of airway from the trachea to the lungs.
Burley tobacco
The main light air-cured tobacco that is grown in Kentucky and surrounding
states, setting the standard for that grown in other parts of the world.
Cambridge filter pad
A glass fiber filter stabilized by an organic binder, originally made by the Cambridge Filter Corporation, Syracuse, New York. It is used in the FTC standard
method to separate the vapor and particulate phases of cigarette smoke.
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Casing
Solution applied to tobacco leaf by dipping or spraying to retain moisture in the
tobacco and improve sensory characteristics of the smoke. Casing solutions
may include sugar, glycerin or glycol, licorice and cocoa.
Char
The remaining carbonized residue following combustion of cigarette tobacco.
Char reacts with oxygen in the air producing carbon monoxide, carbon dioxide
and water leaving ash.
Chronic obstructive pulmonary disease (COPD)
COPD “is a disease characterized by a progressive airflow limitation caused
by an abnormal inflammatory reaction to chronic inhalation of particles.”
(Pauwels, 2000)
Cigarette smoke condensate
See ‘Tobacco smoke condensate’
Compensation
Behavioral responses of smokers after changing to lower yielding cigarettes to
preserve the nicotine intake prior to switching; these responses include larger,
more rapid or frequent puffs, deeper inhalation, blocking of ventilation holes by
the lips/fingers or an increase in cigarettes smoked.
Complex mixture
A heterogeneous group of many chemical substances found together, for example, as in tobacco or in cigarette smoke.
Concordance
Agreement between two sets of data; a confirmation of results.
Condensate
See ‘Tobacco smoke condensate’
Confounder
A factor that can compromise the validity of experimental data and may be
difficult to identify.
Correlation
The simultaneous numerical increase or decrease of two random variables.
Cotinine
A metabolite of nicotine found in the plasma, saliva and urine of smokers and
used as a biomarker of exposure to cigarette smoke.
Deposition
Process resulting in a fraction of the inspired smoke that is not expired (Brain
& Valberg, 1979).
Dilution
See ‘Ventilation’
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Evaluation of Cigarette Ingredients: Feasibility
Direct additive
A chemical substance added to a cigarette during manufacturing.
Distillation
A process that separates a chemical mixture into component fractions by the
application and removal of heat.
DNA adducts
Covalent chemical products formed when an electrophile, typically a metabolite of a mutagen or carcinogen, binds to DNA.
Dose
The amount of a substance absorbed by an organism.
Dosimetry
The accurate measurement of doses. (Pickett, 2000)
Dynamics
The effect of a substance on the body, specifically its mechanism of action,
pathway and biological effect.
Endpoint
Measure of outcome (e.g., cancerous tumor; heart attack; death)
Environmental tobacco smoke (ETS)
The cigarette smoke that is present in the ambient air, consisting of exhaled
mainstream smoke plus sidestream smoke. The highest concentrations are in
near proximity of smokers in confined areas with poor ventilation.
Error
A numerical value that deviates from the true value.
Existing additive
A chemical substance added to a cigarette during manufacturing and already in
use, as a chemical substance listed in Appendix D.
Exposure
The potential dose of a substance, available for absorption (Byrd & Cothern,
2000).
Exposure Biomarker
A chemical substance (for example, a tobacco constituent or its metabolite) in
a biological fluid or tissue that measures internal dose (Institute of Medicine,
2001).
External validity
Confirmation of data/test results from other authoritative sources.
Facilitated diffusion
Movement across a membrane down a concentration gradient involving a transporter and subject to saturation and competitive inhibition.
False Negative
A negative result from a test, when the true outcome was positive.
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293
False Positive
A positive result from a test, when the true outcome was negative.
Filter (cigarette)
A device positioned at the end of cigarette, which serves as a mouthpiece, usually composed of cellulose acetate fibers in the U.S. and encased by a wrapper.
Filter wrapper
Paper wrapping the filter.
599 list
In 1994 U.S. manufacturers published a list of 599 substances that might be
added to some cigarettes. (See Appendix D)
Flue-cured
Tobaccos (Bright or Virginia) that are dried (cured) in tightly constructed barns
with artificial heat beginning at 35 °C and ending at about 75 °C over a 5 to 7
day period.
Gas phase
The nonliquid (vapor) phase of cigarette smoke.
Gold standard
A technique, method, substance or measure considered the most accurate,
precise, or accepted by authoritative experts.
Initiation
“Instantaneous event during exposure, leading to manifest mutation that is irreversible at least until the mutated cell or its offspring is eliminated or escapes
further development by differentiation.” (Tornqvist & Ehrenberg, 2001)
Humectant
Substances or compounds added to cigarette tobacco to retain moisture and
plasticity. The principal humectants in cigarette manufacturing are glycerol
and propylene glycol.
Indirect additive
A chemical substance, detectable in a cigarette, and distinct from its tobacco
paper, introduced into cigarettes through treatment during the growing and handling of the tobacco or manufacture and handling of paper.
Ingredient
Substances except for tobacco and paper used in the manufacture or preparation of cigarettes. Also see (European Parliament, 2001).
Internal Validity
Confirmation of test results based on reference standards or materials employed concurrently with the procedure.
Kinetics
The rates of processes, e.g., pharmacokinetics or toxicokinetics. How rapidly
the body processes a substance, specifically the rates of absorption, distribution, metabolism and excretion of chemical substances.
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Light cigarette
A cigarette designed to lower the delivery of tar when smoked under standard
conditions.
Mainstream smoke
Smoke drawn through the butt end of the cigarette into the mouth as a smoker
puffs on a cigarette
Maryland tobacco
Light bodied, mild, air-cured tobacco grown on the sandy soils in southern
Maryland.
Maximum tolerated dose (MTD)
In theory, an MTD is an estimate of the maximum daily dose or exposure
which a species can experience over a lifetime and experience no adverse
effects. In practice, MTDs for animal species usually are estimated from
experimental data about lifespan or inhibition of body weight gain.
Microexplosions
Small components of a cigarette (e.g., sugar, protein) that break off before
they burn during pyrolysis and are transported in the smoke intact.
Molecular epidemiology
The study of biomarkers in an epidemiological context.
Mouth spill
Smoke lost from the mouth before inhalation (Bentrovato et al., 1995).
Multiplicity
Several approaches or tests designed to establish/validate an effect.
New additive
A chemical substance added to a cigarette during manufacturing not previously
used.
N-Nitrosamine
A chemical substance formed by nitrosation of secondary and tertiary amines.
Tobacco-specific nitrosamines (TSNA) are formed by the nitrosation of the
major tobacco alkaloid, nicotine. (Brunnemann et al., 1996)
Nicotine
A cyclic tertiary amine composed of a pyridine and a pyrrolidine ring. It is a
colorless to pale yellow, water soluble, oily, volatile, and hydroscopic liquid derived from plants of the Nicotiana genus.
Nicotinic receptors
A subclass of cholinergenic receptors, which are ligand-gated and control the
influx of calcium ions into the cell.
Noise
Background interference due to internal vibrations or electronics of instruments.
For internal use of Philip Morris personnel only.
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295
Oriental tobaccos
The varieties of tobaccos derived from the main species Nicotiana tabacum;
specific oils and resins from these tobaccos provide distinctive taste and aroma.
Pack-year
A unit measure of smoking exposure. One pack-year represents the consumption of 20 cigarettes per day (one pack) for one year by one person.
Paper
Cigarette paper (wrapper) is composed of inorganic filler such as CaCO3 and
cellulose fibers from wood pulp and/or flax. The filler holds the fibers apart,
creating pores that allow air permeability.
Parenchyma
Cells of an organ, essential to its function, distinct from the membrane that
encloses it and the connective tissue that supports it.
Particulate matter
In smoke, the solid particles and liquid droplets (typically < 1 µm in diameter) in
vapor, altogether forming an aerosol (Hinds, 1999).
Particulate phase
In smoke, the solid and/or liquid phase of an aerosol, which is suspended in gas.
Liquid particles are referred to as droplets (Hinds, 1999).
Passive diffusion
Molecules or ions that cross permeable membranes to equilibrate concentrations on both sides. Movement occurs predominantly down the concentration
gradient.
Peak puff flow
Maximum rate (in mL per second) of mainstream smoke flow from cigarette to
smoker
Pinocytosis
Transport of small molecules or liquids into cells by invagination of the cell wall
and engulfment of the substance.
Plasticizer
A bonding agent, such as triacetin, used to modify and accelerate the reaction
between chemical subunits that compose a plastic.
Plug wrap
Paper wrapping the cigarette filter.
Polycyclic aromatic hydrocarbons (PAHs)
Organic chemical substances containing fused aromatic ring systems, formed
during incomplete combustion. Some PAHs form DNA adducts.
Potential reduced-exposure products (PREPs)
Pharmaceutical substances or products, such as nicotine replacement therapy
or bupropion, and modified tobacco products with the potential to reduce morbidity and mortality.
For internal use of Philip Morris personnel only.
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Evaluation of Cigarette Ingredients: Feasibility
Precision
Consistent reproduction of a measurement.
Pressure drop
The difference between atmospheric pressure and the pressure inside the cigarette during a standard flow rate. A large pressure drop indicates more difficulty (greater suction) in drawing smoke. Pressure drop indicates dilution. A
reduction in the holes-open pressure drop indicates greater ventilation (dilution).
Processing aids
Ingredients that facilitate the manufacturing procedures and enhance the efficiency of production of cigarettes.
Promoter/Promotion
“…promotion is a reversible effect that, according to experiments, requires the
chronic presence of the promoter. The term promoter is an operational definition for a variety of agents that have cell proliferation and clonal expansion as
a common effect.” (Tornqvist & Ehrenberg, 2001)
Puff duplicator
An automated smoking machine that can be programmed with data, such as
puff profile, from a puff recorder.
Puff interval
Length of time (in seconds) between puffs.
Puff profile
A graphic representation of the rate of the flow of mainstream smoke plotted
as a function of time during the puff. The puff profile can be used to calculate
puff volume. (International Organization for Standardization, 2000).
Puff recorder
A device to measures parameters of smoking behavior while a subject smokes,
such as puff flow and puff duration.
Puff volume
The volume (in mL) of smoke inhaled from one puff.
Pyrolysis
Thermal degradation of a chemical substance, generally resulting in smaller
chemical fragments.
Pyrosynthesis
Formation of additional compounds by the recombination of fragments arising
from incomplete combustion of a parent chemical/compound during pyrolysis.
Reconstituted sheet tobacco (RST)
A processed tobacco widely used in tobacco blends made by a paper makinglike process or from slurry of small scraps not suitable for cut filler. In a typical
American blended cigarette it constitutes 10-25 % of content.
For internal use of Philip Morris personnel only.
Appendices
297
Reference cigarette
Cigarettes prepared under controlled conditions with uniform, documented
source tobaccos and producing standardized yields of tar, nicotine and carbon
monoxide. The Tobacco & Health Research Institute at the University of Kentucky manufactures the 1R2 and 2R4F reference cigarettes.
Relative risk
Expression of a risk in relation to another risk, e.g., the risk of death at work is
approximately twice the risk of death from drowning.
Residence time
The time between inhalation and exhalation when a smoker holds smoke temporarily in the respiratory tract; estimated to vary from 4 sec to 24 sec.
Resistance-to-draw (Draw resistance)
The amount of negative pressure (suction) applied to pull a puff of mainstream
smoke through the butt end of the cigarette in Pascal units (Pa).
Retention
The amount of smoke (and its constituents) found in the lungs at any time,
including deposited and absorbed smoke (Brain & Valberg, 1979).
Returns
A list of all additives and ingredients included in tobacco products of major
manufacturers and the maximum amount (as percentage) used.
Reverse smoking
A method of generating smoke by machine in which air is forced through the
lighted cigarette by applying an elevated pressure to the burning end. This
method has been used in animal exposure systems to minimize the time to
deliver the smoke to the animal (Schultz & Wagner, 1975).
Risk
The probability of a future loss. In this report the probability of a mortality or
morbidity within a stated time (Byrd & Cothern, 2000).
Sales-weighted
Statistical adjustment of data about cigarette brands, such as tar contents, by
their sales volumes.
Sensitivity
The capacity to detect a signal (response) above background interference
(noise).
Sensory impact
The effect of a stimulus which changes behavior dependent on sensate pathways, e.g., an attractant odor or the localized sensation in a smoker’s throat
after inhaling (Junker et al., 2001).
Sidestream smoke
The smoke emitted directly into the air from the burning end of the cigarette,
largely during the smolder interval between puffs.
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Evaluation of Cigarette Ingredients: Feasibility
Signal-to-noise ratio
The ratio of the quality of the signal (data) obtained to the unwanted
interference.
Smoke
Usually a suspension of fine particles in air that scatters light and is physically
visible. A cigarette smoke contains many chemical substances in both gas and
liquid state, suspended in a dynamic aerosol created by incomplete combustion
and changing both physically and chemically with time.
Smolder phase
The part of combustion that is slow burning.
Specificity
Discrimination between the true signal (response) and competing (interfering)
signals.
Surrogate endpoint
A measurement, such as a biomarker, that can be substituted for an endpoint,
such as a disease. Surrogate endpoints are used to compare interventions
(Fleming et al., 1994).
Tar
The Federal Trade Commission refers to tar as the weight in grams of the total
particulate matter collected on a Cambridge filter minus the weight of alkaloids, as nicotine, and water (Federal Trade Commission, 1967a).
Teratogen
A substance causing abnormal fetal development resulting in congenital malformation.
Thorax
Region between neck and abdomen delineated by 12 thoracic vertebrae and by
the diaphragm; contains lungs and heart.
Tip
The portion of the cigarette that separates the body of the cigarette (tobacco
column) from the smoker’s lips. The tip retains the tobacco in the cigarette so
that tobacco particles cannot touch and stick to smoker’s lips (Allseits et al.,
1968).
Tipping paper
The paper (wrapper) that joins the filter plug to the tobacco column covering
the plug and overlapping the tobacco column.
Tobacco smoke condensate
Residual substance trapped on a filter as the smoke passes through a filter;
usually obtained from mainstream smoke.
Total particulate matter
Particles in smoke or smoke condensate larger than 1 µm in diameter.
For internal use of Philip Morris personnel only.
Appendices
299
Tow
The filling of cigarette filters. Most U.S. brands use cellulose diacetate filling
(Norman, 1999).
Tow item
The number of filaments in a tow band calculated by dividing the total weight
of the tow band (total denier) by the weight in grams of a single filament (denier per filament), TD/dpf (Norman, 1999).
Toxicokinetics
The time course of the absorption, distribution, metabolism, and excretion of
chemical substances by the body.
Toxicological
Related to toxic circumstances that alter biological function, such as a change
in morbidity or mortality.
Toxicology
The study of the interactions between chemical, physical, or biological agents
and biological systems.
Transition
A purine-to-purine or pyrimidine-to-pyrimidine mutation.
Transversion
A purine-to-pyrimidine or pyrimidine-to-purine mutation.
True Negative
A negative result from a test when the true outcome was negative
True Positive
A positive result from a test when the true outcome was positive
Vapor phase
Material in the gas state, usually material that passes through a filter.
Ventilation or Dilution
Perforations in the filter tipping paper or paper wrapping the tobacco rod that
dilute total air flow inside the rod and reduce the holes-open pressure drop
measurement; expressed as a percentage with higher numbers indicating greater
ventilation.
Virginia tobacco
A dark, flue-cured tobacco.
Yield or Level
Cigarettes classified based on the amount of a substance, such as tar, nicotine,
or carbon monoxide, as produced in smoke under standard smoking conditions.
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Evaluation of Cigarette Ingredients: Feasibility
LITERATURE CITATIONS
Allseits, F., Prospect, M., and Doblin, J. (8-13-1968). Cigarette Tip. U.S. Patent
No. 3396733
Bentrovato, B., Porter, A., Youssef, M. & Dunn, P. J. (1995) Variations in tar,
nicotine and carbon monoxide deliveries obtained by smokers of the same brand.
In: Proceedings of the CORESTA Smoke and Technology Groups Meeting,
Vienna, Austria. pp. 151-165.
Brain, J. D. & Valberg, P. A. (1979) Deposition of aerosol in the respiratory tract.
Am. Rev. Respir. Dis. 120:1325-1373.
Brunnemann, K. D., Prokopczyk, B., Djordjevic, M. V. & Hoffmann, D. (1996)
Formation and analysis of tobacco-specific N-nitrosamines. Crit. Rev. Toxicol.
26:121-137.
Byrd, D. M. & Cothern, C. R. (2000) Introduction to risk analysis. Government
Institutes, Rockville, MD. pp. 1-433.
European Parliament (2001) EU-Directive 2001/37/EC: On the approximation of
the laws, regulations and administrative provisions of the Member States concerning the manufacture, presentation and sale of tobacco products. Official
Journal L 194:26-35.
Federal Trade Commission (1967a) Cigarettes. Testing for tar and nicotine content. Fed. Reg. 32 (147):11178.
Fleming, T. R., Prentice, R. L., Pepe, M. S. & Glidden, D. (1994) Surrogate and
auxiliary endpoints in clinical trials, with potential applications in cancer and
AIDS research. Stat. Med. 13:955-968.
Hinds, W. C. (1999) Aerosol Technology. Properties, Behavior, and Measurement
of Airborne Particles. 2nd ed.:John Wiley & Sons, Inc., New York, NY. pp. 1483.
Institute of Medicine (2001) Clearing the Smoke. Assessing the Science Base for
Tobacco Harm Reduction. (Stratton, K., Shetty, P., Wallace, R. & Bondurant,
S., eds.). National Academy Press, Washington, D.C.
International Organization for Standardization (2000) Routine Analytical CigaretteSmoking Machine — Definitions and Standard Conditions. Report No. ISO
3308. International Organization for Standardization, Geneva, Switzerland. pp.
1-30.
Junker, M. H., Danuser, B., Monn, C. & Koller, T. (2001) Acute sensory responses
of nonsmokers at very low environmental tobacco smoke concentrations in
controlled laboratory settings. Environ. Health Perspect. 109:1045-1052.
For internal use of Philip Morris personnel only.
Appendices
301
Norman, A. (1999) Cigarette manufacture. 11B. Cigarette design and materials.
In: Tobacco Production, Chemistry and Technology. (Davis, D. L. & Nielsen,
M. T., eds.). CORESTA, Blackwell Science, Inc., Malden, MA. pp. 353-386.
Pauwels, R. A. (2000) National and international guidelines for COPD: the need
for evidence. Chest 117:20S-22S.
Pickett, J. P. (2000) The American Heritage Dictionary of the English Language.
(Pickett, J. P., ed.). Fourth Edition ed. : Houghton Mifflin Company, Boston.
Schultz, F. J. & Wagner, J. R. (1975) A thirty-port smoking machine for continuous
smoke generation. Beitr. Tabakforsch. 8:53-59.
Tornqvist, M. & Ehrenberg, L. (2001) Estimation of cancer risk caused by environmental chemicals based on in vivo dose measurement. J. Environ. Pathol.
Toxicol. Oncol. 20:263-271.
For internal use of Philip Morris personnel only.
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Evaluation of Cigarette Ingredients: Feasibility
APPENDIX C
PUBLIC AND INVITED COMMENTS
Organizations Submitting Information on the Review of
Ingredients Added to Cigarettes
Open Meeting Participants1
The Open Meeting on the Review of Ingredients Added to Cigarettes was held on
August 26, 2002. One oral presentation was made.
Wolf-Deiter Heller, Ph.D.
Faculty for Economic Sciences
University of Karlsruhe
Berlin, Germany
The following individuals submitted written materials for consideration by the
Ad Hoc Expert Panel:
James E. Trosko, Ph.D.
Department of Pediatrics/Human Development
College of Human Medicine
Michigan State University
East Lansing, Michigan
Randall J. Ruch, Ph.D.
Department of Pathology
Medical College of Ohio
Toledo, Ohio
Dr. Thilo Paschke
Verbend der Cigarettenindustrie
Berlin, Germany
1
Copies of the Open Meeting transcript are available from Ace-Federal Reporters, 1120 G
Street, NW, Washington, DC 20005
For internal use of Philip Morris personnel only.
Appendices
303
Abstract of written comments by Trosko and Ruch about gap junctions, as
edited by George J. Christ, Ph.D., of Albert Einstein College of Medicine, in
response to LSRO’s call for comments from the scientific community to guide
our evaluation of the relative risks of non-tobacco ingredients added to cigarettes. May 20, 2003
The development of the most efficacious strategy for the prevention and treatment
of cancers is based on understanding the underlying mechanisms of carcinogenesis. This includes the knowledge that the carcinogenic process is a multi-step,
multi-mechanism process and that no two cancers are alike, in spite of some apparent universal characteristics, such as their inability to have growth control, to terminally differentiate, to apoptose abnormally and to have an apparent extended or
immortalized life span. The multi-step process, involving the “ initiation” of a single
cell via some irreversible process, with the clonal expansion of this initiated cell by
suppressing growth control and inhibiting apoptosis (promotion step), leads to a
situation whereby additional genetic and epigenetic events can take place (progression step) to confer the necessary phenotypes of invasiveness, and metasis (neoplastic stage). While it is clear that, in principle, prevention of each of these three
steps is possible, in practical terms, while it would make sense to minimize the
initiation step, one can never reduce this step to zero. On the other hand, since the
promotion step is the rate-limiting step of carcinogenesis, intervening to block this
step makes the most sense. Also, by understanding the ultimate biological function
that confers growth control, terminal differentiation or apoptosis for cells, there is
even some hope of treating some, but not all, malignant cells such that they can
regain some ability to perform these vital cellular functions.
Gap junctional intercellular communication (GJIC) has been speculated to be a
necessary, if not sufficient, biological function of metazoan cells for the regulation
of growth control, differentiation and apoptosis of normal progenitor cells. Normal,
contact-inhibited fibroblast and epithelial cells have functional GJIC, while most, if
not all, tumor cells have dysfunctional homologous or heterologous GJIC. Cancer
cells are characterized by the lack of growth control, inability to terminally differentiate or apoptose under normal conditions and have extended or immortalized life
spans. Chemical tumor promoters, growth factors and hormones have been shown
to inhibit GJIC. Several oncogenes and anti-sense connexin genes have been shown
to down-regulate GJIC function. Anti-oncogene drugs, anti-tumor promoting natural and synthetic chemicals, as well as GJIC-deficient neoplastic cells, transfected
with various connexin genes, have been shown to re-gain GJIC and growth control
with the loss of tumorigenicity. Therefore, the hypothesis for a rational approach to
identify anti-tumor promoting chemopreventive drugs and anti-carcinogenic treatments is to use the prevention of the down regulation of GJIC by the tumor promoters and the restoration of GJIC in neoplastic cells.
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Evaluation of Cigarette Ingredients: Feasibility
While previous and many current strategies for chemoprevention and therapy have
been based on treating specific oncogene products or cell signalling mechanisms,
as well as advance molecular modifications of older strategies, none have specifically used the prevention of GJIC by agents during the rate limiting step of carcinogenesis or the restoration of GJIC in neoplastic cells which are deficient in GJIC.
Since there are multiple mechanisms by which GJIC is down regulated during the
tumor promotion phase and since stable GJIC deficiencies in neoplastic cells can
be the result of transcriptional, translational or posttranslational mechanisms, it should
be clear there would not be one “golden bullet” approach to resolve either the
chemoprevention or therapeutic approach. Even with the hypothesis that GJIC,
which depends on the transcription of normal connexin genes, their normal translation, trafficking, assembly and function, it should be clear that cells with normal
connexin genes and potentially normal GJIC might not have functional GJIC because of dysfunction of other defects in cancer cells, namely cell-adhesion or cellmatrix problems (both of which are necessary for GJIC to occur). In essence, if
dietary or chemopreventive/therapy is to be effective, the strategy must either
ameliorate the growth stimulatory effects of exogenous chemicals, growth factors
or hormones, that trigger various mitogenic /anti-apoptotic signal transducing systems.
James E. Trosko, Ph.D.
246 Food Safety & Toxicology Building
Department of Pediatrics/Human Development
College of Human Medicine
Michigan State University
Wilson Road
East Lansing, Michigan 48824-1302
and
Randall J. Ruch, Ph.D.
Department of Pathology
Medical College of Ohio
3055 Arlington Avenue
P.O. Box 10008
Toledo, Ohio 43614
For internal use of Philip Morris personnel only.
Appendices
305
Abstract relevant to oral presentation by Wolf-Dieter Heller, Ph.D. at the open
meeting.
This paper presents a literature review of published scientific studies of the effects
of tobacco product ingredients and various experimental additives on cigarette smoke
composition and its biological activity. The format of this work is that of an
uncommented reference paper rather than a critical scientific review. Therefore,
the mention of an ingredient in this survey does not imply that it is used by the
tobacco industry or that it is covered by existing national regulations. A broad
range of scientific papers and patents on tobacco ingredients is included as well as
studies on experimental ingredients. This review may provide public health officials as well as scientists in government agencies and in the tobacco industry with
a helpful overview of published information on tobacco product ingredients, their
transfer into mainstream cigarette smoke, pyrolysis products, and influence on the
biological activity of mainstream cigarette smoke.
Paschke, T., Scherer, G. & Heller, W. D. (2002) Effects of ingredients on cigarette
smoke composition and biological activity: a literature overview. Beitr. Tabakforsch.
Int. 20:107-247.
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Evaluation of Cigarette Ingredients: Feasibility
APPENDIX D
LIST OF INGREDIENTS
BACKGROUND
In 1994 six major cigarette manufacturers reported their most current and complete list of ingredients (Doull et al., 1994). This list, also known as the “599
Ingredients List,” is annotated with references to materials approved for use as
food additives by the U.S. Food and Drug Administration (FDA) as “Generally
Recognized as Safe” (GRAS). The Flavor and Extracts Manufacturers Association of the United States (FEMA) also recognizes many of these ingredients as
safe for ingestion. This list is a subset of approximately 700 ingredients that tobacco companies annually report to the Centers for Disease Control and Preventions (CDC) Office on Smoking and Health (OSH) which is a division of the National Center for Chronic Disease Prevention and Health Promotion. The Department of Health and Human Services (DHHS) list (R.J.Reynolds Tobacco Company, 2000a) is treated as a trade secret or confidential information in accordance
with the Comprehensive Smoking Education Act (U.S. Congress, 2002).
INFORMATION SOURCES
Several versions of the 599 ingredients list can be found via the Internet.
➤ The original report “Cigarette Ingredients: A complete list and background” published by R.J. Reynolds on behalf of six major cigarette companies: The American Tobacco Company, Brown & Williamson Tobacco Corporation, Liggett Group,
Inc., Lorillard Tobacco Company, Philip Morris Incorporated and R.J. Reynolds
Tobacco Company (Doull et al., 1994).
➤ The Tobacco Institute (1994), the Indiana Prevention Resource Center at Indiana University (1998) and the Alternative Cigarettes websites (2002) provide
lists of the 599 ingredients. Of these only the Tobacco Institute numerically lists
the ingredients and indicates whether FEMA and/or GRAS approved the ingredients.
➤ R.J. Reynolds Tobacco Company, Philip Morris USA, Brown & Williamson
Tobacco Corporation, and the Liggett Group Inc. (2000a; 2000; 2002; 2002)
also provide lists of ingredients on their company websites. Of these lists, R.J.
Reynolds, Philip Morris and Brown & Williamson list the “Maximum Use Level”
(MUL), which is the maximum amount of each ingredient that these companies
add to cigarette tobacco. Due to the differences in cigarette brand characteristics, the actual levels of ingredients will vary. Brown & Williamson’s list is
annotated with the Chemical Abstract Services Registry Numbers (CAS Nos.).
For internal use of Philip Morris personnel only.ffice.
Appendices
307
The CAS number is a unique code to identify a chemical, which may have more
than one common or scientific name. Liggett Group’s list indicates tar and
nicotine level, for each of the company cigarette brands but does not annotate
the list with MULs or references to materials that are approved by GRAS or
FEMA. Lorillard Tobacco Company does not provide an ingredients list on its
website.
OBSERVATIONS
Our review of the original 1994 report found spelling errors in the lists predominantly in the amino acids, e.g., dl-alanine, l-leucine listed as d1-alanine and 1leucine, respectively. Since the Indiana Prevention Resource Center and Alternative Cigarettes websites copied the original list to their websites, these errors carried through. Our comparison of the DHHS list with the original 1994 report list
found that the DHHS list contained only 483 ingredients. However, the spelling
errors found in the 599 list are not repeated here. LSRO developed a spreadsheet
to determine which ingredients are on both the 599 list (Doull et al., 1994) and the
DHHS (R.J.Reynolds Tobacco Company, 2000b) lists and which ingredients are
on one list but not the other.
LITERATURE CITATIONS
Alternative Cigarettes. (2002) The 599 ingredients added to tobacco in the manufacture of cigarettes by the five major American cigarette companies. (Access
Date: 7-10-2002). Web/URL: http://www.altcigs.com/ingredients.html
Brown & Williamson Tobacco Corporation. (2000) Cigarette Ingredients. (Access
Date: 8-8-2002). Web/URL: http://www.brownandwilliamson.com/index_
sub2.cfm?ID=11
Doull, J., Frawley, J. P., George, W. J., Loomis, T. A., Squire, R. A. & Taylor, S. L.
(1994) Cigarette ingredients. A complete list and background. R.J. Reynolds Tobacco Company, Winston-Salem, NC. pp. 1-25.
Indiana Prevention Resource Center. (8-2-1998) Additives found in American cigarettes. (Access Date: 4-8-2002). Web/URL: http://www.drugs.indiana.edu/druginfo/
additives.html
Liggett Group Inc. (2002) Tar & nicotine chart and ingredients. (Access Date: 85-2002). Web/URL:http://www.liggettgroup.com/pages/products/chart_and_
ingredients.html
Philip Morris USA. (4-8-2002) Ingredients in cigarettes. (Access Date: 4-5-2002).
Web/URL: http://www.philipmorrisusa.com
For internal use of Philip Morris personnel only.
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Evaluation of Cigarette Ingredients: Feasibility
R.J.Reynolds Tobacco Company. (2000a) RJRT List of ingredients (2000).
(Access Date: 10-1-2002a). Web/URL: http://www.rjrt.com/TI/TIcig_ingred_
summary.asp
R.J.Reynolds Tobacco Company. (2000b) Tobacco companies’ combined ingredients list for HHS (2000). (Access Date: 4-5-2002b). Web/URL:http://www.rjrt.com/
TI/TIcig_ingred_summary.asp
Tobacco Institute. (1994) Tobacco Institute. Ingredients added to cigarettes.
(Access Date: 5-10-2002). Web/URL: http://www.bbc.co.uk/worldservice/sci_tech/
features/health/tobaccotrial/inacigarette599.htm
U.S. Congress. (2002) Comprehensive Smoking Education Act. [U.S. Code, Title
15, Chapter 36, Sec. 1331-1341]. (Access Date: 11-4-2002). Web/URL: http://
www.access.gpo.gov/uscode/uscmain.html
LIST OF INGREDIENTS
Tobacco Companies’ Combined Ingredients List For the U.S.
Department of Health and Human Services (DHHS)
(R.J. Reynolds Tobacco Company, 2000b)
Most of these ingredients are commonly used in foods and beverages, or permitted
for use in foods by the U.S. Food and Drug Administration (FDA), or have been
given the status “Generally Recognized as Safe in Foods” (GRAS) by FDA, the
Flavor and Extract Manufacturers Association (FEMA) or other expert committees.
Ingredient
1. Acetanisole
FDA-approved food additive; FEMA GRAS; found in beef, cranberry, guava, grape,
mango, peppermint; used in frozen dairy products, hard candies.
2. Acetic acid
FDA GRAS; FEMA GRAS; found in banana, beer, beef, apple juice, apricot, blue
cheese, blueberries; used in condiment relishes.
3. Acetoin
FDA GRAS; FEMA GRAS; found in apples, butter, yogurt, asparagus, black currants, blackberry, wheat, broccoli, brussel sprouts, cantaloupe; used in baked goods.
4. Acetophenone
FDA-approved food additive; FEMA GRAS; found in apple, cheese, apricot, banana, beef, cauliflower; used in chewing gum.
For internal use of Philip Morris personnel only.
Appendices
309
5. 2-Acetylpyrazine
FEMA GRAS; found in beef, coffee, popcorn, sesame seed, almond, wheat bread,
cocoa, peanut, pork, potato chips; used in frozen dairy products.
6. 2-Acetylpyridine
FEMA GRAS; found in cocoa, coffee, roasted peanut, potato chips, tea, beer, wheat
bread, hazelnut, lamb/mutton, potato; used in breakfast cereals, ice cream, candy.
7. 3-Acetylpyridine
FEMA GRAS; found in roasted filbert, cocoa; used in nonalcoholic beverages, ice
cream, candy, gelatins & puddings, baked goods.
8. 2-Acetylthiazole
FEMA GRAS; found in bean, potatoes, artichoke, asparagus, beef, beer, brazil
nuts, rice, boiled shrimp; used in snack foods.
9. Aconitic acid
FDA GRAS; FEMA GRAS; found in beet root, sugarcane; used in alcoholic beverages, baked goods, chewing gum.
10. dl-Alanine, l-Alanine
FDA-approved food additive; natural constituent of protein in plants and animals;
found in apple, beef, carob, pea, soybean, wine, zucchini.
11. Alfalfa extract
FDA GRAS; FEMA GRAS; found in alfalfa; used in baked goods.
12. Allspice oil (pimenta berry)
FDA GRAS; FEMA GRAS; used in soups, candies, chewing gum, meats.
13. Allyl Hexanoate
FDA-approved food additive; FEMA GRAS; found in baked potato; used in gelatins & puddings.
14. alpha-Methylbenzyl acetate
FDA-approved food additive; FEMA GRAS; found in grape brandy, rice, tea, tomato, tomato paste; used in chewing gum.
15. Ambergris tincture
FDA GRAS; FEMA GRAS; used in nonalcoholic beverages, ice cream, candy.
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Evaluation of Cigarette Ingredients: Feasibility
16. Ammonium alginate
FDA GRAS; FEMA GRAS; used in confections, frostings, gelatins & puddings,
sauces. Ammonium alginate is used as a binder in the carbon heat source of cigarettes that primarily heat tobacco and also in some tobacco sheets.
17. Ammonium hydroxide
FDA GRAS; found in cured pork.
18. Ammonium phosphate dibasic
FDA GRAS; used in dough, ice cream, gelatins & puddings.
19. Amyl alcohol
FDA-approved food additive; FEMA GRAS; found in apple, banana, cheese,
chicken, coffee, potato, raspberry, strawberry, tomato; used in baked goods, candy,
gelatins & puddings, chewing gum.
20. Amyl butyrate
FDA-approved food additive; FEMA GRAS; found in bananas, beer, apple juice,
apricots, strawberries, wine; used in syrup, candy, chewing gum.
21. Amyl formate
FDA-approved food additive; FEMA GRAS; found in apples, strawberry, brandy,
honey, tomatoes, whiskey; used in nonalcoholic beverages, candy, chewing gum.
22. Amyl octanoate
FDA-approved food additive; FEMA GRAS; found in strawberry, apple, cognac;
used in baked goods, candy, gelatins & puddings.
23. alpha-Amylcinnamaldehyde
FDA-approved food additive; FEMA GRAS; found in black tea, olibanum; used in
candy, baked goods, chewing gum.
24. Amyris oil
FDA-approved food additive; found in brandies, liqueurs, amyris balsamifera; used
in brandies, liqueurs, oriental specialties.
25. trans-Anethole
FDA GRAS; FEMA GRAS; found in cheese, tea, apple, licorice; used in alcoholic
beverages.
26. Angelica root extract and oil
FDA GRAS; FEMA GRAS; used in nonalcoholic beverages, alcoholic beverages,
baked goods, chewing gum.
For internal use of Philip Morris personnel only.
Appendices
311
27. Anise oil
FDA GRAS; FEMA GRAS; found in star anise; used in ice cream, ices, baked
goods, candy, chewing gum, meats, condiments.
28. Anisyl acetate
FDA-approved food additive; FEMA GRAS; found in currant; used in baked goods,
candy, gelatins & puddings, chewing gum.
29. Anisyl alcohol
FDA-approved food additive; FEMA GRAS; found in honey, tomato; used in gelatins & puddings.
30. Anisyl formate
FDA-approved food additive; FEMA GRAS; found in vanilla; used in candy, baked
goods.
31. Apple juice concentrate and extract
Common food item found in apple; used in juices, baked goods.
32. Apricot extract
Common food item found in apricot; used in condiments.
33. l-Arginine
FDA-approved food additive; natural constituent of proteins in plants and animals.
34. Ascorbic acid
FDA GRAS; FEMA GRAS; found in citrus fruit, tea leaves; used in baked goods,
sweet sauce, soups, candy, gelatins & puddings, dairy products.
35. l-Aspartic acid
FDA-approved food additive; FEMA GRAS; found in proteins, licorice; used in
seasonings.
36. Balsam peru and oil
FDA GRAS; FEMA GRAS; found in Peru balsam; used in baked goods, syrups,
candy, chewing gum.
37. Bay oil
FDA GRAS; FEMA GRAS; found in bay leaves; used in condiments, meats.
38. Beeswax resinoid and absolute
FDA GRAS; FEMA GRAS; used in baked goods, candy, honey.
For internal use of Philip Morris personnel only.
312
Evaluation of Cigarette Ingredients: Feasibility
39. Beet juice concentrate
Beets are included among “Miscellaneous Vegetables” in FDA Standards of Identity and are also covered by a USDA Standards for Grades.
40. Benzaldehyde
FDA GRAS; FEMA GRAS; found in apple juice, almond, apricot, artichoke, asparagus, beans, beef, beer; used in baked goods, chewing gum.
41. Benzaldehyde glyceryl acetal
FDA-approved food additive; FEMA GRAS; used in baked foods, candy, gelatins
& puddings, chewing gum.
42. Benzoic acid
FDA GRAS; FEMA GRAS; found in cinnamon, strawberry, tea, apple, beer, bread,
cocoa, honey; used in baked goods, cheese, candy, chewing gum, condiment relish.
43. Benzoin, resin, resinoid, gum, and absolute
FDA-approved food additive; FEMA GRAS; used in baked goods, gelatins & puddings, chewing gum.
44. Benzophenone
FDA-approved food additive; FEMA GRAS; found in grape, apples, papaya; used
in frozen dairy products.
45. Benzyl alcohol
FDA-approved food additive; FEMA GRAS; found in apricot, beef, beer, almonds,
apple, apple juice, asparagus, bananas, black currants, blackberries; used in chewing gum, candy, baked goods.
46. Benzyl benzoate
FDA-approved food additive; FEMA GRAS; found in celery, parsley, black currants, butter, guava, pineapple, papaya; used in ice cream, baked goods, candy.
47. Benzyl butyrate
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, apricot,
banana, parmesan cheese, grape, honey, mango, melon, muskmelon, orange juice,
papaya; used in candy.
48. Benzyl cinnamate
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, baked
goods, candy.
For internal use of Philip Morris personnel only.
Appendices
313
49. Benzyl salicylate
FDA-approved food additive; FEMA GRAS; found in cranberry, apple flowers;
used in baked goods.
50. Bergamot oil
FDA GRAS; FEMA GRAS; found in oranges; used in icings, gelatin, alcoholic
beverages.
51. Bois de Rose (Peruvian) oil
FDA GRAS; FEMA GRAS; used in baked goods, candy, chewing gum.
52. Bornyl acetate
FDA-approved food additive; FEMA GRAS; found in carrot, black currants, gin,
ginger, kiwi fruit, pistacia, plum, sweet potato, soy sauce, black & green tea; used in
gelatins & puddings, candy, ice cream.
53. Brown sugar
Found in food sugar sources; used in baked goods, candy, breakfast cereals.
54. Buchu leaf oil
FDA-approved food additive; FEMA GRAS; used in ice cream, ices, candy, condiments.
55. 1,3-Butanediol
FDA-approved food additive; used as a solvent for natural and synthetic flavors.
56. 4-(2-Butenylidene)-3,5,5-trimethyl-2-cyclohexen-1-one
Found in white-flesh nectarine, starfruit, grapefruit juice.
57. Butter, butter esters, and butter oil
FDA-approved food additive; FEMA GRAS; found in butter; used in frozen dairy
products.
58. Butyl Acetate
FDA-approved food additive; FEMA GRAS; found in apple, banana, beer, black
currant, cashew nuts, cheese, raspberry, apricot, blackberry brandy, cantaloupe;
used in cheeses, baked goods, candy.
59. Butyl Alcohol
FDA-approved food additive; FEMA GRAS; found in apple juice, banana, celery,
cheese (cheddar and Swiss), peach, potato; used in nonalcoholic beverages, alcoholic beverages, ice cream ices, candy, cream, baked goods.
For internal use of Philip Morris personnel only.
314
Evaluation of Cigarette Ingredients: Feasibility
60. Butyl Butyrate
FDA-approved food additive; FEMA GRAS; found in banana, apricot, blackberry,
brandy, parmesan cheese, honey, mango, melon, muskmelon, orange juice, papaya;
used in candy.
61. Butyl butyryl lactate
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, baked
goods, candy, sweet sauces.
62. n-Butyl isovalerate
FDA-approved food additive; FEMA GRAS; found in apple, apricot, banana,
parmesan cheese, olives, pear, plum, strawberry, wine; used in baked goods.
63. Butyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in papaya, alfalfa; used in baked
goods, ice cream, candy.
64. 3-Butylidenephthalide
FEMA GRAS; found in celery, celery stalk; used in soups, condiments, meats.
65. Butyric acid
FDA GRAS; FEMA GRAS; found in apple, beef, beer, black currants, blueberries,
wheat bread, butter, blue cheese; used in snack foods, candy, margarine.
66. Camphene
FDA-approved food additive; FEMA GRAS; found in carrot, cheddar cheese, ginger, apricot, black currants, blackberry, celery, gin, kiwi fruit; used in candy, baked
goods, ice cream.
67. Cananga oil
FDA GRAS; FEMA GRAS; used in nonalcoholic beverages, candy, baked goods.
68. Caramel and caramel color
FDA GRAS; FEMA GRAS; found in sugars; used in gravies, meats, condiments.
69. Caraway oil
FDA GRAS; FEMA GRAS; found in caraway seeds; used in baked goods, condiments.
70. Carbon
Carbons are used in air and water filtration systems. Carbons are also used as
clarifiers and decolorizers in the food and beverage industry. Carbon is the main
heat source constituent in cigarettes that primarily heat tobacco.
For internal use of Philip Morris personnel only.
Appendices
315
71. Carbon dioxide
FDA GRAS; used in beverages, meat products, processed fruits, dairy products.
72. Cardamom oleoresin, oil, extract, seed oil, and powder
FDA GRAS; FEMA GRAS; found in cardamom; used in baked goods, pickles,
meats.
73.Carob bean and extract
FDA GRAS; FEMA GRAS; found in carob beans; used in baked goods, candy,
gelatins puddings, icings & toppings.
74. beta-Carotene
FDA GRAS; found in carrot, pumpkin, spinach, broccoli; used in processed fruit
and fruit juices, dairy products.
75. Carrot oil, seed
FDA GRAS; FEMA GRAS; found in carrots; used in baked goods.
76. Carvacrol
FDA-approved food additive; FEMA GRAS; found in pepper, spearmint, tea; used
in baked goods, condiments, candy, gelatins & puddings, chewing gum.
77. 4-Carvomenthenol
FDA-approved food additive; FEMA GRAS; found in carrot, celery seed, cocoa
powder, grape, grapefruit, orange, tea, wine; used in nonalcoholic beverages, candy,
baked goods, gelatins & puddings, chewing gum.
78. l-Carvone
FDA GRAS; found in grapefruit juice, honey, hops oil, orange juice, beer, cherries,
endive, guava, hazelnuts; used in candy, condiments, baked goods.
79. beta-Caryophyllene oxide
FDA-approved food additive; found in rosemary; used in beverages, ice cream,
candy, condiments.
80. beta-Caryophyllene
FDA-approved food additive; FEMA GRAS; found in carrot, artichoke, banana,
cashews, apples, celery, chervil, chicken, cocoa; used in chewing gum, ice cream,
beverages.
81. Cascarilla oil and bark extract
FDA GRAS; FEMA GRAS; used in baked goods, candy, condiments.
For internal use of Philip Morris personnel only.
316
Evaluation of Cigarette Ingredients: Feasibility
82. Cassia bark, buds, oil and extract
FDA GRAS; FEMA GRAS; found in cassia; used in meat products.
83. Cassie absolute
FDA-approved food additive; FEMA GRAS; found in cassie; used in candy, baked
goods, ice cream.
84. Castoreum extract, tincture, liquid, and absolute
FDA GRAS; FEMA GRAS; found in castor; used in baked goods, condiments,
candy, chewing gum.
85. Cedar leaf oil
FDA-approved food additive; FEMA GRAS; found in cedar tree; used in alcoholic
beverages, meats, candy.
86. Cedarwood oil terpenes
FDA-approved food additive; found in cedarwood, clary sage oil.
87. Celery seed extract, solid, oil, and oleoresin
FDA GRAS; FEMA GRAS; found in celery seeds, celery; used in baked goods,
meats, soups, condiments, pickles.
88. Cellulose and Cellulose fiber
Natural polysaccharide, which is the most abundant carbohydrate in nature; found
in all plant material; used in grated cheese, fruit preserves/jams, fruit jellies. Cellulose fiber is used as a sheet strengthener in tobacco sheets.
89. Chamomile flower oil and extract
FDA GRAS; FEMA GRAS; found in chamomile flowers; used in nonalcoholic
beverages, ice cream, baked goods, gelatins & puddings.
90. Chicory extract
FDA GRAS; FEMA GRAS; found in chicory; used in baked goods, nonalcoholic
beverages, ice cream.
91. Chocolate
Common food item.
92. 1,8-Cineole
FDA-approved food additive; FEMA GRAS; found in black currants, blueberries,
brandy, cantaloupe, cheese, cocoa, grapes, thyme, babaco fruit, bilberry, corn; used
in chewing gum, ice cream, baked goods.
For internal use of Philip Morris personnel only.
Appendices
317
93. Cinnamaldehyde
FDA GRAS; FEMA GRAS; found in beer, brandy, blueberries, cantaloupe, capers,
cranberries, gin, guava, melon; used in gravies, candy, ice cream, meat.
94. Cinnamic acid
FDA-approved food additive; FEMA GRAS; found in beer, blackberry, capers,
cherry, grape, guava, malt, mango, mushroom, passion fruit, strawberry; used in
soft candy.
95. Cinnamon leaf oil, bark oil, and extract
FDA GRAS; FEMA GRAS; found in cinnamon tree; used in baked goods, chewing
gum, candy, meats, condiments, pickles.
96. Cinnamyl acetate
FDA-approved food additive; FEMA GRAS; found in guava; used in candy, ice
cream condiments.
97. Cinnamyl alcohol
FDA-approved food additive; FEMA GRAS; found in blackberry, blueberry, cantaloupe, cranberry, guava, melon, raspberry, strawberry, watermelon; used in nonalcoholic beverages.
98. Cinnamyl cinnamate
FDA-approved food additive; FEMA GRAS; found in storax; used in baked goods,
ice cream, candy.
99. Cinnamyl isovalerate
FDA-approved food additive; FEMA GRAS; found in chestnut flowers; used in
candy, gelatins & puddings, chewing gum.
100. Citral
FDA GRAS; FEMA GRAS; found in grapefruit juice, orange, orange juice, celery,
apricot, black currants, grape, hops, kiwi fruit, mango, mango ginger, melon, plum,
raspberry, rum; used in baked goods, candy, ice cream.
101. Citric acid
FDA GRAS; FEMA GRAS; widely found in fruits and vegetables; used in fruit
juices, meats, poultry, beverages.
102. Citronella oil
FDA GRAS; FEMA GRAS; found in citronella; used in alcoholic beverages, ice
cream, baked goods.
For internal use of Philip Morris personnel only.
318
Evaluation of Cigarette Ingredients: Feasibility
103. dl-Citronellol FDA-approved food additive; FEMA GRAS; found in apple,
apricot, beer, black currants, blackberry, blueberry, orange juice, passion fruit, peach;
used in soft candy.
104. Citronellyl isobutyrate
FDA-approved additive; FEMA GRAS; used in candy, gelatins & puddings, nonalcoholic beverages, baked goods.
105. Civet absolute
FDA GRAS; FEMA GRAS; found in civet; used in ice cream, candy, baked goods,
chewing gum.
106. Clary sage oil and extract
FDA GRAS; FEMA GRAS; found in clary sage; used in alcoholic beverages, baked
goods, condiments.
107. Cocoa, cocoa shells, extract, distillate, powder, alkalized, absolute and tincture
FDA GRAS; found in cocoa, cocoa shells; used in baked goods.
108. Coffee and coffee solid extract
FDA GRAS; found in coffee; used in baked goods, candy, syrups.
109. Cognac white and green oil
FDA GRAS; FEMA GRAS; found in cognac brandy; used in alcoholic beverages,
ice cream, baked goods.
110. Copaiba oil
FDA-approved food additive; found in copaiba.
111. Coriander extract, oil, and seed
FDA GRAS; FEMA GRAS; found in coriander; used in baked goods, meats, condiments.
112. Corn silk
FDA GRAS; FEMA GRAS; found in corn; used in baked goods, beverages, candy,
desserts.
113. Corn syrup
FDA GRAS; found in food sugar sources; used in baked goods, candy, breakfast
cereals.
114. Costus root oil
FDA-approved food additive; FEMA GRAS; found in costus root; used in candy,
baked goods.
For internal use of Philip Morris personnel only.
Appendices
319
115. para-Cymene
FDA-approved food additive; FEMA GRAS; found in apricot, banana, beans, beli,
black currants, brandy apple, carrots, celery; used in chewing gum.
116. l-Cysteine
FDA GRAS; FEMA GRAS; natural constituent of protein in plants and animals;
found in pippali fruit (India); used in condiments relish, beverages, meats, baked
goods, dairy products.
117. Dandelion root solid extract
FDA GRAS; FEMA GRAS; found in dandelions; used in baked goods.
118. Davana oil
FDA-approved food additive; FEMA GRAS; found in Artemisia plant; used in alcoholic beverages.
119. 2,4-Decadienal
FEMA GRAS; found in chicken, cranberry, peanut, tomato; used in vegetables,
baked goods, meat, candy, chewing gum, cereals.
120. delta-Decalactone
FDA-approved food additive; FEMA GRAS; found in apricot, beef, butter, beer,
blue cheese, grape brandy, plum brandy, wheat bread, cantaloupe, cheese; used in
baked goods, frozen dairy products.
121. gamma-Decalactone
FDA-approved food additive; FEMA GRAS; found in apricot, beef fat, butter, black
currants, blackberry, blue cheese, cheddar cheese, chicken, coconut, cranberry,
cream; used in baked goods, margarine, candy.
122. Decanal
FDA GRAS; FEMA GRAS; found in almond, apple, apple flowers, apricot, artichoke, avocado, beef, wheat bread; used in baked goods, beverages, ice cream,
candy.
123. Decanoic acid
FDA-approved food additive; FEMA GRAS; found in apple, apple flowers, banana, beef, beer, blackberries, blue cheese, brandies, wheat bread, butter, heated
butter; used in imitation dairy goods.
124. Decanoic Acid, Ester with 1,2,3 - Propanetriol Octanoate (Coconut oil)
FDA GRAS; found in coconut; used in shortening, candies, chocolate.
For internal use of Philip Morris personnel only.
320
Evaluation of Cigarette Ingredients: Feasibility
125. Dextrin
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, apricot,
asparagus, banana, beer, brandy apple, butter; used in frozen dairy goods, ice cream,
beverages, candy.
126. Diacetyl
FDA GRAS; FEMA GRAS; found in apple, bean, beef, butter, artichoke, avocado,
black currants, blueberry, blue cheese, grape brandy, wheat, brussel sprouts; used
in meat products.
127. Diethyl malonate
FDA-approved food additive; FEMA GRAS; found in whiskey, wine, blackberry,
grape brandy, strawberry wine.
128. 2,3-Diethylpyrazine
FEMA GRAS; found in wheat bread, wheat, hazelnut, baked potato, soy sauce;
used in candy, gelatins & puddings.
129. 5,7-Dihydro-2-methylthieno (3,4-d) pyrimidine
FEMA GRAS; used in breakfast cereals, beverages, ice cream, candy, dairy products.
130. Dill oil
FDA GRAS; FEMA GRAS; found in dill; used in cheese, meats, sauces, dips,
baked goods.
131. meta-Dimethoxybenzene
FDA-approved food additive; FEMA GRAS; found in brandy grape, salami, filberts; used in meat products, beverages, ice cream, candy, baked goods.
132. para-Dimethoxybenzene
FDA-approved food additive; FEMA GRAS; found in tea, hyacinth oil, peppermint
oil; used in gelatins & puddings, beverages, ice cream, candy, baked goods.
133. 2,6-Dimethoxyphenol
FEMA GRAS; found in maple syrup, rum, smoked sausage, wine; used in seafood,
meat, baked goods, candy, soups.
134. 3,4-Dimethyl-1,2-cyclopentadione
FEMA GRAS; found in roasted coffee; used in nut products, beverages, ice cream,
candy.
For internal use of Philip Morris personnel only.
Appendices
321
135. 3,7-Dimethyl-1,3,6-octatriene
FDA-approved food additive; FEMA GRAS; found in apricot, guava, pineapple,
tomato; used in frozen dairy products.
136. 4,5-Dimethyl-3-hydroxy-2,5-dihydrofuran-2-one
FEMA GRAS; found in almond, asparagus, wheat bread, butter, chicken, steamed
clams, cocoa, coconut, coffee, corn; used in baked goods, sweet sauce.
137. 6,10-Dimethyl-5,9-undecadien-2-one
FDA-approved food additive; FEMA GRAS; found in almond, asparagus, beans,
beef, beer, cashew nuts, parmesan cheese, chicken; used in baked goods, candy,
dairy products.
138. 3,7-Dimethyl-6-octenoic acid
FEMA GRAS; found in peppermint oil; used in baked goods.
139. alpha,para-Dimethylbenzyl alcohol
FEMA GRAS; used in nonalcoholic beverages, ice cream, ices, candy.
140. alpha,alpha-Dimethylphenethyl butyrate
FDA-approved food additive; FEMA GRAS; found in sake; used in frozen dairy
goods, beverages, candy, baked goods.
141. 2,3-Dimethylpyrazine
FEMA GRAS; found in asparagus, peanut, coffee, potato; used in gravies, beverages, candy, baked goods.
142. 2,5-Dimethylpyrazine
FEMA GRAS; found in beef, blackberry, grape brandy, cantaloupe, corn, endive,
grapefruit juice; used in breakfast cereal.
143. 2,6-Dimethylpyrazine
FEMA GRAS; found in citronella, camphor oil; used in milk products, meat, candy.
144. delta-Dodecalactone
FDA-approved food additive; FEMA GRAS; found in beef, butter, milk, blue cheese,
cheddar cheese, chicken, coconut, lamb/mutton, peach, plum, pork; used in meat
products, baked goods, candy.
145. gamma-Dodecalactone
FDA-approved food additive; FEMA GRAS; found in apricot, beef, beer, blackberry, blue cheese, butter, carambola (starfruit), cheddar cheese, chervil, chicken;
used in baked goods, beverages, ice cream, candy.
For internal use of Philip Morris personnel only.
322
Evaluation of Cigarette Ingredients: Feasibility
146. Ethyl 2-methylbutyrate
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, beer, bilberry, blackberry, brandy apple, brandy grape, cantaloupe, fig, grape, honeydew
melon; used in hard candy, beverages, ice cream.
147. Ethyl acetate
FDA GRAS; FEMA GRAS; found in apple, apple juice, banana, beans, beef, beer,
blue cheese, blueberry; used in chewing gum, beverages, ice cream, candy.
148. Ethyl acetoacetate
FDA-approved food additive; FEMA GRAS; found in passion fruit, sherry, strawberry, wine; used in soft candy.
149. Ethyl alcohol, including Specially Denatured Alcohol (SDA) No. 4
FDA GRAS; FEMA GRAS; found in apple, banana, bread, coffee, cucumber, potato. As required by the U.S. Bureau of Alcohol, Tobacco and Firearms regulations, nicotine sulfate is used to denature the alcohol, which is used as a solvent to
apply flavors during processing. There is no measurable effect on the nicotine level
of the finished cigarette as a result of this process.
150. Ethyl benzoate
FDA-approved food additive; FEMA GRAS; found in apple, apricot, arctic bramble,
babaco fruit, banana, beer, beli, bilberry, bilberry wine, black currants, blackberry,
brandy apple; used in gelatins & puddings, beverages, ice cream, candy.
151. Ethyl butyrate
FDA GRAS; FEMA GRAS; found in apple, apple juice, banana, beer, apricot, beef,
blue cheese, brandy; used in chewing gum.
152. Ethyl cinnamate
FDA-approved food additive; FEMA GRAS; found in beer, blackberry, brandy
apple; used in baked goods, beverages, ice cream, candy.
153. Ethyl decanoate
FDA-approved food additive; FEMA GRAS; found in apple juice, banana, beef,
wheat bread, butter, cheddar cheese; used in frozen dairy foods, ice cream, candy.
154. Ethyl heptanoate
FDA-approved food additive; FEMA GRAS; found in cashew, apple, cocoa, grape,
grapefruit juice, roasted hazelnut, hops, milk, olive, papaya mountain, passion fruit,
peach; used in chewing gum.
For internal use of Philip Morris personnel only.
Appendices
323
155. Ethyl hexanoate
FDA-approved food additive; FEMA GRAS; found in banana, beer, cheese, beef,
black currants, blackberry, brandies, broccoli; used in baked goods, ice cream, candy.
156. Ethyl isovalerate
FDA-approved food additive; FEMA GRAS; found in banana, celery, apple, beer,
brandy, cantaloupe, cashew apple, parmesan cheese; used in condiments, ice cream,
baked goods.
157. Ethyl lactate
FDA-approved food additive; FEMA GRAS; found in apple, beer, cocoa, pineapple, apricot, bilberry wine, brandy, butter, capers, chicken, meat, elderberry, elderberry juice, grape peas, plum; used in chewing gum, ice cream, baked goods.
158. Ethyl laurate
FDA-approved food additive; FEMA GRAS; found in apple, beer, cheddar cheese,
apricot, bilberry wine, blackberry, brandy, wheat bread, butter; used in baked goods,
candy, ice cream.
159. Ethyl levulinate
FDA-approved food additive; FEMA GRAS; found in bilberry wine, brandy grape,
wheat bread, cherimoya, cocoa, onion roasted, rum, wine; used in frozen dairy
goods, beverages, candy, baked goods.
160. Ethyl maltol
FDA-approved food additive; FEMA GRAS; found in apple juice; used in sweet
sauce, soups, meat, candy.
161. Ethyl methyl phenylglycidate
FDA GRAS; FEMA GRAS; used in condiment relish, beverages, candy, ice cream.
162. Ethyl myristate
FDA-approved food additive; FEMA GRAS; found in cheddar cheese, grape wine,
Bartlett pear; used in nonalcoholic beverages, alcoholic beverages, ice cream, ices,
candy, baked goods.
163. Ethyl nonanoate
FDA-approved food additive; FEMA GRAS; found in apple, apricot, banana, beef,
beer, bilberry wine, brandy apple, wheat bread, cocoa, elderberry, grape, nectarine,
olive, peach; used in baked goods, beverages, ice cream, candy.
164. Ethyl octadecanoate
FEMA GRAS; found in grapes, beer, brandy, maple syrup; used in nonalcoholic
beverages, ice cream, ices, candy, alcoholic beverages.
For internal use of Philip Morris personnel only.
324
Evaluation of Cigarette Ingredients: Feasibility
165. Ethyl octanoate
FDA-approved food additive; FEMA GRAS; found in apple, banana, beer, blue
cheese, apricot, bilberry wine, blackberry, brandy, wheat bread, broccoli, butter,
capers; used in frozen dairy products.
166. Ethyl oleate
FDA-approved food additive; FEMA GRAS; found in melons, grapes, brandy, maple
syrup; used in nonalcoholic beverages, candy, baked goods, gelatins & puddings,
condiments & relishes.
167. Ethyl palmitate
FEMA GRAS; found in cheddar cheese, maple syrup, grape wine; used in nut
products.
168. Ethyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in apple, crisp bread, honey,
beer, beli, bilberry wine, brandy, wheat bread, cantaloupe, chempedak fruit, cocoa,
grape, grapefruit juice, guava, licorice, melon; used in gelatin & puddings, syrups,
baked goods.
169. Ethyl propionate
FDA-approved food additive; FEMA GRAS; found in apples, apricot, banana, beer,
bilberry, blackberry, brandy, cantaloupe, cheddar cheese, cocoa, fig, grape, guava;
used in baked goods, meat products, ice cream.
170. Ethyl valerate
FDA-approved food additive; FEMA GRAS; found in apple, banana, grape, apricot, bilberry wine, black currants, brandy, cashew apples, parmesan cheese, fig,
guava, honey, kiwi fruit, melon, muskmelon; used in chewing gum, baked goods,
beverages.
171. Ethyl vanillin
FDA GRAS; FEMA GRAS; found in vanilla beans; used in alcoholic beverages,
imitation vanilla extract, breakfast cereals.
172. Ethyl vanillin glucoside
Composed of ethyl vanillin, a common flavoring, and glucose, a sugar. Ethyl Vanillin
is FDA/FEMA GRAS; found in vanilla beans and used in alcoholic beverages,
imitation vanilla extract, breakfast cereals. Glucose is found in food sugar sources;
used in baked goods, candy, breakfast cereals.
173. 2-Ethyl-1-hexanol
FEMA GRAS; used in nonalcoholic beverages, ice cream, ices, candy, chewing
gum.
For internal use of Philip Morris personnel only.
Appendices
325
174. 3-Ethyl-2-hydroxy-2-cyclopenten-1-one
FEMA GRAS; found in coffee, maple syrup, peanuts, pork; used in baked goods,
soups, cereals, condiments, milk & dairy products.
175. 2-Ethyl-3,(5 or 6)-dimethylpyrazine
FEMA GRAS; found in beef, coffee, bread; used in baked goods, cereals, candy,
dairy products.
176. 5-Ethyl-3-hydroxy-4-methyl-2(5H)-furanone
FEMA GRAS; nature identical by FEMA; found in beef, beer, wheat bread, cashew
nuts, chicken, cocoa, coconut, coffee, crayfish, eggs, hazelnut; used in chewing
gum, meat products.
177. 2-Ethyl-3-methylpyrazine
FEMA GRAS; found in beef, whole egg, chicken, heated corn oil, krill, lamb/mutton, boiled shrimp, fermented soy sauce; used in candy, ice cream, beverages.
178. 4-Ethylbenzaldehyde
FEMA GRAS; found in oranges, carrots, broccoli, tomatoes; used in baked goods,
meat products, candy, gelatins & puddings, confectionery/frosting, cereals, dairy
products.
179. 4-Ethylguaiacol
FDA-approved food additive; FEMA GRAS; found in coffee, cranberry, smoked
pork, rum, smoked sausage, tea, wine; used in nonalcoholic beverages, ice cream,
ices.
180. para-Ethylphenol
FEMA GRAS; found in cocoa, coffee, peanut, tomato, wine; used in baked goods,
candy, gelatins & puddings, meat products.
181. 3-Ethylpyridine
FEMA GRAS; found in beef, chicken, coffee, corn oil, almond, barley, beer, wheat
bread, cocoa, eggs; used in candy, ice cream, meat, baked goods.
182. Farnesol
FDA-approved food additive; FEMA GRAS; found in oranges, lemongrass; used
in nonalcoholic beverages, ice cream, candy, baked goods, gelatins & puddings.
183. Fenchone
FDA-approved food additive; FEMA GRAS; found in anise, basil, fennel, peppermint, saffron, thyme; used in ice cream, candy, baked goods.
For internal use of Philip Morris personnel only.
326
Evaluation of Cigarette Ingredients: Feasibility
184. Fenugreek, extract, resin, and absolute
FDA GRAS; FEMA GRAS; found in fenugreek; used in nonalcoholic beverages,
gelatins & puddings, syrups.
185. Fig juice concentrate and extract
Common food item; found in figs; used in fruit breads, cookies, cakes, sauces,
fillings and cereals.
186. Food starch modified
FDA-approved food additive; widespread use; used in cured pork.
187. Fructose
Found in food sugar sources; used in baked goods, candy, breakfast cereals.
188. Furfuryl mercaptan
FEMA GRAS; found in coffee, beef, chicken, meat, popcorn; used in nonalcoholic
beverages, ice cream, ices, candy, baked goods, gelatins & puddings, icings.
189. 4-(2-Furyl)-3-buten-2-one
FEMA GRAS; found in coffee; used in nonalcoholic beverages, ice cream, ices,
candy, baked goods, gelatins & puddings, alcoholic beverages.
190. Galbanum oil and resinoid
FDA-approved food additive; FEMA GRAS; found in galbanum; used in meat
products, baked goods, ice cream.
191. Geraniol
FDA GRAS; FEMA GRAS; found in apples, apricot, beer, bilberry, black currants,
blackberries; used in baked goods, candy, ice cream.
192. Geranium rose and bourbon oil
FDA GRAS; FEMA GRAS; found in geranium leaves and stems; used in chewing
gum, ice cream, baked goods.
193. Geranyl acetate
FDA GRAS; FEMA GRAS; found in celery, cocoa, black currants, chervil, gin,
ginger, grape, grapefruit juice, orange juice, passion fruit, pineapple, plum; used in
baked goods, ice cream, syrups.
194. Geranyl butyrate
FDA-approved food additive; FEMA GRAS; found in celery, tomatoes, passion
fruit; used in gelatins & puddings, ice cream, candy.
For internal use of Philip Morris personnel only.
Appendices
327
195. Geranyl formate
FDA-approved food additive; FEMA GRAS; found in hops, black and green tea;
used in baked goods, ice cream, candy.
196. Ginger oil and oleoresin
FDA GRAS; FEMA GRAS; found in ginger; used in candy, baked goods, meats.
197. Glucose/ Dextrose
FDA GRAS; found in food sugar sources; used in baked goods, candy, breakfast
cereals.
198. l-Glutamic acid
FDA GRAS; FEMA GRAS; natural constituent of proteins in plants and animals;
used in baked goods, meat, soups, milk & dairy products, condiments, pickles, cereal.
199. Glycerol
FDA GRAS; FEMA GRAS; found in beer, cherry, wine; used in milk products,
baked goods, meat products.
200. Graphite
Carbons are used in air and water filtration systems. Carbons are also used as
clarifiers and decolorizers in the food and beverage industry. Graphite is a soft,
crystalline form of carbon. Graphite is a heat source constituent in cigarettes that
primarily heat tobacco.
201. Guaiac wood oil
FDA-approved food additive; FEMA GRAS; found in guaiac wood; used in meat
products, ice cream, chewing gum.
202. Guaiacol
FDA-approved food additive; FEMA GRAS; found in celery, cocoa, coffee, rum,
soybean, tea, tomato, whiskey, wine; used in ice cream, ices, baked goods, meat,
chewing gum, dairy products.
203. Guar gum
FDA GRAS; FEMA GRAS; found in the seed of the guar plant, which is similar to
the soybean plant; used in breakfast cereal, dairy products, gravies, processed vegetables, baked goods.
204. 2,4-Heptadienal
FEMA GRAS; found in avocado, beef, black currants, bread, wheat, broccoli, butter, heated butter, cabbage, cauliflower; used in meat products, baked goods, soups,
candy.
For internal use of Philip Morris personnel only.
328
Evaluation of Cigarette Ingredients: Feasibility
205. gamma-Heptalactone
FDA-approved food additive; FEMA GRAS; found in mango, passion fruit, peach,
black tea, asparagus, butter, hazelnut, lamb/mutton, leek, licorice, nectarine, papaya, pineapple; used in candy, baked goods, ice cream.
206. Heptanoic acid
FEMA GRAS; found in apple, beer, banana, beef, dried blue cheese, brandy, bread,
wheat, butter, cheddar cheese; used in baked goods, margarine, ice cream.
207. 2-Heptanone
FDA-approved food additive; FEMA GRAS; found in apples, banana, beer, beef,
apricot, asparagus, beans, blue cheese, wheat butter; used in gravies, ice cream,
condiments, baked goods.
208. 3-Hepten-2-one
FEMA GRAS; found in capsicum peppers, hazelnut, hops, muruci; used in gelatins
& puddings, ice cream, baked goods.
209. 4-Heptenal
FDA-approved food additive; FEMA GRAS; found in butterfat, soybean oil; used
in nonalcoholic beverages, baked goods, meat, candy, cereal, dairy products.
210. Heptyl acetate
FEMA GRAS; found in apples, grapes, bananas, pears, plums, whiskey; used in
baked goods, gelatins & puddings, chewing gum.
211. omega-6-Hexadecenlactone
FDA-approved food additive; FEMA GRAS; found in ambrette seed; used in baked
goods, ice cream, candy.
212. gamma-Hexalactone
FDA-approved food additive; FEMA GRAS; found in apricot, beef, butter, apple,
asparagus, beer, blackberry, brandy, wheat bread; used in candy, baked goods, ice
cream.
213. Hexanal
FDA-approved food additive; FEMA GRAS; found in apples, bananas, beef, bilberries, apricot, artichoke, asparagus, avocado, barley, beer, blackberry, blueberry,
others; used in frozen dairy goods, baked goods, meat.
214. Hexanoic acid
FDA-approved food additive; FEMA GRAS; found in apple, beef, beer, apricot,
banana, barley, blackberry, blue cheese, blueberry, bread, wheat; used in condiment
relish, ice cream, candy.
For internal use of Philip Morris personnel only.
Appendices
329
215. 2-Hexen-1-ol
FDA-approved food additive; FEMA GRAS; found in apples, apricots, bananas,
beer, Swiss cheese, peaches; used in nonalcoholic beverages, candy, baked goods,
gelatins & puddings.
216. 3-Hexen-1-ol
FDA-approved food additive; FEMA GRAS; found in apple, banana, bean, celery,
grape, apricot, cantaloupe, pineapple, honeydew melon; used in chewing gum, ice
cream, candy, baked goods.
217. cis-3-Hexen-1-yl acetate
FEMA GRAS; found in apple juice, apricot, artichoke, asparagus, avocado, banana, beans, beef, beer, blackberry, blueberry, dried bonito, grape brandy, wheat
bread; used in ices, candy, baked goods.
218. Hexen-2-al
FDA-approved food additive; FEMA GRAS; found in apple, banana, raspberry,
strawberry, beer, chicken, fat, grape, guava, hops, peach, pork, black and green tea;
used in frozen dairy goods, baked goods, candy.
219. 3-Hexenoic acid
FEMA GRAS; found in apples, bananas, beer, apple juice, artichoke, beans, beed,
blackberry, blueberry; used in beverages, candy, baked goods, gelatins & puddings,
frozen desserts.
220. trans-2-Hexenoic acid
FEMA GRAS; found in banana, pork fat, raspberry, black tea; used in nonalcoholic
beverages, ice cream, ices, dairy products, candy, gelatins & puddings, chewing
gum.
221. cis-3-Hexenyl formate
FEMA GRAS; found in tea, cognac; used in baked goods, chewing gum, preserves
and spreads.
222. Hexyl acetate
FDA-approved food additive; FEMA GRAS; found in apples, bananas, beer, apricot, beef, black currants, blackberry, blueberry, brandy, cantaloupe, capers, coffee;
used in candy, baked goods, meat products.
223. Hexyl alcohol
FDA-approved food additive; FEMA GRAS; found in apple, banana, beef, chicken,
coffee, pineapple, potato; used in baked goods, gelatins & puddings, dairy products.
For internal use of Philip Morris personnel only.
330
Evaluation of Cigarette Ingredients: Feasibility
224. Hexyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in grape, tea, scotch, spearmint, black and green tea; used in frozen dairy products, candy, baked goods.
225. High Fructose Corn Syrup
FDA GRAS; found in food sugar sources; used in baked goods, candy, breakfast
cereals.
226. Honey
Common food item; used in condiments, sauces, deli meats and salad dressings.
227. Hydrolyzed soy protein
Found in plants; used in baked goods, milk and dairy products, meat products, baby
formulas, soups, condiments and relishes.
228. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone
FEMA GRAS; found in beef, maple syrup, cassia oil; used in frozen dairy products,
baked goods, candy.
229. 2-Hydroxy-3,5,5-trimethyl-2-cyclohexenone
FEMA GRAS; found in rose oil, gardenia; used in frozen dairy goods, candy.
230. 4-Hydroxy-3-pentenoic acid lactone
FEMA GRAS; found in bread, grapes, soy beans; used in ice cream, ices, baked
goods, gelatins & puddings, meat, meat sauces, soups, milk & dairy products, cereals.
231. 4-Hydroxybutanoic acid lactone
FEMA GRAS; found in apricot, bread, coffee, cocoa, mushroom, onion, wine; used
in dairy products, breakfast cereals, meat products.
232. Hydroxycitronellal
FDA-approved food additive; FEMA GRAS; found in beef, mushroom, nectarine,
peach; used in candy, ice cream, baked goods.
233. 6-Hydroxydihydrotheaspirane
FEMA GRAS; found in black tea; used in nonalcoholic beverages, ice cream, candy,
gelatins & puddings.
234. 4-(para-Hydroxyphenyl)-2-butanone
FDA-approved food additive; FEMA GRAS; found in almond, beef, coffee, grape,
guava, hazelnut, pineapple, popcorn, raspberry, soy sauce, strawberry; used in chewing gum, ice cream, baked goods.
For internal use of Philip Morris personnel only.
Appendices
331
235. Hydroxypropyl cellulose
FDA-approved food additive; used in food as an emulsifier, film former, protective
colloid, stabilizer, suspending agent or thickener.
236. Immortelle absolute and extract
FDA GRAS; FEMA GRAS; used in baked goods, candy, gelatins & puddings,
chewing gum, frozen dairy products.
237. Invert sugar
FDA GRAS; found in food sugar sources; used in baked goods, candy, breakfast
cereals.
238. alpha-Ionone
FDA-approved food additive; FEMA GRAS; found in raspberry, almond, banana,
blackberry, grape brandy, raspberry brandy, capers, carrots, celery, cherry, grapefruit juice, kumazasa, mango ginger, peach, peas, plum; used in chewing gum, ice
cream, baked goods.
239. beta-Ionone
FDA-approved food additive; FEMA GRAS; found in carrot, almonds, apricot,
beer, blackberry, brandy, broccoli, capers, cherry, endive; used in candy, baked
goods, ice cream.
240. Isoamyl acetate
FDA-approved food additive; FEMA GRAS; found in apple, banana, beer, apricot,
blackberry, blackberry brandy, wheat bread, butter; used in chewing gum, ice cream,
baked goods.
241. Isoamyl benzoate
FDA-approved food additive; FEMA GRAS; found in beer, cherries, cocoa, papaya; used in baked goods, candy, gelatins & puddings.
242. Isoamyl butyrate
FDA-approved food additive; FEMA GRAS; found in banana, blue cheese, grape,
apple, apricot, beer, apple brandy, grape brandy, guava, honey, mango; used in nonalcoholic beverages, ice cream, baked goods, chewing gum.
243. Isoamyl cinnamate
FDA-approved food additive; FEMA GRAS; found in wine, cinnamon, styrax; used
in baked goods, candy, gelatins & puddings.
For internal use of Philip Morris personnel only.
332
Evaluation of Cigarette Ingredients: Feasibility
244. Isoamyl formate
FDA-approved food additive; FEMA GRAS; found in apple, beer, chicken, honey,
eggs, rum, strawberries, tea, vinegar, grape brandy, whiskey, wine; used in baked
goods, candy.
245. Isoamyl hexanoate
FDA-approved food additive; FEMA GRAS; found in apple, apricot, banana, grapefruit juice, plums, strawberries, beer, grape brandy, plum brandy, rum, sherry; used
in candy, chewing gum.
246. Isoamyl isovalerate
FDA-approved food additive; FEMA GRAS; found in banana, tomato, beer, sherry,
spearmint, scotch; used in frozen dairy goods, candy.
247. Isoamyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in peppermint oil; used in baked
goods, candy, chewing gum.
248. Isobutyl acetate
FDA-approved food additive; FEMA GRAS; found in apples, bananas, cantaloupe,
cocoa, figs, honeydew melon, beer, grape, guava, mango, melon; used in chewing
gum, gelatins & puddings.
249. Isobutyl alcohol
FDA-approved food additive; FEMA GRAS; found in beef, blackberry, apple, apricot, banana, barley, brandy; used in gelatin & puddings, candy, baked goods.
250. Isobutyl cinnamate
FDA-approved food additive; FEMA GRAS; found in coffee, tomato, mullein leaves;
used in candy, ice cream, baked goods.
251. Isobutyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in cocoa; used in baked goods,
ice cream, candy.
252. 2-Isobutyl-3-methoxypyrazine
FEMA GRAS; found in bean, coffee, pea, pepper, potato, spinach, grape; used in
nonalcoholic beverages, ice cream, dairy products.
253. alpha-Isobutylphenethyl alcohol
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, alcoholic beverages, ice cream, ices, candy, gelatins & puddings, baked goods.
For internal use of Philip Morris personnel only.
Appendices
333
254. Isobutyraldehyde
FDA-approved food additive; FEMA GRAS; found in apple, banana, barley, beans,
beef, beer, blue cheese, brandy, bread, wheat, butter; used in gelatins & puddings,
candy, frozen dairy products.
255. Isobutyric acid
FDA-approved food additive; FEMA GRAS; found in apple, beef, beer, celery,
banana, blue cheese, grape brandy, wheat bread, cashews, apples, cheddar cheese;
used in baked goods, candy, gelatins & puddings.
256. 2-Isopropylphenol
FEMA GRAS; found in Japanese whiskey; used in meat, soups, condiments.
257. Isovaleric acid
FDA-approved food additive; FEMA GRAS; found in apple, beer, banana, blue
cheese, grape brandy, wheat bread, heated butter, capers, cashews, apples, cheddar cheese; used in frozen dairy goods, candy, cheese.
258. Jasmine absolute
FDA GRAS; FEMA GRAS; found in jasmine flowers; used in baked goods, chewing gum, candy.
259. Kola nut extract
FDA GRAS; FEMA GRAS; found in kola nut; used in gelatins & puddings, ice
cream, candy.
260. Labdanum oils, absolute
FDA-approved food additive; FEMA GRAS; used in baked goods, frozen dairy
products, gelatins & puddings.
261. Lactic acid
FDA GRAS; FEMA GRAS; found in apple juice, beef, beer, bread, cocoa, coffee,
wheat bread, cherry, grape, guava, mango milk, papaya, dry salami, sherry, tomato;
used in cheese, candy, chewing gum, baked goods.
262. Lauric acid
FDA-approved food additive; FEMA GRAS; found in apple, beer, blue cheese,
banana, beef, blackberry, brandy, wheat bread, butter, heated butter, cantaloupe,
cashew nuts; used in baked goods, candy, ice cream.
263. Lavandin oil
FDA GRAS; FEMA GRAS; found in lavender plant; used in baked goods, candy,
chewing gum.
For internal use of Philip Morris personnel only.
334
Evaluation of Cigarette Ingredients: Feasibility
264. Lemon oil
FDA GRAS; FEMA GRAS; found in lemons; used in candy, breakfast cereals,
frozen dairy products.
265. Lemongrass oil
FDA GRAS; FEMA GRAS; found in lemongrass; used in chewing gum, ice cream,
baked goods.
266. Levulinic acid
FDA-approved food additive; FEMA GRAS; found in wheat bread, papaya; used
in reconstituted vegetables, ice cream, baked goods.
267. Licorice root, fluid extract and powder
FDA GRAS; FEMA GRAS; found in glycyrrhizia; used in candy, baked goods,
meat products.
268. Lime oil and lime oil terpeneless
FDA GRAS; FEMA GRAS; found in lime; used in frozen dairy goods, candy.
269. Linalool
FDA GRAS; FEMA GRAS; found in banana, beer, blackberry, beans, blueberry,
apple, apricot, arctice bramble, artichoke, grape brandy, plum brandy; used in meat
products.
270. Linalool oxide
FDA-approved food additive; FEMA GRAS; found in oranges, apricot, coffee;
used in ice cream, baked goods, candy.
271. Linalyl acetate
FDA GRAS; FEMA GRAS; found in bergamot, clary sage, lemon oil, pepper, tomato, lavender; used in baked goods, dairy products, candy.
272. L-Lysine
FDA-approved food additive; amino acid, natural constituent of plant and animal
proteins; used in meat products, breakfast cereals.
273. Lovage oil and extract
FDA-approved food additive; FEMA GRAS; found in levisticum; used in sweet
sauce, alcoholic beverages, ice cream.
274. Mace powder and oil
FDA GRAS; FEMA GRAS; found in mace; used in alcoholic beverages, candy,
frozen dairy products.
For internal use of Philip Morris personnel only.
Appendices
335
275. l-Malic acid
FDA GRAS; FEMA GRAS; found in celery, cocoa, orange juice, grape brandy,
sour cherry, gin, grapefruit juice, honey, hops, kiwi fruit, mango, mushroom; used in
frozen dairy goods, candy, baked goods.
276. Malt and malt extract
FDA GRAS; found in barley; used in beer, frozen dairy products, baked goods.
277. Maltodextrin
FDA GRAS; used in baked goods, candy, frozen dairy products.
278. Maltol
FDA-approved food additive; FEMA GRAS; found in barley, cocoa, coffee, beef,
wheat bread, butter, hazelnut, licorice, malt, milk, peanut; used in frozen dairy goods,
jellies, baked goods.
279. Mandarin and tangerine oil
FDA GRAS; FEMA GRAS; found in tangerines, mandarin oranges; used in candy,
frozen dairy products.
280. Maple syrup
Common food item; used in sauces, dressings, frostings, icings, candies and desserts.
281. Mate leaf, absolute, extract and oil
FDA GRAS; found in mate leaves; used in flour, meat, poultry.
282. para-Mentha-8-thiol-3-one
FEMA GRAS; used in frozen dairy, soft candy, gelatins & puddings, baked goods.
283. Menthol and L-Menthol
FDA-approved food additive; FEMA GRAS; found in peppermint plant, honey,
mint, rum, cocoa, eggs, guava, raspberry, rice, spearmint; used in candy, mouthwash.
284. Menthone
FDA-approved food additive; FEMA GRAS; found in celery, clams, cocoa, peppermint, raspberries, rice, spearmint; used in baked goods, candy.
285. Menthyl Acetate
FEMA GRAS; found in peppermint oil, orange juice, raspberries; used in baked
goods, frozen dairy, soft candy, gelatins & pudding.
For internal use of Philip Morris personnel only.
336
Evaluation of Cigarette Ingredients: Feasibility
286. Menthyl Isovalerate
FDA-approved food additive; FEMA GRAS; found in nutmeg, peppermint oil; used
in alcoholic beverages, baked goods, chewing gum, frozen dairy goods, gelatins &
puddings, soft candy.
287. 2-,5-, or 6-Methoxy-3-methylpyrazine (mixture of isomers)
FEMA GRAS; found in beef, coffee, bread; used in baked goods, cereals, candy,
dairy products.
288. 2-Methoxy-4-methylphenol
FDA-approved food additive; FEMA GRAS; found in cocoa, sausage, banana,
beer, coffee; used in baked goods, meat products.
289. 2-Methoxy-4-vinylphenol
FDA-approved food additive; FEMA GRAS; found in bean, coffee, sherry, whiskey, banana, beer, cocoa, cured ham, malt; used in meat products, ice cream, baked
goods.
290. para-Methoxybenzaldehyde
FDA-approved food additive; FEMA GRAS; found in coffee, tea, tomato, beer,
krill; used in baked goods, candy, dairy products.
291. 1-(para-Methoxyphenyl)-1-penten-3-one
FDA-approved food additive; FEMA GRAS; found in jasmine, ylang ylang; used in
soft candy, sweet sauce, baked goods.
292. 1-(para-Methoxyphenyl)-2-propanone
FDA-approved food additive; FEMA GRAS; found in chervil; used in baked goods,
frozen dairy products, soft candy, gelatins & puddings.
293. Methoxypyrazine
FEMA GRAS; found in beef, cocoa; used in meat products, soups, gravies, baked
goods.
294. Methyl 2-furoate
FEMA GRAS; found in almond, cocoa, coffee, filbert, peanut, wine; used in nonalcoholic beverages, ice cream, candy, baked goods.
295. Methyl 2-octynoate
FDA-approved food additive; FEMA GRAS; used in baked goods, frozen dairy
products, gelatins & puddings.
For internal use of Philip Morris personnel only.
Appendices
337
296. Methyl 2-pyrrolyl ketone
FEMA GRAS; found in apple juice, cocoa, coffee, onion, peanut; used in baked
goods, candy, gelatins & puddings, meat products.
297. para-Methyl anisate
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, frozen dairy products, baked goods, soft candy, gelatins & puddings.
298. Methyl anthranilate
FDA GRAS; FEMA GRAS; found in cocoa, grape, tea, wine, coffee, strawberry;
used in baked goods, candy, frozen dairy products.
299. Methyl benzoate
FDA-approved food additive; FEMA GRAS; found in banana, cherry, coffee,
brandy, butter, cashew, apple; used in chewing gum, ice cream, candy.
300. Methyl cinnamate
FDA-approved food additive; FEMA GRAS; found in guava, strawberry, cranberry, pineapple, plum; used in baked goods, candy, gelatins & puddings.
301. Methyl dihydrojasmonate
FEMA GRAS; used in nonalcoholic beverages, ice cream, ices, baked goods, candy.
302.Methyl ester of rosin, partially hydrogenated
FDA-approved food additive; used in baked goods, candy.
303. Methyl isovalerate
FDA-approved food additive; FEMA GRAS; found in apple, peach, pineapple, banana, blackberry, parmesan cheese, coffee, honey, nectarine, olives, peas, strawberries; used in candy, baked goods.
304. Methyl linoleate (48%) methyl linolenate (52%) mixture
FEMA GRAS; found in banana, grape, grapefruit juice, melon, strawberries; used
in ice cream, baked goods, candy.
305. Methyl phenylacetate
FDA-approved food additive; FEMA GRAS; found in coffee, cocoa; used in candy,
syrups, baked goods.
306. Methyl salicylate
FEMA GRAS; found in blackberry, broccoli, butter, cherry cake, coffee; used in
candy, ice cream, syrups.
For internal use of Philip Morris personnel only.
338
Evaluation of Cigarette Ingredients: Feasibility
307. Methyl sulfide
FDA-approved food additive; FEMA GRAS; found in asparagus, bean, beef, beer,
white bread, brussel sprouts, butter, cabbage, carrot, cauliflower, broccoli; used in
meat products, candy, ice cream.
308. 5-Methyl-2-phenyl-2-hexenal
FEMA GRAS; found in Romano cheese, potato chips; used in soft candy.
309. 6-Methyl-3,5-heptadien-2-one
FDA-approved food additive; FEMA GRAS; found in almonds, asparagus, beef,
beer, wheat bread, cashew nuts, chicken; used in snack foods.
310. 1-Methyl-3-methoxy-4-isopropylbenzene
FEMA GRAS; found in tangerine peel, thyme; used in nonalcoholic beverages,
baked goods, meat, candy, condiments.
311. Methyl-3-methylthiopropionate
FDA-approved food additive; FEMA GRAS; found in cantaloupe, pineapple; used
in candy, baked goods, beverages, syrups.
312. 2-Methyl-4-phenylbutyraldehyde
FEMA GRAS; used in nonalcoholic beverages, ice cream, candy, gelatins & puddings.
313. 6-Methyl-5-hepten-2-one
FDA-approved food additive; FEMA GRAS; found in guava, mango, potato, rum;
used in gravies, candy, baked goods.
314. 4-Methyl-5-thiazoleethanol
FEMA GRAS; found in cocoa, malt, roasted peanuts; used in meat products.
315. 4'-Methylacetophenone
FDA-approved food additive; FEMA GRAS; found in hop oil, cocoa powder, black
currant; used in chewing gum.
316. Methyl-alpha-ionone
FDA-approved food additive; FEMA GRAS; used in baked goods.
317. para-Methyl-anisole
FDA-approved food additive; FEMA GRAS; found in cocoa, malt, peanut, pork,
potato chips, sesame seeds; used in baked goods.
For internal use of Philip Morris personnel only.
Appendices
339
318. 2-Methylbutyraldehyde
FDA-approved food additive; FEMA GRAS; found in beef, apple, cheddar cheese,
coffee, cranberry, eggs, fish, lettuce, olive, onion, peas, tomato; used in gelatins &
puddings.
319. 3-Methylbutyraldehyde
FDA-approved food additive; FEMA GRAS; found in apple, banana, bread, tomato, rice, blackberry; used in baked goods.
320. 2-Methylbutyric acid
FDA-approved food additive; FEMA GRAS; found in apple, apricot, avocado, beef,
beer, blackberry, brandy, butter, cantaloupe, carrots; used in cheese, ice cream,
candy.
321. alpha-Methylcinnamaldehyde
FDA-approved food additive; FEMA GRAS; found in blackberry, cauliflower,
cherry, cocoa, endive, guava, honey, peach; used in candy.
322. Methylcyclopentenolone
FDA-approved food additive; FEMA GRAS; found in carambola (starfruit), cheese,
tomato; used in breakfast cereals, baked goods, candy.
323. 2-Methylheptanoic acid
FDA-approved food additive; FEMA GRAS; found in gardenia flower oil, almonds,
cocoa, coffee, soy sauce, onions; used in baked goods, ice cream, candy.
324. 2-Methylhexanoic acid
FEMA GRAS; found in apple, avocado, banana, barley, beans, beef, beer, blackberry, blue cheese, wheat bread, butter; used in baked goods, candy, gelatins &
puddings.
325. 3-Methylpentanoic acid
FEMA GRAS; found in apple, apricot, beer, blackberry, blueberry, wheat bread,
Romano cheese, chicken; used in baked goods.
326. (Methylthio)methylpyrazine (mixture of isomers)
FEMA GRAS; used in baked goods, candy.
327. 2-Methylpyrazine
FEMA GRAS; found in peppermint oil, tomato, popcorn; used in milk products,
baked goods, candy.
For internal use of Philip Morris personnel only.
340
Evaluation of Cigarette Ingredients: Feasibility
328. 5-Methylquinoxaline
FEMA GRAS; found in roasted almonds, coffee; used in frozen dairy, beverages,
candy, gelatin.
329. 3-(Methylthio)propionaldehyde
FEMA GRAS; found in bean, bread, cheese, cocoa bean, roasted nuts, milk, soy
sauce, tomato; used in ice cream, ices, baked goods, dairy products, fats/oils.
330. Methyl-trans-2-butenoic acid
FEMA GRAS; found in celery oil, orange juice crystals, coffee, strawberry; used in
baked goods, meat products, soups.
331. 2-Methylvaleric acid
FDA-approved food additive; FEMA GRAS; found in almond, barley, wheat bread,
cocoa, coffee, hazelnut, licorice, malt, peanut, soy sauce, lamb/mutton; used in frozen dairy goods, candy.
332. Mimosa absolute
FDA-approved food additive; FEMA GRAS; found in mimosa flowers; used in
frozen dairy products.
333. Molasses, blackstrap, sugarcane and extract
FDA GRAS; found in refined sugars; common food item.
334. Mountain maple solid extract
FDA-approved food additive; FEMA GRAS; found in mountain-maple-tree sap;
used in baked goods.
335. Myristic acid
FDA-approved food additive; FEMA GRAS; found in apple, banana, beef, beer,
blackberry, brandy grape, butter, cantaloupe, cashew nuts, cheese (blue, cheddar);
used in nonalcoholic beverages, candy, baked goods.
336. Myrrh oil and absolute
FDA-approved food additive; FEMA GRAS; found in myrrh; used in alcoholic
beverages.
337. Nerol
FDA-approved food additive; FEMA GRAS; found in apricot, beer, blackberry,
blueberry, brandy grape, cranberry, gin, grape, grapefruit juice, honey, hops, wine;
used in frozen dairy goods.
For internal use of Philip Morris personnel only.
Appendices
341
338. Neroli bigarade oil
FDA GRAS; FEMA GRAS; found in oranges; used in baked goods, candy.
339. Nerolidol
FDA-approved food additive; FEMA GRAS; found in grapefruit, hops, lime, grapefruit oil; used in nonalcoholic beverages, ice cream, ices, candy, baked goods.
340. Nona-2-trans,6-cis-dienal
FEMA GRAS; found in apple, banana, beef, beer, blue cheese, cheddar cheese,
brandy plum; used in frozen dairy goods.
341. 2,6-Nonadien-1-ol
FDA-approved food additive; FEMA GRAS; found in cucumber, frozen peas, whole
soybean, tomato; used in baked goods, candy, gelatins & puddings, gravies.
342. gamma-Nonalactone
FDA-approved food additive; FEMA GRAS; found in beer, wheat bread, capers,
cherry, chicken, clam; used in candy, baked goods, ice cream.
343. Nonanal
FDA-approved food additive; FEMA GRAS; found in apricot, asparagus, beef,
blackberry, wheat bread, cantaloupe, cocoa; used in baked goods.
344. Nonanoic acid
FDA-approved food additive; FEMA GRAS; found in apple, apricot, artichoke,
avocado, banana, beef, beer, wheat bread; used in baked goods, candy, meat products.
345. 2-Nonanone
FDA-approved food additive; FEMA GRAS; found in strawberry; used in dairy
products, condiments.
346. trans-2-Nonen-1-ol
FEMA GRAS; found in asparagus, brandy grape, cucumber, melon, nectarine, plum,
prickly pear, wine; used in baked goods.
347. Nonyl alcohol
FDA-approved food additive; FEMA GRAS; found in apples, bananas, and oranges; used in baked goods, soft candy, frozen dairy, and chewing gum.
348. Nutmeg powder and oil
FDA GRAS; FEMA GRAS; found in nutmeg; used in condiments, baked goods.
For internal use of Philip Morris personnel only.
342
Evaluation of Cigarette Ingredients: Feasibility
349. Oak moss absolute
FDA-approved food additive; FEMA GRAS; found in essential oil of lichen, oak
moss; used in meat products, candy, ice cream.
350. 9,12-Octadecadienoic acid (48%) and 9,12,15-octadecatrienoic acid (52%)
(mixture) FEMA GRAS; found in potato, tomato, apple, beer, cheese, country ham;
used in preserves, spreads, candy, gelatins & puddings.
351. delta-Octalactone
FEMA GRAS; found in apricot, beef, blackberry, butter, cheese (blue, cheddar,
parmesan), cranberry, cream, coconut; used in candy, margarine, baked goods.
352. gamma-Octalactone
FDA-approved food additive; FEMA GRAS; found in apricot, asparagus, beef,
beer, blackberry, blue cheese, brandy grape, cantaloupe, cherry, chicken, cranberry;
used in baked goods, candy, ice cream.
353. Octanal
FDA-approved food additive; FEMA GRAS; found in apple, apricot, artichoke,
avocado, beef, beer, blackberry, brandy, wheat bread, butter; used in frozen dairy
products, beverages, baked goods, candy.
354. Octanoic acid
FDA GRAS; FEMA GRAS; found in apple, banaa, beef, blackberry, plum brandy,
wheat butter, beer, blue cheese; used in snack foods, baked goods, candy.
355. 2-Octanone
FDA-approved food additive; FEMA GRAS; found in apple, banana, beef, cheese,
coffee, cocoa, milk, peanut, tea, wine; used in baked goods, candy, gelatins & puddings, dairy products.
356. 1-Octen-3-ol
FDA-approved food additive; FEMA GRAS; found in mushroom, peppermint,
spearmint; used in processed vegetables.
357. 2, trans-Octenal
FEMA GRAS; found in asparagus, beer, banana, beans, blue cheese, butter, cantaloupe, capers, chicken; used in snack foods, baked goods, dairy products.
358. Octyl isobutyrate
FDA-approved food additive; FEMA GRAS; found in hops, plum; used in baked
goods, beverages, ice cream, candy.
For internal use of Philip Morris personnel only.
Appendices
343
359. Oleic acid
FDA-approved food additive; FEMA GRAS; found in apple, banana, grape, ginger,
potato, strawberry, tomato; used in condiment relish, citrus fruit, yeast, sugar beets.
360. Olibanum oil
FDA-approved food additive; FEMA GRAS; found in gum-resin exudate; used in
nonalcoholic beverages, ice cream, ices, candy, baked goods.
361. Opoponax oil
FDA-approved food additive; found in opoponax, opoponas; natural flavor; used in
alcoholic beverages.
362. Orange leaf absolute
FDA GRAS; FEMA GRAS; found in oranges; used in nonalcoholic beverages, ice
cream, ices, candy, baked goods.
363. Orange Oil (sweet orange oils terpeneless, sour/ bitter orange oils)
FDA GRAS; FEMA GRAS; found in oranges; used in nonalcoholic beverages, ice
cream, ices, baked goods, condiments, candy, gelatins & puddings, chewing gum.
364. Orange oil and extract (Sweet, distilled orange oils and terpeneless)
FDA GRAS; FEMA GRAS; found in oranges; used in nonalcoholic beverages, ice
cream, ices, baked goods, condiments, candy, gelatins & puddings, chewing gum.
365. Origanum oil
FDA GRAS; FEMA GRAS; found in origanum flowers; used in soups.
366. Orris concrete oil and root extract
FDA-approved food additive; FEMA GRAS; found in orris roots; used in gelatins
& puddings, alcoholic beverages.
367. Palmarosa oil
FDA GRAS; FEMA GRAS; found in geranium; used in baked goods.
368. Palmitic acid
FDA-approved food additive; FEMA GRAS; found in apple, beer, celery, cheddar
cheese, milk, potato, tomato; used in meat products, baked goods.
369. Parsley seed oil
FDA GRAS; FEMA GRAS; found in parsley; used in soups.
For internal use of Philip Morris personnel only.
344
Evaluation of Cigarette Ingredients: Feasibility
370. Patchouly oil and absolute
FDA-approved food additive; FEMA GRAS; found in dried leaves of Pogostemon
cablin Benth; used in nonalcoholic beverages, ice cream, ices, baked goods, candy,
chewing gum.
371. Pectin
FDA GRAS; found in plants; used in frozen desserts, canned fruits, jellies, preserves.
372. omega-Pentadecalactone
FDA-approved food additive; FEMA GRAS; used in baked goods, ice cream, candy.
373. 2,3-Pentanedione
FDA-approved food additive; FEMA GRAS; found in nuts, beef, beer, bread,
chicken, cocoa, coffee, tomato, yogurt; used in baked goods, candy, gelatins &
puddings.
374. 2-Pentanone
FDA-approved food additive; FEMA GRAS; found in apple juice, banana, beef,
butter, oil, cheese, chicken, grape, ham, honey, peanut; used in nonalcoholic beverages, ice cream, ices, candy, baked goods.
375. Pepper oil, black
FDA GRAS; FEMA GRAS; found in pepper corns; used in condiments, ice cream,
baked goods.
376. Peppermint oil and peppermint oil terpeneless
FDA GRAS; FEMA GRAS; found in peppermint; used in chewing gum, meat
products, ice cream, baked goods.
377. Petitgrain absolute, oil, and terpeneless oil
FDA GRAS; FEMA GRAS; found in bitter orange tree, leaves, twigs, oranges;
used in baked goods, condiments, candy.
378. alpha-Phellandrene
FDA-approved food additive; FEMA GRAS; found in apple, gin, hops, mango,
nectarine, papaya, paprika, parsley, beans, carrots; used in milk products, baked
goods, candy.
379. 2-Phenethyl acetate
FDA-approved food additive; FEMA GRAS; found in apple, banana, beer, brandy,
raspberry, wheat bread, butter, cantaloupe; used in candy, ice cream, baked goods.
For internal use of Philip Morris personnel only.
Appendices
345
380. Phenethyl alcohol
FDA-approved food additive; FEMA GRAS; found in apple juice, banana, beef,
beer, blackberry, blueberry, apple, apricot, asparagus; used in chewing gum, ice
cream, baked goods.
381. Phenethyl butyrate
FDA-approved food additive; FEMA GRAS; found in beer, banana, apple brandy,
grape, strawberry, wine; used in baked goods, candy, ice cream.
382. Phenethyl cinnamate
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, alcoholic beverages, ice cream, ices, baked goods, candy, gelatins & puddings.
383. Phenethyl isobutyrate
FDA-approved food additive; FEMA GRAS; found in beer, brandy, grape, olive,
rum; used in chewing gum, baked goods, candy.
384. Phenethyl isovalerate
FDA-approved food additive; FEMA GRAS; found in peppermint, spearmint, banana, beer, brandy, grape; used in chewing gum, candy, frozen dairy products.
385. Phenethyl phenylacetate
FDA-approved food additive; FEMA GRAS; used in baked goods, candy, cheese.
386. 3-Phenyl-1-propanol
FDA-approved food additive; FEMA GRAS; found in cinnamon, honey, tea; used
in frozen dairy, baked goods, soft candy, gelatins & puddings, chewing gum.
387. 2-Phenyl-2-butenal
FEMA GRAS; found in beer, chicken, tomato, almonds, asparagus, cocoa, tea,
hazelnuts; used in gelatin and puddings, candy, ice cream.
388. 4-Phenyl-3-buten-2-one
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, frozen dairy, baked goods, soft candy; gelatins & puddings.
389. Phenylacetaldehyde
FDA-approved food additive; FEMA GRAS; found in chicken, strawberry; used in
baked goods, ice cream, candy.
390. Phenylacetic acid
FDA-approved food additive; FEMA GRAS; found in almond, asparagus, cocoa,
coffee, mushroom, peanut, pork, potato chips, sesame seed, tea; used in sweet
sauce, baked goods, candy.
For internal use of Philip Morris personnel only.
346
Evaluation of Cigarette Ingredients: Feasibility
391. l-Phenylalanine
FDA-approved food additive; FEMA GRAS; found in meats, eggs, breads, cereals,
milk, cheese, fish, corn, beans, potatoes, asparagus, peas; used in frozen dairy,
baked goods, candy, condiments, meat products.
392. 3-Phenylpropionaldehyde
FDA-approved food additive; FEMA GRAS; found in beer, chicken, tomato; used
in baked goods, candy, condiments.
393. 3-Phenylpropionic acid
FDA-approved food additive; FEMA GRAS; found in beef, beer, blue cheese, grape
brandy, wheat bread, broccoli, apricot, artichoke, asparagus, banana, beans; used in
baked goods, candy.
394. 3-Phenylpropyl acetate
FDA-approved food additive; FEMA GRAS; used in nonalcoholic beverages, ice
cream, baked goods, candy, chewing gum, condiments.
395. Phosphoric acid
FDA GRAS; FEMA GRAS; component of living organisms; used in cheese, baked
goods, candy, gelatins & puddings, meat products.
396. Pine needle oil
FDA-approved food additive; FEMA GRAS; found in pine tree needles; used in
candy, baked goods, ice cream.
397. Pine oil, scotch
FDA-approved food additive; FEMA GRAS; found in pine trees; used in candy,
baked goods, nonalcoholic beverages.
398. Pineapple juice concentrate
Found in pineapple; defined as a fruit juice under FDA Standards of Identity.
399. alpha-Pinene
FDA-approved food additive; FEMA GRAS; found in apple, blueberry, plum brandy,
carrots, celery, cheddar cheese, chicken; used in condiments, candy, meat products.
400. beta-Pinene
FDA-approved food additive; FEMA GRAS; found in apricot, plum brandy, butter,
cantaloupe, carrots, celery, cheddar cheese, cocoa, cranberry; used in baked goods,
candy, meat products.
For internal use of Philip Morris personnel only.
Appendices
347
401. Piperonal
FDA GRAS; FEMA GRAS; found in cantaloupe, capers, melon, sherry; used in
candy, baked goods.
402. Pipsissewa leaf extract
FDA GRAS; FEMA GRAS; used in nonalcoholic beverages, candy.
403. Plum juice, concentrate and extract
Found in plums.
403. Potassium carbonate
FDA GRAS; used in wine and juice prior to or during fermentation. Potassium
carbonate is a flavor enhancer that facilitates the release of natural tobacco aroma
and flavor.
405. Potassium sorbate
FDA GRAS; FEMA GRAS; found in mountain-ash-tree berries; used in cheese.
406. l-Proline
FDA-approved food additive; FEMA GRAS; essential amino acid, found in proteins, plants and animals; used in breakfast cereals, baked goods.
407. Propenylguaethol
FDA-approved food additive; FEMA GRAS; used in sweet sauce, baked goods,
candy.
408. Propionic acid
FDA GRAS; FEMA GRAS; found in apple, apple juice, beef, beer, blueberry juice,
bread, cheese, coffee, grape juice, maple syrup, orange juice, raspberry, rum; used
in fruit, candy, gelatins & puddings, dairy products.
409. Propyl acetate
FDA-approved food additive; FEMA GRAS; found in banana, grape, apple juice,
beer, wheat bread, cantaloupe, capers, cocoa, guava, honey, fig, honeydew, melon,
heated corn oil; used in beverages, ice cream, baked goods.
410. Propyl para-hydroxybenzoate
FDA-approved food additive; FEMA GRAS; found in licorice; used in processed
vegetables.
411. Propylene glycol
FDA GRAS; FEMA GRAS; found in sesame seed, mushroom; used in confection
frostings, cheese, candy.
For internal use of Philip Morris personnel only.
348
Evaluation of Cigarette Ingredients: Feasibility
412. 3-Propylidenephthalide
FDA-approved food additive; FEMA GRAS; found in lovage; used in frozen dairy
products, baked goods, candy.
413. Prune juice and concentrate
Common food item; found in prunes; used in beverages, bread and other bakery
products.
414. Pyridine
FDA-approved food additive; FEMA GRAS; found in bean, bread, cheese, cocoa,
coffee, fish, onion, peanut, pecan, popcorn, potato, rum, tea, tomato; used in ice
cream, baked goods, condiments, meat products.
415. Pyroligneous acid and extract
FDA-approved food additive; FEMA GRAS; found in birch tree; used in alcoholic
beverages, baked goods, meat products.
416. Pyruvic acid
FDA-approved food additive; FEMA GRAS; found in beer, wheat bread, celery,
asparagus, milk, onion, sake; used in frozen dairy products, baked goods, candy.
417. Raisin juice concentrate and extract
Common food item; found in raisins; used in baked goods.
418. l-Rhamnose
FEMA GRAS; found in food-sugar sources; used in baked goods, candy, breakfast
cereals.
419. Rhodinol
FDA-approved food additive; FEMA GRAS; found in geranium flowers; used in
chewing gum, baked goods, ice cream.
420. Rose absolute and oil
FDA GRAS; FEMA GRAS; found in roses; used in chewing gum, ice cream,
baked goods.
421. Rosemary oil and extract
FDA GRAS; FEMA GRAS; found in rosemary; used in condiments, meat products, baked goods.
422. Rum and rum extract
Alcoholic beverages; natural flavor.
For internal use of Philip Morris personnel only.
Appendices
349
423. Rum ether
FDA-approved food additive; FEMA GRAS; found in rum; used in nonalcoholic
beverages, alcoholic beverages, frozen dairy products, baked goods, candy.
424. Rye Extract
Found in rye plant; used in rye bread.
425. Sage oil, oleoresin, and powder
FDA GRAS; FEMA GRAS; found in sage; used in baked goods, condiments, meat
products.
426. Salicylaldehyde
FDA-approved food additive; FEMA GRAS; found in beer, butter, chicken, coffee,
cranberry, grape, potato, rum, sherry, tea, tomato, whiskey; used in baked goods,
condiments, candy.
427. Sandalwood oil, yellow
FDA-approved food additive; FEMA GRAS; found in sandalwood; used in candy,
baked goods, ice cream.
428. Sclareolide
FEMA GRAS; found in clary sage; used in milk products, baked goods, candy,
meat products, breakfast cereals.
429. Sodium benzoate
FDA GRAS; FEMA GRAS; used in baked goods, margarine, dietary supplements.
430. Sodium bicarbonate
FDA GRAS; natural mineral; main component of baking powder and baking soda.
431.Sodium carbonate
FDA GRAS; used in baked goods, desserts, margarine, poultry. Sodium carbonate
is a heat source constituent in cigarettes that primarily heat tobacco.
432. Sodium citrate
FDA GRAS; FEMA GRAS; used in evaporated milk, general purpose food
additive.
433. Sorbic acid
FDA GRAS; found in mountain-ash-tree berries (Merck); used in flavorings.
434. d-Sorbitol
FDA GRAS; FEMA GRAS; found in cherry, plum, apple; used in soft candy, chewing
gum, frozen dairy desserts.
For internal use of Philip Morris personnel only.
350
Evaluation of Cigarette Ingredients: Feasibility
435. Spearmint oil
FDA GRAS; FEMA GRAS; found in spearmint; used in chewing gum, ice cream,
candy, baked goods.
436. Storax and styrax, extract, gum, and oil
FDA-approved food additive; FEMA GRAS; found in storax; used in baked goods,
candy, jellies.
437. Sucrose
FDA GRAS; found in food sugar sources; used in baked foods, candy, breakfast
cereals.
438. Sucrose octaacetate
FDA-approved food additive; FEMA GRAS; found in ginger ale; used in candy,
gelatins & puddings, baked goods.
439. Tagetes oil
FDA-approved food additive; FEMA GRAS; found in marigold flowers; used in
condiment relish.
440. l-Tartaric acid, dl-Tartaric acid
FDA GRAS; FEMA GRAS; found in wine grapes; used in fruit juices, baked goods,
ice cream.
441. Tea extract
FDA GRAS; natural food extractive.
442. alpha-Terpineol
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, apricot,
artichoke, beans, beef, beli, bilberry, blueberry, plum brandy; used in chewing gum,
baked goods, ice cream.
443. Terpinolene
FDA-approved food additive; FEMA GRAS; found in thyme, valencia oranges;
used in baked foods, ice cream, candy.
444. alpha-Terpinyl acetate
FDA-approved food additive; FEMA GRAS; found in apricot, beer, blackberry,
carrots, celery, cranberry, gin, ginger; used in meat products, baked goods, candy.
445. 5,6,7,8-Tetrahydroquinoxaline
FEMA GRAS; found in beef, wheat bread, cocoa, peanut, pork; used in candy,
baked goods, dairy products.
For internal use of Philip Morris personnel only.
Appendices
351
446. 1,5,5,9-Tetramethyl-13-oxatricyclo (8.3.0.0(4,9)) tridecane
FEMA GRAS; found in clary sage oil; used in nonalcoholic beverages, ice cream,
ices, baked goods, candy, gelatins & puddings.
447. 2,3,5,6-Tetramethylpyrazine
FEMA GRAS; found in wheat bread, sake, shrimp, beef, beer, coffee, peanut; used
in baked goods, dairy products, candy.
448. Thyme oil
FDA GRAS; FEMA GRAS; found in thyme; used in condiments, meats, soups,
baked goods.
449. Thymol
FDA-approved food additive; FEMA GRAS; found in blueberry, Romano cheese,
papaya, peppermint, pistacia, fruit tea, wine; used in candy, baked goods, ice cream.
450. Tolu balsam gum, resinoid, and extract
FDA-approved food additive; FEMA GRAS; found in balsam tolu; used in baked
goods, candy, syrups.
451. Tolualdehydes (ortho,meta,para)
FDA-approved food additive; FEMA GRAS; found in beef, beer, butter, coffee,
endive, rum, tea; used in chewing gum, baked goods, ice cream.
452. para-Tolyl 3-methylbutyrate
FEMA GRAS; found in raspberry, coffee, tea, rum; used in baked goods, ice cream,
candy.
453. para-Tolyl acetate
FDA-approved food additive; FEMA GRAS; found in cananga, ylang ylang; used
in candy, ice cream, baked goods.
454. para-Tolyl isobutyrate
FDA-approved food additive; FEMA GRAS; used in baked goods, ice cream, candy.
455. para-Tolyl phenylacetate
FDA-approved food additive; FEMA GRAS; used in baked goods, candy, cheese,
ice cream.
456. Triacetin
FDA GRAS; FEMA GRAS; found in papaya; used in candy, baked goods, ice
cream.
For internal use of Philip Morris personnel only.
352
Evaluation of Cigarette Ingredients: Feasibility
457. 2-Tridecanone
FEMA GRAS; found in cheese, coffee, coconut oil, hops; used in ice cream, ices,
baked goods, margarine, candy, gelatins & puddings.
458. Triethyl citrate
FDA GRAS; FEMA GRAS; found in red currant; used in chewing gum, baked
goods, ice cream.
459. 3,5,5-Trimethyl-1-hexanol
FEMA GRAS; used in baked goods, condiments, pickles.
460. 4-(2,6,6-Trimethylcyclohex-1-enyl)but-2-en-4-one
FEMA GRAS; found in rose, rum, brandy, tea; used in baked goods, candy, chewing gum.
461. 2,6,6-Trimethylcyclohex-2-ene-1,4-dione
FEMA GRAS; found in apricot, beer, blackberry, grape, hops, kiwi fruit; used in
soft candy.
462. 4-(2,6,6-Trimethylcyclohexa-1,3-dienyl)but-2-en-4-one
FEMA GRAS; found in apples, black tea, Riesling wine; used in beverages, frozen
dessert, baked goods, candy, gelatins & puddings, preserves, condiments.
463. 2,2,6-Trimethylcyclohexanone
FEMA GRAS; found in bilberry, passion fruit, tea; used in nonalcoholic beverages,
frozen dessert, confectionery, ice cream, ices, candy.
464. 2,3,5-Trimethylpyrazine
FEMA GRAS; found in barley, almond, asparagus, beef, wheat bread, chicken,
cocoa, coffee; used in baked goods, candy, dairy products, cereals.
465. delta-Undecalactone
FEMA GRAS; found in beef, butter, coconut, milk; used in baked goods, candy,
dairy products, cereals.
466. gamma-Undecalactone
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, apricot,
heated butter, peach, plum, pork, rice; used in chewing gum, candy, ice cream,
baked goods.
467. 2-Undecanone
FDA-approved food additive; FEMA GRAS; found in coconut oil, banana, beer,
beef, cheese, cocoa, coffee, wine, milk, mushroom, peanut, strawberry; used in
nonalcoholic beverages, ice cream, baked goods, candy, dairy products.
For internal use of Philip Morris personnel only.
Appendices
353
468. Urea
FDA GRAS; found in mushrooms; used in baked goods.
469. Valeraldehyde
FDA-approved food additive; FEMA GRAS; found in apple, apple juice, apricot,
artichoke, asparagus, avocado, banana, beef, blue cheese; used in candy, baked
goods, ice cream.
470. Valerian root extract, oil and powder
FDA-approved food additive; FEMA GRAS; found in valerian root; used in baked
goods, chewing gum.
471. Valeric acid
FDA-approved food additive; FEMA GRAS; found in banana, beef, beer, blue
cheese, blueberry, wheat bread, butter; used in imitation dairy goods.
472. gamma-Valerolactone
FEMA GRAS; found in beef, beer, cocoa, coffee, mushroom, peach, peanut, wheat
bread, heated butter, honey; used in candy, meat products, baked goods.
473. l-Valine
FDA-approved food additive; FEMA GRAS; found in plants, lemons, oranges,
grapefruits; used in ice cream, candy, baked goods.
474. Vanilla extract and oleresin
FDA GRAS; FEMA GRAS; found in vanilla bean; used in baked goods, gelatins &
puddings, condiments.
475. Vanillin
FDA GRAS; FEMA GRAS; found in asparagus, barley, beer, brandy, blackberry,
blueberry, coffee, cranberry; used in confection frosting, baked goods, candy.
476. Veratraldehyde
FDA-approved food additive; FEMA GRAS; found in coffee, raspberry; used in
baked goods, ice cream, candy.
477. Vetiver oil
FDA-approved food additive; found in vetiver flowers.
478. Violet leaf absolute
FDA GRAS; FEMA GRAS; found in violets; used in baked goods, ice cream,
candy.
For internal use of Philip Morris personnel only.
354
Evaluation of Cigarette Ingredients: Feasibility
479. Walnut hull extract
FDA-approved food additive; FEMA GRAS; found in walnuts; used in breakfast
cereals, ice cream, candy.
480. Water
Common food item.
481. Wheat absolute
Common food component.
482. Wine and wine sherry
Common beverages, found in grape fermentation/distillation.
483. Ylang ylang oil
FDA GRAS; FEMA GRAS; used in baked goods, soft candy, frozen diary, and
chewing gum.
For internal use of Philip Morris personnel only.
Appendices
355
APPENDIX E
LEGAL DEFINITION OF “CIGARETTE”
The Food and Drug Administration and the Bureau of Alcohol, Tobacco and Firearms define a cigarette as “any product which contains nicotine, is intended to be
burned under ordinary conditions of use, and consists of: (1) Any roll of tobacco
wrapped in paper or in any substance not containing tobacco; or (2) Any roll of
tobacco wrapped in any substance containing tobacco which, because of its appearance, the type of tobacco used in the filler, or its packaging and labeling, is
likely to be offered to, or purchased by, consumers as a cigarette” (21CFR897.3,
1999; 27CFR290.11, 1999).
LITERATURE CITATIONS
21CFR897.3 (1999) Cigarettes and smokeless tobacco (rev. 4-1-99). Code of
Federal Regulations Title 21 Volume 8:495-496.
27CFR290.11 (1999) Part 290—Exportation of tobacco products and cigarette papers
and tubes, without payment of tax. Code of Federal Regulations Title 27 Volume
2:230-232.
For internal use of Philip Morris personnel only.
356
Evaluation of Cigarette Ingredients: Feasibility
APPENDIX F
STANDARD METHODS FOR TOBACCO
AND SMOKE ANALYSIS1
Method
Tobacco
Alkaloids (total as nicotine)
in tobacco
Chlorides in tobacco
Determination of silica
content
Determination of
chlorophyll residues content
(green index)
Determination of
dithiocarbamate pesticide
residues
Determination of maleic
hydrazide residues
Determination of nitrate in
tobacco by continuous flow
analysis
Determination of
organochlorine pesticide
residues
Determination of reducing
carbohydrates in tobacco by
continuous flow analysis
Determination of reducing
substances in tobacco by
continuous flow analysis
Determination of residual
stem content
Determination of residues
of the suckercide
Flumetralin (Prime Plus,
CGA-41065) on tobacco
AOAC2
ISO3
CORESTA4
960.07 (1964)
960.08 (1964)
963.05 (1964)
2881-1992
20-1968
35-1994
1
2817-1999
8452-1992
6466-1983
1-1978
4876-1980
4-1976
36-1994
4389-2000
38-1994
37-1994
12195-1995
Adapted and modified from (Green & Rodgman, 1996)
Association of Official Analytical Chemists (2000)
3
International Organization of Standardization (Bialous & Yach, 2001; 2002)
4
Cooperation Centre for Scientific Research Relative to Tobacco (1994)
2
For internal use of Philip Morris personnel only.
2-1994
17-1991
30-1991
Appendices
Method
AOAC5
Tobacco
Determination of residues
of suckercide Off-Shoot-T
(N-alkanol mixture) on
tobacco
Determination of residues
of suckercide Off-Shoot-T
(N-alkanol mixture) on
tobacco
Determination of starch
content
Determination of strip
particle size
Glycerol, propylene glycol, 971.02 (1985)
and triethylene glycol in
cased cigarette cut filler and
ground tobacco
Menthol in cigarette filler
968.02 (1970)
968.03 (1970)
Moisture in tobacco
966.02 (1968)
Nitrogen in tobacco
959.04 (1964)
Oriental leaf tobacco –
Baling
Oriental leaf tobacco –
Determination of form and
size characteristics
Potassium in tobacco
966.03 (1968)
Preparation and constitution
of identical samples from
the same lot (Code practice,
for collaborative studies for
evaluating methods of test)
Sampling batches of raw
material – General
principles
Fine-cut tobacco-Sampling
5
ISO6
32-1991
8451-1991
12194-1995
For internal use of Philip Morris personnel only.
16-1991
6488-1981
10919-1994
8043-1990
7821-1982
4874-2000
15592-1-2001
15592-2-2001
357
CORESTA7
32-1991
Association of Official Analytical Chemists (2000)
International Organization of Standardization (Bialous & Yach, 2001; 2002)
7
Cooperation Centre for Scientific Research Relative to Tobacco (1994)
6
43-1997
358
Evaluation of Cigarette Ingredients: Feasibility
Method
AOAC8
Smoke
Determination of alkaloid
retention by the filters
Determination of alkaloids
in smoke condensates
Determination of carbon
monoxide in the vapour
phase of smoke – NDIR
method
Determination of nicotine
in cigarette filters by GC
Determination of smoke
condensate retention index
of a filter – Direct
spectrometric method
Determination of total and
nicotine-free dry
particulate matter using a
routine analytical smoking
machine
Determination of water in
smoke condensates
Nicotine on Cambridge
979.01 (1984)
filter pads, GC method
Routine analytical cigarette
smoking machine –
Definitions and standard
conditions
Smoking machines for
tobacco and tobacco
products – Non-routine test
methods
Atmosphere for
conditioning and testing
fine-cut tobacco and finecut smoking articles
Cigarettes
Determination of loss of
tobacco from the ends
8
ISO9
CORESTA10
3401-1991
13-1968
3400-1989
12-1968
8454-1995
5-1993
9-1989
4388-1991
4387-2000
23-1991
10362-1-1999
10362-2-1994
10315-2000
10315-cor1-2000
3308-1991
8-1991
15-1990
7-1991
7210-1997
42-1997
3550-1-1997
3550-2-1997
Association of Official Analytical Chemists (2000)
International Organization of Standardization (Bialous & Yach, 2001; 2002)
10
Cooperation Centre for Scientific Research Relative to Tobacco (1994)
9
For internal use of Philip Morris personnel only.
22-1991
Appendices
Method
AOAC11
Cigarettes
Determination of
ventilation – Definitions
and measurement
principles
Cigarette-Sampling
Determination of draw
resistance of cigarettes and
filter rods
Determination of acetate in
cigarette paper
Determination of air
permeability
Determination of citrate in
cigarette paper
Draw resistance of
cigarettes and filter rodsDefinitions, standard
conditions, and general
aspects
Atmosphere for
conditioning and testing
Other
Determination of the purity
of nicotine and nicotine
salts by gravimetric
analysis-Tungstosilicic
acid method
Nicotine in environmental 990.01 (1997)
tobacco smoke
991-50 (1997)
11
For internal use of Philip Morris personnel only.
359
ISO12
CORESTA13
9512-2002
6-1983
8243-1991
24-1991
41-1995
33-1993
2965-1997
40-1994
34-1993
6565-2002
3402-1999
Association of Official Analytical Chemists (2000)
International Organization of Standardization (Bialous & Yach, 2001; 2002)
13
Cooperation Centre for Scientific Research Relative to Tobacco (1994)
12
21-1991
39-1994
14-1993
360
Evaluation of Cigarette Ingredients: Feasibility
LITERATURE CITATIONS
AOAC (2000) Tobacco. In: AOAC Official methods of analysis. 17 ed. (Horowitz,
W., ed.). AOAC International, Gaithersburg, MD. pp. 30-37.
Bialous, S. A. & Yach, D. (2001) Whose standard is it, anyway? How the
tobacco industry determines the International Organization for Standardization
(ISO) standards for tobacco and tobacco products. Tob. Control 10:96-104.
CORESTA (1994) List of CORESTA Recommended Methods. Coresta Inf. Bull.
94:12-14.
Green, C. R. & Rodgman, A. (1996) The Tobacco Chemists’ Research Conference: A half century forum for advances in analytical methodology of tobacco and
its products. Rec. Adv. Tob. Sci. 22:131-304.
International Organization for Standardization. (2002) International Organization
for Standardization. (Access Date: 5-1-2002). Web/URL: http://www.iso.ch/iso/en/
CatalogueListPage.CatalogueList?ICS1=65&ICS2=160&ICS3=
For internal use of Philip Morris personnel only.
Appendices
APPENDIX G
For internal use of Philip Morris personnel only.
361
362
Evaluation of Cigarette Ingredients: Feasibility
APPENDIX H
POWER CALCULATIONS
One question for clinical study to address is whether an ingredient in a cigarette
induces a change in the number smoked. This change is determined by comparing
cigarettes containing a specific ingredient to essentially identical cigarettes that
lack that ingredient. The simplest approach to this question is to randomize smokers between two groups in a double-blind study and after an appropriate interval,
enumerate and compare cigarette consumption between the two groups. Subsequent crossover studies, based on these study groups, can compare each subject
with themselves (the same subject), as well as replicate the original study with the
subject groups reversed (Senn, 2002).
Various additional factors in this study design merit attention, particularly assurance
of subject compliance. For example, investigators will want to monitor whether
subjects continue to smoke cigarettes as fully in both groups, by collecting and
measuring butt length. However, a second question becomes important. If the
ingredient does not induce a meaningful difference in cigarette consumption, how
can a study design investigate smoking behavior to understand whether the smoking exposure changed?
A simple method to determine whether an ingredient has altered a smoker’s exposure to cigarette smoke would be to monitor a biomarker of smoking exposure, for
example, cotinine levels in urine. Urine collection is non-invasive. Also, investigators may want to validate twenty-four hour samples by collecting first morning void
samples from the same subject for compliance comparisons.
This design brings up the issue of how many smokers we should study. However,
establishing a specific number of smokers to study raises two questions: 1) Are
there any reasons for using that particular number? 2) How large a difference can
we detect? A statistical method to predict these data is a power analysis. Broadly,
a power analysis is the prospective examination of the expected properties of proposed studies, even though the quantity to be calculated might be the number of
samples needed, or the sensitivity of study (Gad & Rousseaux, 2002). To conduct
a power analysis, the investigator must select answers to four of the five questions
below, and the answer to the fifth emerges by calculation. The five questions are:
1. How large a difference (or other measure) is important?
2. What is the standard deviation of the thing that matters?
For internal use of Philip Morris personnel only.
Appendices
363
3. How sure do you wish to be that a declared difference is not due to chance?
4. How sure do you wish to be that the difference declared in question 1
would in fact be detected if it were truly present?
5. How many samples must be collected?
Having answered all four of these questions, the fifth may be calculated.
Apart from the five questions above, two other questions arise in the planning. One
is, “Does the measure give rise to a one-sample test or a two-sample test?” The
other is, “Do we use a one-tail rejection region or a two-tail rejection region?”
1. How large a difference (or other measure) is important?
The larger the difference of interest the easier will be the study. If we are interested only if the ingredient causes a 100% increase in blood cotinine levels this will
be an easier study than if we declare that we wish to be sensitive to a 1% increase.
If the study fails to find a difference due to the ingredient then we would like to be
able to declare that we are pretty sure there was not a 100% increase in cotinine
levels. It would be more reassuring to be able to say we are pretty sure there was
not a 5% or even a 1% increase. We will not be able to say there was no increase.
We will be able to say there was no detectable increase, but the reflective person
will naturally wish to know what we might reasonably have been able to detect (the
answer to question 4).
2. What is the standard deviation of the thing that matters?
Surrogates for smoke exposure show different degrees of scatter. One of the reasons cotinine is often chosen is that the standard deviation associated with repeated
measurements is less than that of other surrogates. Generally, a number for the
reproducibility will be known from the literature. Small standard deviations are
good, larger ones require larger studies, or the tolerance of greater uncertainty.
If a new surrogate is to be used, a preliminary study will be required to determine
the reproducibility under conditions of the study planned. For example, blood levels
of the surrogate are measured at 1, 2 and 3 months following a switch to cigarettes
containing the ingredient. A preliminary study using measurements at 0, 1, 2, and 3
months without a change in smoking habits will give us the information needed to
calculate the standard deviation of paired differences from 0. This can be used to
answer question 2 for a study following a switch to a cigarette containing the ingredient.
For internal use of Philip Morris personnel only.
364
Evaluation of Cigarette Ingredients: Feasibility
3. How sure do you wish to be that a declared difference is
not due to chance?
This is usually taken to be 0.05, called alpha, and referred to as a type I error. The
type I error is the probability that one would reject the null hypothesis by chance
even though it is true. This is the chance of declaring a significant difference even
though the true difference is zero. In the present setting where it will be important
to assert confidently that we really should have been able to detect a difference if
it was truly there, consideration might be given to using an alpha of 0.1 or larger. In
accepting a larger risk of declaring a difference by chance we would also be increasing the probability of detecting a true difference if it is present. (Question 4)
4. How sure do you wish to be that the difference declared in
question 1 would in fact be detected if it were truly present?
This is less standardized than the choice for type I errors although in many clinical
trials a value of 0.8 is used. This is called the power of the test and is also 1 minus
the probability of a type II error. The type II error is the probability of not rejecting
the null hypothesis though it is false. There really is a difference but we couldn’t
find it. Naturally we wish this number to be large. Making the number of measurements larger, reducing the standard deviation, increasing the sought for difference,
or increasing the risk of a type I error will all reduce type II errors.
5. How many samples must be collected?
This is often chosen as the question to be answered by calculation, though occasionally, costs, time or just availability of subjects constrains this number. One
seeks instead to answer question 1 (how large a difference are we likely to detect),
or perhaps 2 (how small must we make the standard deviation to accomplish our
aims), or 3 or 4 (what sort of uncertainty are we forced to tolerate).
One-Sample or Two-Sample?
One-sample tests compare the mean or median of a single set of data to some fixed
reference, for example zero. Paired differences give rise to one-sample tests. Twosample tests compare two sets of data with one another. The reason this matters is
that computer programs for calculating power will be expecting the standard deviation of a single set of data for the one-sample test, but will be expecting a common
standard deviation for the two sets of data for the two-sample, and will calculate
the standard deviation of the difference between the two means.
One-tail or Two-tail?
If the interest is in detecting any change whose magnitude exceeds the answer to
question one above, then a two-tail rejection region is appropriate. On the other
hand, one might argue that if smoke exposure declines with the use of an ingredi-
For internal use of Philip Morris personnel only.
Appendices
365
ent, this is of no consequence since no claim of improvement is intended. It will
also suffice to say that no increased exposure was detected and that we would
have expected a 90% chance of detecting an increase exceeding 10%. By sacrificing the detection of a decrease, we can increase the sensitivity of detection of an
increase for the same sample size.
Calculations
Calculations will be made using commercial software that requires data from the
previous questions. If the software does not require the information above, then it is
making assumptions about whatever is omitted. Many software programs are
based on normal theory, which is usually appropriate if it is anticipated that 10 or
more patients will be entered in each of two groups, or 10 or more pairs of observation are expected (Desu & Raghavarao, 1990). As a practical requirement, it is
likely that at least 20 subjects in each group will be considered useful for experiments that compare smoke exposure in subjects smoking a cigarette with or without an ingredient. In such a case normal theory should be quite adequate for study
planning and analysis.
If for some reason the normal theory is deemed not suitable, then the chief nonparametric approaches would be the Wilcoxon rank sum test for comparison of
unpaired samples or the Wilcoxon signed rank test for paired observations. Both
approaches assume there will be no ties in the data, and the signed rank test makes
the further assumption of asymmetry. If either of these conditions is likely to be
violated, then the power calculations available in commercial software should be
avoided.
If a sample of residuals is available for data similar to that to be collected, then
bootstrap estimates of the power can be obtained (Efron & Tibshirani, 1993).
LITERATURE CITATIONS
Desu, M. M. & Raghavarao, D. (1990) Sample Size Methodology. Academic
Press, Boston.
Efron, B. & Tibshirani, R. J. (1993) An Introduction to the Bootstap. Chapman &
Hall, New York.
Gad, S. C. & Rousseaux, C. G. (2002) Use and misuse of statistics in the design
and interpretation of studies. In: Handbook of Toxicological Pathology. 2nd Ed. Vol. 1.
Academic Press, New York, pp. 327-418.
Senn, S. (2002) Cross-over trials in clinical research. In: Statistics in Practice 2nd
ed., John Wiley & Sons Ltd., Chichester, England.
For internal use of Philip Morris personnel only.
366
Evaluation of Cigarette Ingredients: Feasibility
Index
A
C
Absolute risk 7
Cambridge filter pad 46
Absorption 63, 65, 74–79, 155,
170–180, 193
Cancer 8–11, 52, 54–58, 97, 103–116,
120, 128, 130–133, 142–144, 149,
159–160, 174–177, 180–182
Added ingredient 6–8, 10–11, 13–14,
20, 23–24, 29, 31–33, 50–55, 139–
156, 167–168, 194–195, 289–293,
306–354
Additive 6–9, 11–13, 24–25, 113, 118–
121, 139–140, 150–151, 172, 175,
186–189, 193–195, 289
Air-cured 43, 289
Aldehydes 50–51
Animal model 11, 55, 58, 94–98,
121, 135, 139–140, 142–144, 148–149,
154–155, 183, 193–194, 290
Ash 25, 27, 290
Carbon monoxide 21–22, 27, 33–36,
40–42, 48, 50–51, 78, 87, 90–92,
96–99, 168, 183
Cardiovascular diseases 2, 103, 110,
114, 116, 163, 175–176, 178
Casing 24, 51, 290
Chemical substances 9, 18, 23–24,
28–30, 36–44, 48–51, 53–58, 78,
128–131, 143, 167–170, 187, 193, 195
Chronic obstructive pulmonary disease
2, 102, 115, 176, 291
Cigarette definition 18, 355
B
Behavior 7, 9, 14, 19, 25, 63, 66–72,
75, 77, 97, 104, 113, 116, 145, 147, 157,
171– 173, 178–183, 191–194
Biological activity 9, 62, 123, 125–126,
141, 145, 174, 290
Biologically effective dose 174– 175,
290
Biomarker 9, 54, 63, 69, 71, 86, 90– 92,
98, 132, 141, 148, 155, 171, 174–175,
183, 193–194, 290
Bright tobacco 36, 290
Burley tobacco 20, 24, 36, 144, 190,
290
Cigarette smoke 6, 9–10, 14, 18–19,
22–59, 73–98, 101, 102, 110–113, 121,
125, 131, 134, 139, 141, 143–159,
165–177, 183, 187, 189, 191–193, 298
Clearance 74, 88, 147, 155, 174, 182
Compensation 69–72, 81, 99, 172, 192,
291
Complex mixture 18, 23, 25, 36, 62, 140,
166, 187, 195, 291
Concordance 128–129, 131, 137–138,
291
Cotinine 69, 71, 87–90, 95, 155–156,
161, 178, 180, 194, 291
For internal use of Philip Morris personnel only.
Index
D
Deposition 2, 51, 72–83, 85–86, 89–90,
123–124, 140, 155, 169, 172–174,
181–182, 291
Dilution 20, 22, 26, 30, 32, 42, 47, 59,
69–70, 77, 96, 155, 291, 299
Direct additive 1, 291
Distillation 26, 28–29, 291
Dose 69, 85–90, 111–119, 128, 143,
145–147, 150, 173–175, 188, 190, 195,
292
Dosimetry 63, 73, 85, 95, 98, 142–143,
161, 167, 292
367
H
Haber’s rule 63–66, 100–111, 113
Humectant 24, 144, 293
I
Ingredient 1–14, 23–25, 118, 121,
137–155, 161, 186–195, 293, 306–354
Inhalation 8, 63–68, 101–102, 139–143,
172, 183, 188–189
Ink 23
Internal validity 2, 123, 129, 133, 136,
293
K
E
Kinetics 123, 135, 143, 293
Environmental tobacco smoke 7,
25–26, 118, 292
Excursion limit 4, 15, 135, 137
Existing additive 24, 292
Exposure 4, 6–8, 10–15, 63–66, 69–70,
73, 78, 80–97, 109–113, 118–121, 125,
128–138, 140–148, 150–158, 170,
172–175, 186, 188, 189, 191–194, 292
External validity 2, 52, 123, 128, 129,
133, 292
F
Filtration 26, 30, 39, 44, 46, 51
Flavoring 12, 24–25, 151, 155
Flue-cured 36, 53, 88, 144–145, 293
Free radicals 29, 32, 42, 43, 163,
177– 178
G
Gas phase 2, 29, 45, 58, 98, 153, 167,
193, 293
L
Lung cancer 2, 9–10, 55, 58, 97, 105,
108–116, 132–133, 149, 159–160,
174, –176, 181–182
M
Mainstream smoke 19, 22–23, 25–26,
29, 32, 35–36, 39, 42–47, 49–51,
53–55, 58–59, 66, 76–77, 80, 89,
152–156, 161, 168, 294
Maryland tobacco 144, 190, 294
Menthol 25, 29, 126, 150–162, 171, 174,
180, 183, 195
Morbidity 1, 3, 7–10, 13–14, 65, 98,
104–105, 117–118, 122, 125, 129, 165,
169, 180, 194–196
Mortality 1–3, 7–10, 13–14, 65, 96–98,
102–105, 107–109, 114–118, 125, 129,
143, 145– 146, 163, 165, 169, 175–
176, 180, 195
Genetics 164, 171, 178
Gold Standards 123, 126, 293
For internal use of Philip Morris personnel only.
368
Evaluation of Cigarette Ingredients: Feasibility
N
Nicotine 18, 20–23, 30–31, 33, 35–36,
39–42, 45–50, 54, 68–72, 87–90, 95,
153–158, 168–169, 171–174, 294
Nitrosamines 9, 22, 37, 46, 54– 55, 294
No detectable change 9, 103, 121, 135,
165, 187, 189, 190–191, 194
R
Reference tobaccos 169, 297
Relative risk 4, 7, 9, 14, 98, 104, 109,
116–117, 125, 135, 141, 160–161, 165,
167, 171, 173, 184, 186, 188–189, 191,
193, 196, 297
Retention 23, 75, 82, 158, 174, 297
Pack-year 63–66, 83, 115, 122, 160,
194, 295
Risk 1, 4, 6, 11, 14, 55, 101, 103–104,
107–109, 114–122, 128–129, 141–149,
159–161, 165– 173, 176–184, 186,
188–189, 191, 193–194, 196, 297
Paper 18–19, 22–23, 27–30, 39, 51, 59,
69, 134, 166, 295
Risk factors 8, 103, 104, 164, 176, 178,
182, 184
Particulate matter 21, 23, 25, 31–32,
39, 42, 44–46, 76, 78, 152–155, 158,
193, 295
S
P
Particulate phase 22, 30–31, 39,
42–44, 46, 49–50, 53–54, 58, 65,
73–74, 78, 87, 295
Peak puff flow 66, 158, 183, 295
Pharmacokinetics 12, 63, 88, 90, 96
Phenols 17, 21–22, 29, 48, 49, 53, 166
Polycyclic aromatic hydrocarbons
53–55, 295
Potential reduced-exposure products
10, 295
Sensitivity 53, 96, 108, 127, 128, 130,
131, 150, 174, 181, 297
Sidestream smoke 22–26, 31, 35, 39,
43–46, 49–51, 58–59, 95–153, 298
Signal-to-noise ratio 132–133, 141, 167,
192, 298
Smoke chemistry 32, 36–46, 124, 126,
134, 140, 153, 161, 165–166, 188–190,
193
Smoke deposition 140, 163, 173
Smoke inhalation 2, 64, 66, 77, 172
Proprietary mixture 4
Smoke physics 169
Puff interval 26, 31, 48, 59, 66, 72, 157,
158, 183, 296
Smoking behavior 2, 14, 63, 66–69, 72,
101, 121, 147, 157–158, 171–172,
178–184, 191–192, 194
Puff profile 36, 66–68, 71
Puff volume 48, 66–68, 70–72, 89, 91,
156–158, 161, 174, 181, 183, 296
Pyrolysis 2–3, 7, 12, 14–16, 26, 28– 30,
43, 53, 72, 81, 87, 101, 135, 140–141,
152, 161, 166–167, 185, 187–191, 193,
296
Smoking cessation 2, 9, 102–103, 108,
112, 115, 170–172, 181
Smoking compensation 69–72, 291
Specificity 53, 107, 128, 130, 131, 138,
174, 298
Pyrosynthesis 16, 20, 26, 29, 30, 37, 49,
141, 170, 296
For internal use of Philip Morris personnel only.
Index
T
Tar 21–23, 33, 35–36, 39, 42, 44–46,
48, 50–51, 53, 64, 66, 70–72, 75, 78,
81–82, 122, 140, 153, 156, 168–169,
177, 298
Total particulate matter 21, 31, 32, 39,
42, 45, 78, 152–155, 158, 299
Toxicity testing 139–140, 149
Transfer 3, 14, 29–30, 44, 46, 74, 87,
101, 139, 152, 161, 165–168, 187–190
V
Variation 19–21, 39, 59, 63, 68, 73,
82–83, 89, 107, 111, 120, 147, 168,
182–183, 192
Ventilation 22, 23, 39, 42, 51, 70, 72, 77,
80, 140, 182, 299
Virginia tobacco 20, 33, 299
For internal use of Philip Morris personnel only.
369