GREEN BOOK 4
Alkyl Benzoates
CIR EXPERT PANEL MEETING
AUGUST 30-31, 2010
July 30, 2010
MEMORANDUM
To:
CIR Expert Panel and Liaisons
From:
Lillian C. Becker, M.S.
Scientific Analyst and Writer
Subject:
Draft Report for C12-15 Alkyl Benzoate and related Alkyl Benzoates
The Cosmetic Ingredient Review (CIR) announced the Scientific Literature Review (SLR) for
alkyl benzoates in June, 2010.
C12-15 alkyl benzoate is the lead ingredient of this safety assessment. Related alkyl benzoate
ingredients are included.
CIR has been informed that a comprehensive dossier on the C12-15 alkyl benzoates being
prepared for the European REACH program will be completed and provided to CIR in late
September or early October.
The Panel should review the Draft Report and decide: 1) if it is reasonable to include the other
listed ingredients with C12-15 alkyl benzoate in this report and 2) whether any additional data
are needed in order to reach a safety conclusion for C12-15 alkyl benzoates and the related
ingredients. If no additional data are required, then the Panel may issue a Tentative Report.
Alternatively, the Panel may choose to table the report to await the receipt of the dossier
mentioned above.
CIR Panel Book Page 1
CIR Panel Book Page 2
History of Alkyl Benzoates
June, 2010 – SLR issued.
August, 2010 -
CIR Panel Book Page 3
Search Strategy for Benzoates EXPORATORY SEARCH: PUBMED: “alkyl benzoate” – 7 hits, 1 useful; CAS No. – 0 hits. Internet (Dogpile) – “alkyl benzoate” ‐ 1 MSDS FULL SEARCH: PUBMED: “lauryl alcohol” – 53 hits, 6 ordered. Learned that Valerie was doing this ingredient. “tridecyl alcohol” – 0 hits. CAS No. – 0 and 19 hits. 1 useful. “Amyl benzoate” ‐0 hits. CAS no – no hits. “benhyl benzoate” – 0 hits. CAS no – no hits. “Butyl Benzoate” – 9 hits, 1 useful. CAS no. – no hits. “Butyloctyl Benzoate” – 0 hits. No CAS no. “Ethyl Benzoate” – 44 hits, 4 useful. “Ethylhexyl Benzoate” – no hits. “Hexyldecyl Benzoate” – no hits. “Isobutyl Benzoate” – not hits. CAS no. – no hits. “939‐48‐0” OR “34364‐24‐4” OR “Lauryl/Myristyl Benzoate” OR “112‐53‐8” OR “Octyldodecyl Benzoate” OR ”2315‐68‐6” OR “10578‐34‐4” – 224 hits, 19 useful TOXNET: 68411‐27‐8 – 0 hits; 112‐53‐8 – 253 hits, 47 useful; 112‐70‐9 – 33 hits, 2 useful; 26248‐42‐0 ‐ 10 hits, 4 useful; 629‐76‐5 – 15 hits, 1 useful (already have); 2049‐96‐9 – 6 hits, 0 useful; “amyl alcohol” ‐ 798 hits, 86 + 64 hits so far; 103403‐38‐9 – no hits; 136‐60‐7 ‐ Butyloctyl Benzoate – no hits; butyloctyl alcohol – no hits; C16‐17 Alkyl Benzoate – no hits; palmyl alcohol – no hits; heptadecyl alcohol – no hits; 93‐89‐0 – 56 hits, 64‐17‐5 alcohol – 50000 hits, did not explore yet; Hexyldecyl Benzoate – no hits; Hexyldecyl Benzoate – No hits; 120‐50‐3 – 3 hits, 0 useful; 939‐48‐0 – 2 hits, 0 useful; 34364‐24‐4 – no hits, Isopropyl Benzoate – No hits; Isostearyl Benzoate – no hits; Lauryl/Myristyl Benzoate ‐ no hits; 112‐
53‐8 [print toxnet] – 248 hits, 112‐53‐8 – 248 hits, 61 useful; 93‐58‐3 – 135 hits, 14 useful; Octyldodecyl Benzoate – no hits; Octyldodecyl Alcohol – no hits; 2315‐68‐6 – 7 hits, 2 useful; 10578‐34‐4 – no hits. EPA – HPV – One relevant report that included methyl benzoate. Data added to report under original citations. CIR Panel Book Page 4
Report
Draft Report
Alkyl Benzoates as used in Cosmetics
August 30, 2010
The 2010 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.;
Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G.
Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR
Director is F. Alan Andersen, Ph.D. This report was prepared by Lillian C. Becker, Scientific Analyst/Writer.
© Cosmetic Ingredient Review
1101 17th Street, NW, Suite 412 " Washington, DC 20036-4702 " ph 202.331.0651 " fax 202.331.0088 "
[email protected]
CIR Panel Book Page 5
TABLE OF CONTENTS
TABLE OF CONTENTS........................................................................................................................................................... iii INTRODUCTION ....................................................................................................................................................................... 1 CHEMISTRY .............................................................................................................................................................................. 1 Definition and Structure .......................................................................................................................................................... 1 Physical and Chemical Properties ........................................................................................................................................... 1 Manufacture and Production ................................................................................................................................................... 1 Impurities ................................................................................................................................................................................ 2 Analytical Methods ................................................................................................................................................................. 2 USE.............................................................................................................................................................................................. 2 Cosmetic .................................................................................................................................................................................. 2 Non-Cosmetic.......................................................................................................................................................................... 2 GENERAL BIOLOGY ................................................................................................................................................................ 2 Absorption, Distribution, Metabolism, and Excretion............................................................................................................. 2 Growth Inhibition .................................................................................................................................................................... 3 TOXICOLOGY ........................................................................................................................................................................... 3 Acute Toxicity ......................................................................................................................................................................... 3 Methyl Benzoate ................................................................................................................................................................. 3 Ethyl Benzoate .................................................................................................................................................................... 3 Isopropyl Benzoate ............................................................................................................................................................. 3 Isobutyl Benzoate................................................................................................................................................................ 3 Short-Term Toxicity ................................................................................................................................................................ 3 Benzoic Acid and Sodium Benzoate ................................................................................................................................... 3 Subchronic Toxicity ................................................................................................................................................................ 3 Benzoic Acid....................................................................................................................................................................... 3 Chronic Toxicity ..................................................................................................................................................................... 3 Sodium Benzoate ................................................................................................................................................................ 3 Ocular/Mucosal Irritation ........................................................................................................................................................ 3 Isostearyl Benzoate ............................................................................................................................................................. 3 Dermal Irritation ...................................................................................................................................................................... 3 Methyl Benzoate ................................................................................................................................................................. 3 Ethyl Benzoate .................................................................................................................................................................... 4 Propyl Benzoate .................................................................................................................................................................. 4 Butyl Benzoate .................................................................................................................................................................... 4 Dermal Sensitization ............................................................................................................................................................... 4 Reproductive and Developmental Toxicity ............................................................................................................................. 4 iii
CIR Panel Book Page 6
Sodium Benzoate ................................................................................................................................................................ 4 Benzoic Acid....................................................................................................................................................................... 4 GENOTOXICITY ....................................................................................................................................................................... 4 Methyl Benzoate ................................................................................................................................................................. 4 Benzoic Acid and Sodium Benzoate ................................................................................................................................... 4 CARCINOGENICITY ................................................................................................................................................................ 5 Methyl Benzoate ................................................................................................................................................................. 5 Benzoic Acid....................................................................................................................................................................... 5 CLINICAL ASSESSMENT OF SAFETY .................................................................................................................................. 5 Absorption, Distribution, Metabolism, and Excretion............................................................................................................. 5 Toxicity ................................................................................................................................................................................... 5 Benzoic Acid....................................................................................................................................................................... 5 Dermal Irritation ...................................................................................................................................................................... 5 C12-15 Alkyl Benzoate....................................................................................................................................................... 5 Benzoic Acid....................................................................................................................................................................... 5 Dermal Sensitization ............................................................................................................................................................... 5 C12-15 Alkyl Benzoate....................................................................................................................................................... 5 Isostearyl Benzoate ............................................................................................................................................................. 5 Octyldodecyl Benzoate ....................................................................................................................................................... 5 Benzoic Acid....................................................................................................................................................................... 6 Phototoxicity ........................................................................................................................................................................... 6 Benzoic Acid....................................................................................................................................................................... 6 SUMMARY................................................................................................................................................................................. 6 TABLES AND FIGURES ........................................................................................................................................................... 7 REFERENCES .......................................................................................................................................................................... 13 iv
CIR Panel Book Page 7
INTRODUCTION
This is a safety assessment of alkyl benzoate esters that are used in cosmetics. The ingredients included in this
literature review are: methyl benzoate, ethyl benzoate, propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl
benzoate, C12-15 alkyl benzoate, C16-17 alkyl benzoate, stearyl benzoate, behenyl benzoate, isopropyl benzoate, isobutyl
benzoate, isostearyl benzoate, ethylhexyl benzoate, butyloctyl benzoate, hexyldecyl benzoate, and octyldodecyl benzoate.
The alkyl benzoate ingredients are esters of benzoic acid and a corresponding alcohol, with the shorter chain alkyl
benzoates (methyl, ethyl, propyl, isopropyl, butyl, isobutyl and amyl benzoate) ranging in MW from 136 to 192 and the
longer chain alkyl acetates (lauryl/myristyl, C12-15 alkyl, C16-17 alkyl, stearyl, isostearyl, behenyl, ethylhexyl, butyloctyl,
hexyldecyl, and octyldodecyl benzoate) ranging in MW from 234 to 431.
It is unclear as to whether the alkyl benzoates in this report penetrate the skin. If they do, these compounds may be
metabolized in the skin into benzoic acid and the parent alcohol.
The conclusions from prior assessments of benzoic acid, sodium benzoate, and the parent alcohols (methyl alcohol,
ethyl alcohol, butyl alcohol, myristyl alcohol, behenyl alcohol, isostearyl alcohol) by the Cosmetic Ingredient Review (CIR)
Expert Panel are listed below. These conclusions may be relevant to this report if significant penetration and metabolism do
occur.
Benzoic acid and sodium benzoate - “…safe for use in cosmetic formulations at concentrations up to 5%. The
available data are insufficient to support the safety of [benzoic acid and sodium benzoate] in cosmetic products in which a
primary route of exposure is inhalation”.1
Methyl alcohol - safe for use as a denaturant in ethyl alcohol for cosmetic products, with qualifications. The Panel
has not stated that methyl alcohol is safe or unsafe as a solvent.2
Ethyl alcohol – (as “Alcohol Denat.” with methyl alcohol) - safe in the present practices of use and concentration.3
Butyl alcohol - safe as a cosmetic ingredient in the present practices of use.4 In 2005, the panel looked at new data
and the safety conclusion in the report was confirmed.
Myristyl alcohol - safe as a cosmetic ingredient in the present practices of use.5
Cetyl alcohol - safe as a cosmetic ingredient in the present practices of use.6 In 2005, the panel looked at new data
and the conclusion in the report was confirmed.
Stearyl alcohol - safe as currently used in cosmetics.7 In 2006, the panel looked at new data and the conclusion in
the report was confirmed.
Isostearyl alcohol - safe as cosmetic ingredients in the present practices of use. 6 In 2005, the panel looked at new
data and the conclusion in the report was confirmed.
Behenyl alcohol - safe as a cosmetic ingredient in the present practices of use.5 In 2005, the panel looked at new
data and the conclusion in the current report was confirmed.
Propyl alcohol and isopropyl alcohol are concurrently under review by the CIR expert Panel in the report titled
Methyl Acetate.
Summaries of the data from the benzoic acid and sodium benzoate safety assessment are included in this safety
assessment.
The potential metabolites of ethylhexyl benzoate, butyloctyl benzoate, hexyldecyl benzoate, isobutyl benzoate, amyl
benzoate, pentadecyl benzoate, heptadecyl benzoate, and octyldecyl benzoate are not current cosmetic ingredients in the
dictionary, thus have not been reviewed by CIR.
CHEMISTRY
Definition and Structure
Alkyl benzoates are mostly used as skin-conditioning agents, preservatives, solvents, and plasticizers. The CAS
numbers, definitions, functions, as well as technical and trade names of the ingredients under review are presented in Table 1.
Structures and potential metabolic pathways of these ingredients are presented in Figures 1 and 2.
Physical and Chemical Properties
The shorter chain alkyl benzoate esters are colorless liquids. Viscosity generally increases as the molecular mass
(chain length) increases.8 The physical and chemical properties of the benzoates are shown in Table 2.
At room temperature and pressure, methyl benzoate, ethyl benzoate, butyl benzoate, and isobutyl benzoate are
fragrant, colorless oils, and are insoluble in water.9
Manufacture and Production
In general, the alkyl benzoates can be produced industrially via esterification of benzoic acid.8 The manufacture of
butyl benzoate, for example, is traditionally accomplished via an acid catalyzed (e.g., sulfuric acid) reactive distillation
process between benzoic acid and butyl alcohol (Figure 3).10
Methanol and ethanol are normally obtained via fermentation of natural sources. However, some alcohols with
chains longer than ethanol are often produced synthetically. An important process for producing C3- C22 industrial alcohols
involves a process known as oxo-synthesis (a process for the production of aldehydes which occurs by the reaction of olefins
(which can be natural or petroleum sourced) with carbon monoxide, hydrogen and a catalyst [typically cobalt based]),
followed by hydrogenation of the aldehyde products, to form the alcohols.11 Recently, a biocatalytic process developed
1
CIR Panel Book Page 8
specifically for the manufacture of esters for use in the formulation of cosmetic and personal care ingredients (i.e. for
producing cosmetic grade esters) was developed.12
Impurities
The manufacturing processes of the benzoic esters are typically high yielding (>90%) and easily purified (e.g., by
distillation). Therefore, the starting materials and water, at least, may be expected to be present in preparations of these esters
as the major impurities.8 For example, methyl benzoate is available with a minimum of 99.2% purity, wherein the major
contaminants are water (<0.1%) and benzoic acid (<0.02%).13
Analytical Methods
The benzoic esters can be analyzed using gas chromatography/mass spectroscopy (GCMS), nuclear magnetic
resonance (NMR) spectroscopy, ultraviolet (UV) spectroscopy and infrared (IR) spectroscopy.8,11,14
USE
Cosmetic
According to the Voluntary Cosmetic Registration Program (VCRP) administered by the Food and Drug
Administration (FDA), the total number of uses of C12-15 alkyl benzoate was 971 (858 leave-on and 113 rinse-off
products).15 A survey conducted by the Personal Care Products Council (Council) found that C12-15 alkyl benzoate was
used at 0.0008% - 35% (tonics, dressings, and other hair grooming aids) in leave-on products and 0.0008% - 50% (paste
masks [mud packs]) in rinse-off products.16 There were 2 uses reported of C16-17 alkyl benzoates at 0.7% (bath soaps and
detergents). Stearyl benzoate at 2% was reported to have 3 uses (face and neck creams, lotions, and powders). While there
were no uses reported by VCRP, the Council reported behenyl benzoate use at 0.04% (lipstick), ethyl benzoate use at
0.0008% - 0.01% (foot powders and sprays), isobutyl benzoate use at 0.01% (perfumes), isostearyl benzoate use at 1% (body
and hand creams, lotions, and powders), methyl benzoate use at 0.0005% – 0.3% (perfumes), and octyldodecyl benzoate at
3% - 4% (shaving cream) The uses and concentrations are provided in Table 3. No uses or concentrations of use were
reported for propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl benzoate, isopropyl benzoate, ethylhexyl
benzoate, butyloctyl benzoate, and hexyldecyl benzoate.
These ingredients are used in aerosol/spray products, and effects on the lungs that may be induced by aerosolized
products containing these ingredients may be of concern when these ingredients are vaporized.
In the EU, methyl benzoate, ethyl benzoate, propyl benzoate and butyl benzoate may be used as preservatives in
cosmetics up to 0.5% (acid).17
Non-Cosmetic
Alkyl benzoate esters are typically used as solvents in paints, lacquers and coatings, and as intermediates in various
chemistry processes.8 Methyl benzoate is used in flavoring and perfumery, and as a solvent in resins.9 Ethyl benzoate is used
in flavoring and perfumery, and as a solvent in lacquers and resins.9 Butyl benzoate is used as a solvent for cellulose ether, as
a plasticizer, perfume ingredient and for dyeing of textiles.9 Isobutyl benzoate is used in flavoring and perfumery.9
GENERAL BIOLOGY
Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to
benzyl glucuronide and benzoyl CoA.18 Benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted in the
urine. Dermally applied benzoic acid is excreted in the urine within 24 h.
Absorption, Distribution, Metabolism, and Excretion
There are limited data found on the absorption, distribution, metabolism, and excretion of any of the alkyl benzoates
in this safety assessment. However, data on benzyl alcohol show that it is broken down into benzoic acid by simple
oxidation.1 Orally consumed benzoic acid is absorbed from the gastrointestinal tract and conjugated with glycine in the liver.
The resulting hippuric acid is excreted in the urine (75% - 100% within 6 h). Dermally applied benzoic acid is also excreted
in the urine within 24 h.
Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to
benzyl glucuronide and benzoyl CoA.18 The benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted
in the urine.
In general, esters can be hydrolyzed to the parent alcohol and acid.8 The rate of this reaction can be increased by
raising the temperature and decreased by lowering pH. Secondary and tertiary esters are hydrolyzed more slowly than
primary esters. Enzymatic hydrolysis occurs via enzymes called esterases. These enzymes are present in the respiratory
tract, skin, blood and gastrointestinal tract.19,20
In general, alcohols can be metabolized by alcohol dehydrogenases to aldehydes or ketones. The aldehydes may be
further metabolized by aldehyde dehydrogenases to the corresponding acids.
Low molecular weight alkyl esters readily penetrate the skin and mucous membranes. However, benzoic esters are
not absorbed through the skin as rapidly as alkyl esters.21 If alkyl benzoates are absorbed and metabolized, the resulting
alcohols can be oxidized via alcohol dehydrogenases to produce the corresponding aldehyde or ketone. As noted above,
these aldehydes can then be further oxidized via aldehyde dehydrogenases to the corresponding acids.
2
CIR Panel Book Page 9
Growth Inhibition
In a protein count assay of methyl benzoate, the EC50 (50% of the concentration of maximum effect) was 1506.58
(C.I. 1349.27 - 168.22) mM, the NI50 (the concentration that reduced the uptake of neutral red by 50%) was 683.30 (466.46 –
1000.91) mM in a neutral red uptake assay, and the (the concentration that inhibited growth by 50%) ID50 was 987.19
(605.15-1610.43) mM in a growth inhibition assay using HeLa cells.22
Methyl benzoate (2.5 and 5.0 mg/ml) inhibited mycelia growth and aflatoxin release by Aspergillis flavus and A.
parasiticus.23
TOXICOLOGY
Acute Toxicity
The oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for rats, and
3000-3330 mg/kg for mice. The oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats. The
oral LD50 of isopropyl benzoate was 3730 mg/kg and 3685 mg/kg for isobutyl benzoate for rats. The dermal LD50 of methyl
benzoate was ≥ 2000 mg/kg for rabbits.
Methyl Benzoate
The reported oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for
rats, and 3000-3330 mg/kg for mice.24-27
The dermal LD50 of methyl benzoate was ≥ 2000 mg/kg for New Zealand white rabbits (n = 5).28 There was fecal
staining for 3 days after treatment. Irritation was observed at the application site. There was weight loss for 1 – 7 days after
treatment. There were no gross findings at necropsy. There were no mortalities.
Ethyl Benzoate
The reported oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats.24,27
Isopropyl Benzoate
The reported oral LD50 of isopropyl benzoate was 3730 mg/kg for rats.29
Isobutyl Benzoate
The reported oral LD50 of isobutyl benzoate was 3685 mg/kg for rats.18
Short-Term Toxicity
Benzoic acid and sodium benzoate were toxic to rats and mice at oral doses > 1%.
Benzoic Acid and Sodium Benzoate
In multiple-dose oral toxicity studies on rats and mice, decreased feed consumption, depressed growth, and toxic
effects were observed at doses > 1% benzoic acid or sodium benzoate.1 A neurobiological study on rats and mice was
negative.
Subchronic Toxicity
Benzoic acid was toxic to mice at oral doses of 80 mg/kg/d.
Benzoic Acid
Cross bred white mice (n = 100) were orally administered benzoic acid at 80 mg/kg/d for 3 months.30 The treated
group had decreased weight gain. Mortality was increased (68% vs. 60 % in the control group).
Chronic Toxicity
Sodium benzoate at 880 mg/kg/d incorporated into the feed of rats for 18 – 24 months was not toxic.
Sodium Benzoate
Sodium benzoate (0, 1%, or 2%; 735 or 880 mg/kg/d) was incorporated into the feed of Fischer 344 rats (n = 102; 50
males, 52 females) for 18 – 24 months.31 There were no differences in mortality between groups. Necropsies were
unremarkable.
Ocular/Mucosal Irritation
Isostearyl benzoate was rated as a slight ocular irritant in two in vitro tests.
Isostearyl Benzoate
In an ocular irritation assessment of a body lotion containing isostearyl benzoate (0.95%) using neutral red release
(NRR) assay, the hen’s egg test on the chorio-allantoic membrane (HET-CAM) assay, and the reconstituted human epithelial
culture (REC) assay, the authors rated the lotion as slightly irritating.32
Dermal Irritation
Methyl benzoate, ethyl benzoate, propyl benzoate, and butyl benzoate at 100% are dermally irritating to rabbits.
Methyl Benzoate
Methyl benzoate was applied to the clipped dorsum (100%; 0.5 ml) and external surface of the outer ear (0.2 ml)
daily for 6 days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reactions and
dermal edema beginning on day 2 followed by dermal hemorrhages, desquamated crust, and thickening of the malpighian
stratum beginning on day 5. On the inner ear, there was slight hyperkeratosis at day 6.
3
CIR Panel Book Page 10
Ethyl Benzoate
Ethyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6
days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there was marked cellular changes, edema, desquamated
crusts, and thickening of the malpighian stratum beginning on day 1. On the inner ear, there were slight cellular reaction, no
edema or hemorrhages, no necrosis, slight to marked desquamated crusts, marked thickening of malpighian stratum,
hyperkeratosis, and slight hyperplasia of sebaceous glands beginning day 1.
Propyl Benzoate
Propyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6
days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reactions, necrosis,
thickening of the malpighian stratum beginning on day 1 followed by dermal hemorrhages, desquamated crusts beginning on
days 3 or 4.. On the inner ear, there were slight cellular reactions, necrosis, and moderate thickening of the malpighian
stratum beginning on day 1 or 3.
Butyl Benzoate
Butyl benzoate was applied to the clipped dorsum (0.5 ml) and external surface of the outer ear (0.2 ml) daily for 6
days to male New Zealand albino rabbits (n = 14).33 On the dorsum, there were marked cellular reaction, necrosis, and slight
detachment of the dermo-epidermis beginning on day 1 followed by desquamated crusts and thickening of the malpighian
stratum beginning on day 3. On the inner ear, there were slight cellular reaction, necrosis beginning on day 1 and moderate
desquamed crusts and hyperplasia sebaceous glands on day 2 or 3.
Dermal Sensitization
There were no animal dermal sensitization data discovered on alkyl benzoates in this safety assessment.
Reproductive and Developmental Toxicity
Studies on sodium benzoate and benzoic acid did not show reproductive or developmental toxicity. Where effects of
the fetus were noted, they occurred at maternally toxic concentrations (> 4% sodium benzoate in rats).
Sodium Benzoate
Sodium benzoate (0, 1%, 2%, 4%, or 8%; 0, 667, 1333, 1600, or 710 [sic] mg/kg/d) was orally administered in the
feed of female Wistar rats (n = 27-30) during gestation.34 On day 20, 20-25 of each group were killed and necropsied. The
rest were allowed to live through pregnancy and nurse for 3 or 8 weeks and then killed. Half of the pups were then killed at
each of these times and necropsied. The 2 highest dose groups had an increase in the number of dead fetuses and resorbed
embryos. The body weights of the viable pups were decreased, there was mild systemic edema observed. The number of
fetal abnormalities was increased in a dose-dependent manner. The number of pups born was decreased, the number of
perinatal deaths increased to 100%, lactation rate decreased, and survival rate decreased to 0 in the 2 highest dose groups.
The effects on the fetus occurred only at maternally toxic concentrations of ≥ 4% sodium benzoate.
Sodium benzoate (up to 5mg/egg) was injected twice into the air sac of fertilized chicken eggs at 0 and 96 h and
incubated to hatching.35 Surviving chicks were killed and necropsied. There were no teratogenic effects reported. The LD50
was 4.74 mg/egg.
Female Wistar rats (n = 20) were orally administered sodium benzoate (0, 1.75, 8.0, or 175 mg/kg/d) during days 6 –
15 of gestation.36 On day 20 of gestation, the pups were delivered by Caesarean section. There were no differences in the
types or amounts of abnormalities observed in any of the treatment groups compared to the control. The fetal and maternal
NOAEL was 175 mg/kg.
The study above was repeated with mice (n = 20), hamsters (n = 21, 22; gestation days 6 - 10), and rabbits (n = 10).
Similar results were reported.
Benzoic Acid
In oral teratogencity studies, benzoic acid increased the number of resorptions at ≥ 30 mg/kg/d and increased the
number of fetal malformations at > 600 mg/kg/d results in hamsters. Results were negative in 2 rat studies up to 500
mg/kg/d.1
Cross bred white mice (n = 25 males, 25 females) were orally administered benzoic acid (40 mg/kg/d) for 8 months
before breeding.30 This was continued for 5 generations. The parental and F1 cohorts had increased mortality compared to
controls after a 5-day 100% food restriction test. Otherwise, there were no effects on reproduction.
GENOTOXICITY
In an Ames test, methyl benzoate was not genotoxic to S. typhimurium or E. coli. Benzoic acid and sodium benzoate
were not genotoxic in several assays.
Methyl Benzoate
In an Ames test using S. typhimurium (TA97, TA98, TA100, TA1535, and TA1537), methyl benzoate (6666
µg/plate) was not mutagenic with or without metabolic activation.37 Methyl benzoate (dose not reported) was not found to be
mutagenic using E. coli (Sd-4-73).38
Benzoic Acid and Sodium Benzoate
Benzoic acid was negative in several Ames tests using Salmonella typhimurium (including TA98, TA100, TA1535,
4
CIR Panel Book Page 11
TA1537 and TA 1538) with and without metabolic activation.39-43
In one Ames test using S. typhimurium (TA98 and TA100), benzoic acid (0.1 mg/plate) and sodium benzoate (0.1
mg/plate) were genotoxic with activation.42 In a reverse mutation test, benzoic acid (5 mg/disc) was positive for
genotoxicity.
In a sister chromatid exchange assay using human lymphocytes, benzoic acid (0 – 2.0 mM) was not genotoxic with
metabolic activation.40
In a chromosomal aberration test using Chinese hamster fibroblasts, benzoic acid (1.5 mg/ml) and sodium benzoate
(2.0 mg/ml) had positive results without metabolic activation.46
Sodium benzoate was positive without metabolic activation and negative with metabolic activation in a reverse
mutation assay using Bacillus subtilis.41 Benzoic acid (1.5 mg/ml) was positive in a chromosomal aberration test without
metabolic activation.
In a sister chromatid exchange assay using hamster lung fibroblasts, sodium benzoate was not clastogenic without
metabolic activation.44
CARCINOGENICITY
Orally administered methyl benzoate (80 mg/kg/d) to mice increased tumor growth compared to controls. Benzoic
acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair dye.
Methyl Benzoate
In a 1970 review, it was reported that cross bred white mice (n = 100) orally administered methyl benzoate (80
mg/kg/d) had increased tumor growth compared to controls.30
Benzoic Acid
Benzoic acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair
dye.1
CLINICAL ASSESSMENT OF SAFETY
Absorption, Distribution, Metabolism, and Excretion
There were no human absorption, distribution, metabolism, and excretion data discovered for alkyl benzoates.
Toxicity
Benzoic Acid
In clinical studies, toxic symptoms (metabolic acidosis, central neural depression, respiratory distress progressing to
gasping respiration, hypotension, renal failure, and occasional seizures and intracranial hemorrhages) were observed
following doses far exceeding the acceptable daily intake (ADI) established by the World Health Organization.1
Dermal Irritation
C12-15 Alkyl benzoate was not irritating at 100%. In multiple clinical studies, the benzoates were recognized to
produce non-immunologic contact uriticaria or non-immunologic immediate contact reactions, but it was not clear whether
the reactions are histamine or prostaglandin mediated.
C12-15 Alkyl Benzoate
In an irritation study, C12-15 alkyl benzoate (0, 3%, 10%, 30, and 100% in vegetable oil) was applied to the backs
of subjects (n = 21) under occlusion for 48 h.45 No signs of irritation were observed at 48 and 72 h.
Benzoic Acid
In multiple clinical studies, the benzoates were recognized to produce non-immunologic contact uriticaria or nonimmunologic immediate contact reactions, but it was not clear whether the reactions are histamine or prostaglandin
mediated.1
Dermal Sensitization
In HIRPTs, C12-15 alkyl benzoate at 100%, isostearyl benzoate at 0.95%, and octyldodecyl benzoate at 0.4% were
not sensitizing. In 4 studies, test for the sensitization of benzoic acid were negative.
C12-15 Alkyl Benzoate
A human repeated insult patch test (HRIPT; n = 101) was conducted on C12-15 alkyl benzoate (100%).46 There
were no visible reactions to the test substance observed.
Isostearyl Benzoate
A HRIPT (n = 107) was conducted on a body lotion product containing isostearyl benzoate (0.95%) under semiocclusion.47 Except for one subject, who also reacted to several other test substances on the shared panel, there were no
visible reactions to the product containing isostearyl benzoate at 0.95%.
Octyldodecyl Benzoate
An HRIPT (n = 105) was conducted on a shaving cream product containing octyldodecyl benzoate (4%) under semiocclusion.48 The product was diluted to a 10% aqueous solution. There were no visible reactions to the product containing
octyldodecyl benzoate at 0.4%.
5
CIR Panel Book Page 12
Benzoic Acid
In 4 studies, tests for the sensitization of benzoic acid were negative.1
Phototoxicity
Benzoic Acid
In several studies, phototoxicity and photosensitivity tests of benzoic acid were negative up to 0.2%.1
SUMMARY
This is a safety assessment of alkyl benzoates that are used in cosmetics. Alkyl benzoates are mostly used as skinconditioning agents, preservatives, solvents, and plasticizers. In general, the alkyl benzoates can be produced industrially via
esterification of benzoic acid. The manufacturing processes of the benzoic esters are typically high yielding (>90%) and
easily purified (e.g., by distillation). The esters, acids and alcohols can be analyzed using gas chromatography/mass
spectroscopy (GCMS), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectroscopy and infrared (IR)
spectroscopy.
The total number of uses of C12-15 alkyl benzoate was 971 (858 leave-on and 113 rinse-off products) at
concentrations up to 35% and 50% in leave-on and rinse-off products, respectively. The highest concentrations of use for
C16-17 alkyl benzoates, stearyl benzoate, behenyl benzoate, ethyl benzoate, isobutyl benzoate, isostearyl benzoate, methyl
benzoate, and octyldodecyl benzoate were reported to be from 0.01% to 4%. No uses or concentrations of use were reported
for propyl benzoate, butyl benzoate, amyl benzoate, lauryl/myristyl benzoate, isopropyl benzoate, ethylhexyl benzoate,
butyloctyl benzoate, and hexyldecyl benzoate.
Benzoate esters are metabolized into benzoic acid (and the corresponding alcohols) and further metabolized to
benzoyl glucuronide and benzoyl CoA.18 The benzoyl CoA metabolizes into hippuric acid, the principal metabolite excreted
in the urine. Dermally applied benzoic acid is also excreted in the urine within 24 h. There were no human absorption,
distribution, metabolism, and excretion data discovered for alkyl benzoates.
The oral LD50 of methyl benzoate was 2170 mg/kg for rabbits, 4100 mg/kg for guinea pigs, 1350-3500 for rats, and
3000-3330 mg/kg for mice. The oral LD50 of ethyl benzoate was 2630 mg/kg for rabbits and 2100-6480 mg/kg for rats. The
oral LD50 of isopropyl benzoate was 3730 mg/kg and 3685 mg/kg for isobutyl benzoate for rats. The dermal LD50 of methyl
benzoate was ≥ 2000 mg/kg for rabbits.
Benzoic acid and sodium benzoate were toxic to rats and mice at doses > 1% in short-tem oral studies . Benzoic
acid was toxic to mice at 80 mg/kg/d in chronic oral studies. Sodium benzoate at 880 mg/kg/d incorporated into the feed of
rats for 18 – 24 months was not toxic.
Isostearyl benzoate was rated as a slight ocular irritant in two in vitro tests. Methyl benzoate, ethyl benzoate, propyl
benzoate, and butyl benzoate at 100% are dermally irritating to rabbits.There were no animal dermal sensitization data
discovered on alkyl benzoates in this safety assessment.
Studies of sodium benzoic acid and sodium benzoate showed no reproductive or developmental toxicity. Effects
noted were at a maternally toxic concentration of > 4% sodium benzoate.
Methyl benzoate was not genotoxic to S. typhimurium or E. coli. Benzoic acid and sodium benzoate were not
genotoxic in several assays. Orally administered methyl benzoate (80 mg/kg/d) to mice increased tumor growth compared to
controls. Benzoic acid was negative for carcinogenicity when dermally applied to mice at 0.016% in a non-oxidative hair
dye.
In humans, C12-15 Alkyl benzoate was not irritating at 100%. In multiple clinical studies, the benzoates were
recognized to produce non-immunologic contact uriticaria or non-immunologic immediate contact reactions, but it was not
clear whether the reactions are histamine or prostaglandin mediated. In HIRPTs, C12-15 alkyl benzoate at 100%, isostearyl
benzoate at 0.95%, and octyldodecyl benzoate at 0.4% were not sensitizing. In 4 studies, tests for the sensitization of benzoic
acid were negative.
6
CIR Panel Book Page 13
TABLES AND FIGURES
Ingredient
Alkyl Benzoates
Methyl Benzoate
Table 1. Definitions, functions and structures of alkyl benzoate and alcohol ingredients in this safety assessment.
CAS No.
Definition
Function(s)
Technical names
Trade names
93-58-3
Methyl benzoate is the
ester of methyl alcohol
and benzoic acid that
conforms to the formula
in Figure 1.
Ethyl benzoate is the
ester of ethyl alcohol and
benzoic acid.
Propyl benzoate is the
ester of n-propyl alcohol
and benzoic acid.
Butyl benzoate is the
ester of butyl alcohol and
benzoic acid.
Amyl benzoate is the
ester of amyl alcohol and
benzoic acid that
conforms to the formula
in Figure 1.
Lauryl/Myristyl benzoate
is the organic compound
that conforms to the
formula in Figure 1.
Benzoic Acid, Methyl
Ester;
Methyl
Benzenecarboxylate
Methyl benzoate (RIFM)
Benzoic Acid, Ethyl
Ester;
Ethyl benzoate (RIFM)
Benzoic Acid, n-Propyl
Ester;
Propyl benzoate (RIFM)
Benzoic Acid, n-Butyl
Ester;
Butyl benzoate (RIFM)
Benzoic Acid, Pentyl
Ester;
Pentyl Benzoate
Pentyl benzoate (RIFM)
Morflex Methyl Benzoate
Skin-conditioning
agentmiscellaneous
-
Corum 5014
AEC C12-15 Alkyl
Benzoate;
Botanester AB;
Cetiol AB;
Corum 5012;
Crodamol AB;
Crodamol AB;
Dub B1215;
Finsolv TN;
Hest 25B;
Liponate NEB;
OriStar AKB;
Saboderm AB;
Sterol B 125;
Tegosoft TN;
Tegosoft TN 2
Finsolv G-2
Fragrance
ingredient, skinconditioning agentemollient, solvent
Fragrance
-
Ethyl Benzoate
93-89-0
Propyl Benzoate
2315-68-6
Butyl Benzoate
136-60-7
Amyl Benzoate
2049-96-9
Lauryl/ Myristyl
Benzoate
No CAS No.
C12-15 Alkyl
Benzoate
68411-27-8
C12-15 alkyl benzoate is
the mixture of esters of
benzoic acid and C12-15
alcohols.
Skin-conditioning
agents - emollient
Alkyl (C12-C15)
Benzoate;
Benzoic Acid, C12-15
Alkyl Esters;
C12-15 Alcohols
Benzoate
C16-17 Alkyl
Benzoate
669700-05-2
Skin-conditioning
agents-emollient,
solvent
-
Stearyl Benzoate
10578-34-4
Skin-conditioning
agent-emollient,
solvent
103403-38-9
Benzoic Acid, Octadecyl
Ester;
Benzoic Acid, Stearyl
Ester;
Octadecyl Benzoate
Benzoic Acid, Docosyl
Ester
Dub PG;
Finsolv 116
Behenyl Benzoate
C16-17 alkyl benzoate is
a mixture of esters of
C16-17 alcohols and
benzoic acid that
conforms generally to the
formula in Figure 1.
Stearyl benzoate is the
ester of stearyl alcohol
and benzoic acid that
conforms to the formula
in Figure 1.
Behenyl benzoate is the
ester of behenyl alcohol
and benzoic acid that
conforms to the formula
in Figure 1.
Isopropyl benzoate is the
ester of isopropyl alcohol
and benzoic acid.
Fragrance
ingredient
Benzoic Acid, Isopropyl
Ester;
Benzoic Acid, 1Methylethyl Ester;
Isopropyl benzoate
(RIFM);
1-Methylethyl Benzoate
-
Branched Alkyl Benzoates
Isopropyl
939-48-0
Benzoate
Fragrance
ingredient,
preservative
Fragrance
ingredient,
preservative
Fragrance
ingredient
Skin-conditioning
agent – emollient
7
CIR Panel Book Page 14
-
Finsolv 137
Ingredient
Isobutyl Benzoate
Isostearyl
Benzoate
Ethylhexyl
Benzoate
Butyloctyl
Benzoate
Hexyldecyl
Benzoate
Octyldodecyl
Benzoate
Table 1. Definitions, functions and structures of alkyl benzoate and alcohol ingredients in this safety assessment.
CAS No.
Definition
Function(s)
Technical names
Trade names
Fragrance
Benzoic Acid, Isobutyl
120-50-3
Isobutyl benzoate is the
ingredient, solvent
Ester;
ester of isobutyl alcohol
Benzoic Acid, 2and benzoic acid.
Methylpropyl Ester;
Isobutyl benzoate
(RIFM);
2-Methylpropyl Benzoate
Finsolv SB
34364-24-4
Isostearyl benzoate is the
Skin-conditioning
Benzoic Acid,
ester of isostearyl alcohol
agent-emollient
Isooctadecyl Ester;
and benzoic acid.
Benzoic Acid, Isostearyl
Ester
Bernel Ester OB;
Benzoic Acid, 2Skin-conditioning
5444-75-7
Ethylhexyl benzoate is
Finsolv EB
Ethylhexyl Ester;
agent-emollient,
the ester of 22-Ethylhexyl Benzoate
solvent
ethylhexanol and benzoic
Octyl Benzoate
acid.
Benzoic Acid, 21888038-97-3 Butyloctyl benzoate is the Plasticizer; skinconditioning agentButyloctyl Ester
organic compound that
emollient, solvent
conforms to the formula
in Figure 2.
Plasticizer, skin163883-40-7
Hexyldecyl benzoate is
Benzoic Acid, 2conditioning agentthe organic compound
Hexyldecyl Ester
emollient, solvent
that conforms to the
formula in Figure 2.
Skin-conditioning
108347-89-3
Octyldodecyl benzoate is
Benzoic Acid, 2Finsolv BOD
agent-emollient
the ester of
Octyldodecyl Ester
octyldodecanol and
benzoic acid.
8
CIR Panel Book Page 15
Methyl
Benzoate
Table 2. Physical and Chemical properties of the acetate ingredients.9,9,11,49,49
Butyl Benzoate
Amyl Benzoate
Ethyl
Propyl
Benzoate
Benzoate
Lauryl/Myristal
Benzoate
CAS No.
93-58-3
93-89-0
2315-68-6
136-60-7
2049-96-9
-
Molecular
Weight (g/mol)
Boiling Point
(°C)
Density (g/cm3)
136.15
150.17
164.20
178.23
192.25
290.44/318.49
198.6
212.9
230.0
247.3
248
225 (Lauryl at 20 mmHg)
1.09
1.04
1.04
1.00
0.95
0.93(Lauryl)
Vapor pressure
(mm Hg @
20°C)
Solubility
(g/1000g water
@ 20°C)
Log Kow
0.38
0.267
0.136
0.01
0.009
-
2.1
0.72
0.351
0.059
0.028
-
2.12
2.64
3.01
3.84
4.16 (est.)
7.23 (est. Lauryl)
C12-15 Alkyl
Benzoate
C16-17 Alkyl
Benzoate
Stearyl
Benzoate
Behenyl
Benzoate
Isopropyl Benzoate
Isobutyl Benzoate
68411-27-8
667900-05-2
10578-34-4
103403-38-9
939-48-0
120-50-3
Molecular
Weight (g/mol)
Boiling Point
(°C)
Density (g/cm3)
290.44-332.52
346.55-360.57
374.60
430.71
164.20
178.23
363 (est.)
-
433 (est.)
518.3
266
237
-
-
-
0.908
-
1.02
Vapor pressure
(mm Hg @
20°C)
Solubility
(g/1000g water
@ 20°C)
Log Kow
0.00001
(est.)
-
0.00000006
(est.)
0.00000000007
0.161 (est.)
0.0417 (est.)
0.000009
(est.)
-
0.000009
(est.)
0.0000007
0.126 (est.)
0.098 (est.)
7.23 (est.)
-
10.18 (est.)
13.35
3.18
3.23 (est.)
Isostearyl
Benzoate
Ethylhexyl
Benzoate
Butyloctyl
Benzoate
Hexyldecyl
Benzoate
Octyldodecyl Benzoate
34364-24-4
5444-75-7
188038-97-3
163883-40-7
108347-89-3
374.60
234.33
290.44
346.55
402.65
426 (est.)
169-170 (at 20
mmHg)
376.9
434.8
449 (est.)
-
0.91
0.939
0.923
-
CAS No.
CAS No.
Molecular
Weight (g/mol)
Boiling Point
(°C)
Density (g/cm3)
Vapor pressure
0.0000001
0.0005 (est.)
0.000007
0.00000009
0.00000002 (est.)
(mm Hg @
20°C)
0.00058
0.000015
0.000001 (est.)
0.00001
0.0011
Solubility
(g/1000g water
@ 25°C)
Log Kow
10.10 (est.)
5.7 (est.)
7.857
9.982
11.09 (est.)
est.= Values were estimated using the EPI Suite, Version 4.0 program or Advanced Chemistry Development (ACD/Labs) Software V11.02.
- Not found
9
CIR Panel Book Page 16
Table 3. Frequency of use according to duration and exposure.15,16
Use type
Total/range
Duration of use
Leave-on
Rinse-off
Exposure type
Eye area
Possible
ingestion
Inhalation
Dermal
Deodorant
(underarm)
Hair –
noncoloring
Hair – coloring
Nail
Mucous
Membrane
Bath products
Infant
Total/range
Duration of use
Leave-on
Rinse-off
Exposure type
Eye area
Possible
ingestion
Inhalation
Dermal
Deodorant
(underarm)
Hair –
noncoloring
Hair – coloring
Nail
Mucous
Membrane
Bath products
Infant
Total
uses/use
type
36,808
Concentration
Uses
(%)
C12-15 Alkyl benzoate
971
0.0008-50
Concentration
Uses
(%)
C16-17 Alkyl benzoate
2
0.7
Concentration
Uses
(%)
Ethyl benzoate
0.0008-0.01
Concentration
Uses
(%)
Isobutyl benzoate
0.01
23,788
13,020
858
113
0.0008-35
0.3-50
2
0.7
-
0.0008-0.01
-
-
0.01
-
3663
69
0.0008-11
-
-
-
-
-
-
872
66
3-16
-
-
-
-
-
-
3447
26,863
25
870
0.3-12
0.0008-50
2
0.7
-
0.003-0.01
0.0008-0.01
-
0.01
0.01
623
6
0.004
-
-
-
-
-
-
5687
98
0.3-35
-
-
-
-
-
-
2808
674
2
0.5-2
0.008-10
-
-
-
-
-
-
3732
12
0.01-0.04
2
0.7
-
-
-
-
745
357
9
10
-
-
-
-
-
-
36,808
Isostearyl benzoate
1
1
-
Methyl benzoate
0.0005-0.3
23,788
13,020
1
-
-
-
0.007-0.3
-
3-4
3
-
2
-
3663
-
-
-
0.0005-0.3
-
-
-
-
872
-
-
-
-
-
-
-
-
3447
26,863
1
1
-
-
0.3
0.0005-0.3
-
3-4
1
2
623
-
-
-
0.004
-
-
-
-
5687
-
-
-
-
-
-
-
-
2808
674
-
-
-
-
-
-
-
-
3732
-
-
-
-
-
3
-
-
745
357
-
-
-
-
-
2
-
10
CIR Panel Book Page 17
Octyldodecyl benzoate
3-4
Stearyl benzoate
3
2
Figure 1. Straight chain alkyl benzoates: structures, esterase metabolism, and metabolites.
11
CIR Panel Book Page 18
Figure 2. Branched-chain alkyl benzoates: structures, esterase metabolism, and metabolites.
Legend
Ingredients which are part of this
review
Safe as used
* Not in ICI Dictionary and
Handbook, 13th Ed.
**under
Ingredients which are concurrently
review in another report
Result of Esterase metabolism
O
Benzoic acid
**
OH
**Isopropyl alcohol
Isopropyl benzoate
O
CH3
HO
CH3
CH3
O
Isobutyl alcohol
HO
CH3
O
*
Isobutyl benzoate
CH3
O
CH3
CH3
CH3
Isostearyl benzoate
O (one example of the mixture of branched chains)
CH3
O
CH3
Isostearyl alcohol
CH3
HO
CH3
O
Ethylhexyl alcohol
*
HO
Ethylhexyl benzoate
O
CH3
CH3
O
Butyloctyl alcohol
Butyloctyl benzoate
CH3
O
*
HO
CH3
CH3
CH3
CH3
CH3
O
Hexyldecyl alcohol
*
HO
Hexyldecyl benzoate
CH3
O
CH3
CH3
CH3
O
Octyldodecyl alcohol
*
HO
Octyldodecyl benzoate
CH3
O
CH3
CH3
CH3
Figure 3. The synthesis of butyl benzoate.
O
O
OH
HO
CH3
H2SO4
Δ
12
CIR Panel Book Page 19
O
CH3
REFERENCES
1.
Andersen FA. Final report on the safety assessment of benzyl alcohol, benzoic acid, and sodium benzoate. International Journal of
Toxicology. 2001;20(Suppl 3):23-50.
2.
Andersen, F. A. Final Report on the Safety Assessment of Methyl Alcohol. International Journal of Toxicology. 2001;20((Suppl. 1)):57-85.
3.
Andersen, F. A. Alcohol Denat., including SD Alcohol 3-A, SD Alcohol 30, SD Alcohol 39, SD Alcohol 39-B, SD Alcohol 39-C, SD Alcohol
40, SD Alcohl 40-B, and SD Alcohol 40-C, and the Denatonium Bezoate, Quassin, and Brucine Sulfate/Brucine. 2008 CIR
Compedium. 2005;9-12.
4.
Andersen, F. A. n-Butyl Alcohol Amended Report. 2008 CIR Compedium. 2005;49-51.
5.
Elder RL. Final report on the safety assessment of cetearyl alcohol, cetyl alcohol, isostearyl alcohol, myristyl alcohol, and behenyl alcohol.
Journal of the American College of Toxicology. 1988;7(3):359-413.
6.
Elder, R. L. Final Report on the Safety Assessment of Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl
Alcohol. Journal of the American College of Toxicology. 1988;7(3):359-413.
7.
Elder, R. L. and El. Final Report on the Safety Assessment of Stearyl Alcohol, Oleyl Alcohol, and Octyl Dodecanol. Journal of the American
College of Toxicology. 1985;4(5):1-29.
8.
Riemenschneider, W. Organic Esters. 2002. 6th:(12): pp.305-328. New York: Wiley-VCH.
9.
Richard J Lewis Sr. Hawley's Condensed Chemical Dictionary. 2007.
10.
Robert S.Huss, Fengrong Chen, Michael F.Malone, and Michael F.Doherty. Reactive Distillation for Methyl Acetate Production. Computers
and Chemical Engineering. 2003;271855-1866.
11.
Falbe, J., Bahrmann, H., Lipps, W., and Mayer, D. Aliphatic Alcohols. 2002. 6:(2): pp.19-46. New York: Wiley-VCH.
12.
T.Veit. Biocatalysis for the Production of Cosmetic Ingredients. Engineering in Life Sciences. 11-2-2004. 4:(6): pp.508-511.
13.
Sales specification sheet. Vertellus Specialties. 2010.
14.
International Programme on Chemical Safety.Butyl Acetates. http://www.inchem.org/documents/cicads/cicads/cicad64.htm. Accessed 1120-2009.
15.
Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database. 2010. Washington, DC: FDA.
16.
Personal Care Products Council. 7-8-2010. Concentration of use C12-15 alkyl benzoate, amyl benzoate, behenyl benzoate, butyl benzoate,
butyloctyl benzoate, C16-17 benzoate, ethyl benzoate, ethylhexyl benzoate, hexydecyl benzoate, isobutyl benzoate, isopropyl
benzoate, isostearyl benzoate, lauryl/myristyl benzoate, methyl benzoate, octyldodecyl benzoate, propyl benzoate and
stearylbenzoate.
17.
Commission of the European Comunities. COMMISSION DIRECTIVE 2007/17/EC of 22 March 2007 amending Council Directive
76/768/EEC, concerning cosmetic products, for the purposes of adapting Annexes III and VI thereto to technical progress . 2007.
Commision Directive 2007/17/EC:
18.
Adams TB, Cohen SM, Doull J, Feron VJ, Goodman JI, Marnett LJ, Munro IC, Portoghese PS, Smith RL, Waddell WJ, and Wagner BM.
The FEMA GRAS assessment of benzyl derivatives used as flavor ingredients. Food and Chemical Toxicology. 2005;431207-1240.
19.
Dahl, A. R., Miller, S. C., and Petridou-Fischer, J. Carboxylesterases in the respiratory tracts of rabbits, rats and Syrian hamsters. Toxicol
Lett. 1987;36(2):129-136.
20.
Longland, R. C., Shilling, W. H., and Gangolli, S. D. The hydrolysis of flavouring esters by artificial gastrointestinal juices and rat tissue
preparations. Toxicology. 1977;8(2):197-204.
21.
Kirk-Othmer Concise Encyclopedia of Chemical Technology. 4 ed. New York, NY: Wiley, 2001.
22.
Shen, Y. and West, C. Toxicity of aromatic aerobic biotransformation products of toluene to hela cells. Bulletin.of
Environmental.Contamination.and Toxicology. 1998;60(2):177-184.
23.
Chipley, J. R. and Uraih, N. Inhibition of Aspergillus growth and aflatoxin release by derivatives of benzoic acid.
Appl.Environ.Microbiol.%1980., Aug. 40(2):352-7.(2:352-7):Applied.
24.
Graham BE and Kuizenga MH. Toxicity studies on benzyl benzoate and related benzyl compounds. Journal of Pharmacology and
Experimental Therapeutics. 1945;84358-362.
13
CIR Panel Book Page 20
25.
Jenner PM, Hagan EC, Taylor JM, Cook EL, and Fitzhugh OG. Food flavorings and compounds of related structure I. Acute oral toxicity.
Food Cosmet.Toxicol. 1964;2327-343.
26.
Kravets-Bekker AA and Ivanova OP. Sanitary-toxicological characteristics of methyl benzoate and potassium benzoate. Farmacevtski
Vestnik. 1970;2125-129.
27.
Smyth Jr.HF, Carpenter CP, and Weil CS. Range finding toxicity data: List V. Archives of Industrial Hygiene and Occupational Medicine.
1954;4119-122.
28.
Merriman TN. An acute dermal toxicity study in rabbits with methyl benzoate (C-2000) Final report. Springborn Laboratories, Inc. 1995.
Report No. 3206.347.
29.
Smyth Jr.HF, Carpenter CP, and Weil CS. Range finding toxicity data: List IV. Archives of Industrial Hygiene and Occupational Medicine.
1951;4119-122.
30.
Shtenberg AJ and Ignat'ev AD. Toxicological evaluation of some combinations of food preservatives. Food Cosmet.Toxicol. 1970;8369-380.
31.
Sodemoto Y and Enomoto M. Report of carcinogenesis bioassay of sodium benzoate in rats: absence of carcinogenicity of sodium benzoate
in rats. Journal of Environmental Pathology and Toxicology. 1980;487-95.
32.
Skin Research Dpt. 2005. Assessment of the eye irritating potential of a cosmetic product (body lotion containing 0.95% isostearyl benzoate)
through alternative methods to the Draize test.
33.
Branca, M., Garcovich, A., Linfante, L. D., Macr&igrave, A, Mantovani, A., Olivetti, G., and Salvatore, G. Macro- and microscopic
alterations in 2 rabbit skin regions following topically repeated applications of benzoic acid n-alkyl esters. Contact
Dermatitis.%1988., Nov. 1988;19(5):320-34.(5:320-34):Contact.
34.
Onodera H, Ogiu T, Matsuoka C, Furuta K, Takeuchi M, Oono Y Kubota T, Miyahara M, Maekawa A, and Odashima S. Studies on effects
of sodium benzoate on fetuses and offspring of Wistar rats. Bull Nat Inst Hyg Sci. 1978;9647-55.
35.
Verrett MJ, Scott WF, Reynaldo EF, Alterman EK, and Thomas CA. Toxicity and teratogencity of food additive chemicals in the
developing chicken embryo. Toxicol Appl Pharmacol. 1980;56265-273.
36.
Morgareidge K. Teratologic evaluation of FDA 71-37 (sodium benzoate). U.S. Food and Drug Administration. 1972. Report No. PB-221
777.
37.
Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. Salmonella mutagenicity tests: V. Results from the testing of 311
chemicals. Environ.Mol.Mutagen.%1992.;19.Suppl 21:2-141. 1992.
38.
Szybalski W. Special microbial systems. II. Observations on chemical mutagenesis in microorganisms. Annals of the New York Academy of
Sciences. 1958;475-489.
39.
Andersen FA. Final report on the safety assessment of benzyl alcohol, banzoic acid, and sodium benzoate. International Journal of
Toxicology. 2001;20(Suppl. 3):23-50.
40.
Jansson T, Curvall M, Hedin A, and Enzell CR. In vitro studies of biological effects of cigarette smake condensate. II. Induction of sisterchromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents. Mutation Research. 1986;169129-139.
41.
Results of recent studies on the relevance of various short-term screening tests in carcinogenicity evaluation. 1980. The Predictive Value of
Short-Term Screening Tests in Carcinogencity Evaluation. Williams GM, Kroes R, Waaijers HW, and van de Pol KW.
42.
Kuboyama N and Fujii A. Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal
species. J Nihon Univ Sch Dent. 1992;34(3):183-195.
43.
McCAnn J, Choi E, Yamasaki E, and Ames BN. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300
chemicals. Proceedings of the National Academy of Sciences USA. 1975;72(12):5135-5139.
44.
Cooperative programe on short-term assays for carcinogencity in Japan. 1980. Molecular and Cellular Aspect of Carcinogen Screening
Tests. Montesano R, Bartsch H, and Tomatis L. Lyon, France: International Agency for Research on Cancer Scientific
Publications.
45.
Consumer Product Testing Co. 48 Hour patch test of C-SAT 020093 (C12-15 Alkyl benzoate). 2003. Report No. C02-1224.01.04. pp. 1-10.
46.
Consumer Product Testing Co. Repeated insult patch test of C-SAT 020093 (C12-15 alkyl benzoate). 2003. Report No. C02-1224.01.04. pp.
1-14.
47.
Consumer Product Testing Co. 2005. Repeated insult patch test of a body lotion containing 0.95% isostearyl benzoate. Experiment
reference number C05-0728.01.
14
CIR Panel Book Page 21
48.
Personal Care Products Council. 2010. Summary of an HRIPT of a Shaving Cream Product containig 4% octyldodecyl benzoate.
49.
The Merck Index. http://themerckindex.cambridgesoft.com/TheMerckIndex/index.asp. Accessed 10-20-2009.
15
CIR Panel Book Page 22
Data
Personal Care
Products Council
Committed to Safety,
Quality & Innovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDIENT REVIEW (CIR)
-
FROM:
John Bailey, Ph.D.
Industry Liaison to the Cifi Expert Panel
DATE:
July 9, 2010
SUBJECT:
Summary of an HRIPT of a Shaving Cream Product containing 4% Octyldodecyl
Benzoate
A shaving cream product, containing 4% Octyldodecyl Benzoate, was tested in an NRIPT by a 3rd
party testing facility. The test included 105 panelists, was semi-occlusive, and the product was diluted
to a 10% aqueous solution before application. The results of HR]PT “did not demonstrate a potential
for eliciting dermal irritation or sensitization.” More specifically, for all 105 panelist no visible skin
reaction was observed over the testing period.
11011 7th Street, N.W., Suite 300
Washington, D.C. 20036-4702
202.331.1770
CIR Panel Book Page 23
202.331.1969 (fax)
www.personalcarecouncil.org
Personal Care
Products Council
Committed to Safety,
ua ty nnovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDIENT REVIEW (CIR)
-
-‘FROM:
‘JohnBai1ey,Ph.D
Industry Liaison to the CIR Expert Panel
DATE:
July9, 2010
SIJEJECT:
Studies of C 12-15 Alkyl Benzoate
Cognis. 2007. Cetiol AB (C 12-15 Alkyl Benzoate) data sheet.
Cognis. 2002. UV absorption spectrum of Cetiol AB (C 12-15 Alkyl Benzoate).
Consumer Product Testing Co. 2003. 48 Hour patch test of C-SAT 020093 (C 12-15 Alkyl Benzoate).
Experiment Reference Number C02- 1224.01.04.
Consumer Product Testing Co. 2003. Repeated insult patch test of C-SAT 020093 (C 12-15 Alkyl
Benzoate). Experiment Reference Number C02- 1225.01.
11011 7th Street, N.W., Suite 3O0 Washington, D.C. 20036-4702
202.331.1770
CIR Panel Book Page 24
202.331.1969 (fax)
www.personalcarecouncil.org
cgnisUom
caIs
CETIOL® AB
Labeling information
INCI name(s)
Cl 2-15 Alkyl Benzoate (EU 200612571EC)
Cl 2-15 Alkyl Benzoate (CTFA)
Registrations
Ingredient
CASR-No.
6841.1 -27-8
EINECS/ELINCS-No.
270-112-4
Officially listed in I Quality conforms to
JCIC:
012-15 Alkyl Benzoate (Ingredient Code 512004)
Product properties
Appearance
Cetiol® AB is a clear, almost colourless, almost odourless oil.
Example of use
Cetiol® AB is a traditional emollient for modern skin care applications. Particularly suitable for suncare
formulations due to its outstanding solubilizing capacities for crystalline UV filters as well as dispersing
properties for pigments.
Characteristic values
The specifications stated in the paragraphs ‘Quality control data and Additional product descriptive
data’ finally and conclusively describe the properties of the Product.
Provisional quality control data
(Data which is used for quality release and is certified for each batch.)
Appearance
conforms to standard
Odour
conforms to standard
Acid value
max. 0.5 mg KOH/g
ISO 660
Saponification value
172- 182 mg KOH/g
ISO 3657
Colour value (APHA)
max. 50
ISO 6271
Density (20°C)
0.920 0.940 g/ml
ISO 2811-3
Refractive index (20°C)
1.4830 1.4870
ISO 6320
Water (Karl Fischer)
max. 0.3
ISO 4317
-
-
Provisional additional product descriptive data
(Data which is proven statistically but not determined regularly.)
Viscosity (20°C)
max. 30 mPas
Cloud point
max. 5°C
-
ISO 12058-1
ISO 3015
Revision-No. 2-08.2007
All products in the text marked with an ® are trademarks of the Cognis group.
The information on product specifications provided herein is only binding to the extent confirmed by Cognis in a written Sales
Agreement. COGNIS EXPRESSLY DISCLAIMS ANY RESPONSIBILITY FOR THE SUITABILITY OF THE PRODUCTS FOR ANY
SPECIFIC OR PARTICULAR PURPOSES INTENDED BY THE USER. Suggestions for the use and application of the products and
guide formulations are given for information purposes only and without commitment. Such suggestions do not release cognis’
customers from testing the products as to their suitability for the customer’s intended processes and purposes. Cognis does not
assume any liability or risk involved in the use of its products as the conditions of use are beyond its control. The user of the
products is solely responsible for compliance with all laws and regulations applying to the use of the products, including intellectual
property rights of third parties.
IB.08.2008
CETIOLrABE
CIR Panel Book Page 25
25
30
35
40
45
50,0
ABS
20
Is
10
5
200,0
250
300
-
S
350
I
450
NM
I
500
——
Cog-02-07141, Cetiol AB
400
_
C 1
__
550
650
A I k yI iS
600
2
—
e
700,0
CIR Panel Book Page 26
Consumer Product Testing Co.
L&E I)75
FINAL REPORT
COLQOO
—‘p’
CLIENT:
Cognis Deutsehiand GmbH & Co. KG.
Henkelstr. 67
D-4055 1
Duesseldorf, Germany
ATTENTION:
Mr. Peter Wierich
TEST:
48 Hour Patch Test
Protocol No.: CZKH-OOl
TEST MATERIALS:
.01
.02
.03
.04
C-SAT
C-SAT
C-SAT
C-SAT
020093 (diluted 3% in vegetable oil)
020093 (diluted 10% in vegetable oil)
020093 (diluted 30% in vegetable oil)
020093
j t3e,.oci+C
1
/.Ik
EXPERIMENT
REFERENCE NUMBER:
C02-1224.01-.04
Richard R. Eisenberg,
Board Certified Dermatologist
-
Robert W. Shanahan, Ph.D.
Principal Investigator
4yFfank, R.N.
‘study Director
This report is submitted for the exclusive use at the person, partnership, or corporation to whom it is addressed, and neither the report nor tie
name of these Laboratories nor any member of its staff. may be used in connection with the advertising or sale of any product or process
without written authorization.
7() Ntv 1)uth I,rlll(’
•
IfirfiC1(I. Ncv ,Jcrsc\’ 07004-2514
•
CIR Panel Book Page 27
(973) 808-7111
•
1x (973) 808-7234
Consumer Product Testing Co.
[Si.
75
QUALITY ASSURANCE UNIT STATEMENT
Study No.: C02-1224.0l-.04
The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical
laboratory studies. These studies have been performed with adherence to ICH Guideline E6 for Good
Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to
standard operating procedures and applicable protocols. The QAU maintains copies of study protocols
and standard operating procedures and has inspected this study on the date(s) listed below. The
findings of these inspections have been reported to management and the Study Director. All materials
and data pertinent to this study will be stored in the Archive Facility at 70 New Dutch Lane, Fairfield,
New Jersey, 07004, unless specified otherwise, in writing by the Sponsor.
January 14, 2003
Date(s) of inspection:
Senior personnel involved:
Laura A. Artiles, M.A.
-
Manager, Quality Assurance
Marie Terlizzese, M.S.
Quality Assurance Associate
The representative signature of the Quality Assurance Unit signifies that this study has been performed
in accordance with standard operating procedures and study protocol as well as government regulations
regarding such procedures and protocols.
7() New Dutch Lane
•
Fairfield. New Jersey 07004-2514 • (973) 808-71 1 I • Fax 197:3) 808-7234
• Toxicology • Analytical Chemistry • Microbiology
Clinical
CIR Panel Book Page 28
Cognis Deutscbland GmbH & Co. KG.
C02-1224.0l-.04
Page 3
Objective:
To determine by epidermal contact the primary irritation potential of a
test material.
Participants:
Twenty-two (22) subjects, male and female, ranging in age from 20 to 73
years, who qualified were selected for this evaluation. Twenty-one (21)
subjects completed this study. The remaining subject discontinued her
participation for personal reasons unrelated to the use of the test
materials.
Inclusion Criteria:
a. Male and female subjects, age a and over.
b. Absence of any visible skin disease which might be confused with a
skin reaction from the test materials.
c. Prohibition of use of topical or systemic steroids andlor antihistamines
for at least seven days prior to study initiation.
d. Completion of a Medical History form and the understanding and
signing of an Informed Consent form.
e. Considered reliable and capable of following directions.
Exclusion Criteria:
a. Ill health.
b. Under a doctor’s care or taking medication(s) which could influence
the outcome of the study.
c. Females who are pregnant or nursing.
d. A history of adverse reactions to cosmetics or other personal care
products.
Test Materials:
.01
.02
.03
.04
Study Schedule:
Panel #
Initiation Date
Completion Date
20020613
December 31, 2002
January 3, 2003
C-SAT 020093 (diluted 3% in vegetable oil)
C-SAT 020093 (diluted 10% in vegetable oil)
C-SAT 020093 (diluted 30% in vegetable oil)
C-SAT 020093
aWjth parental or guardian consent
CIR Panel Book Page 29
Cognis Deutschland GmbH & Co. KG.
C02-1224.0l-.04
Page 4
Methodology:
Prior to the initiation of this study, the test material was prepared as 3%,
10%, and 30% dilutions, using vegetable oil.
The upper back between the scapulae served as the treatment area.
Approximately 0.2 ml of the diluted, as well as an undiluted sample of the
test material, or an amount sufficient to cover the contact surface, was
applied to the 3/4” x 3/4” absorbent pad portion of adhesive dressings*.
When secured to the appropriate treatment site, these dressings formed
occlusive patches.
The test materials remained in contact with the skin for a total of fortyeight hours. These sites were then evaluated for gross changes. Absence
of any visible skin change was assigned a zero value. The test sites were
re-evaluated at seventy-two hours.
Evaluation Key:
0
=
+
=
1
2
3
4
Results:
=
=
=
=
No visible skin reaction
Barely perceptible or spotty erythema
Mild erythema covering most of the test site
Moderate erythema, possible presence of mild edema
Marked erythema, possible edema
Severe erythema, possible edema, vesiculation, bullae andlor
ulceration
The results of each participant are appended (Tables 1-4).
Observations of all treated areas remained negative throughout the test
interval.
Summary:
Under the conditions of this study, the test material, C-SAT 020093,
applied neat and at 3%, 10%, and 30% dilutions in vegetable oil, did not
indicate a potential for dermal irritation.
*Manufactured by TruMed Technologies, Inc., Bumsville, MN
CIR Panel Book Page 30
Cognis Deutschland GmbH & Co. KG.
C02-1224.01-.04
Page 5
Table 1
Panel #20020613
Individual Results
C-SAT 020093 (diluted 3% in vegetable oil)
Subject
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
DNC
=
Observations
48 Hours
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Did not complete study
CIR Panel Book Page 31
72 Hours
0
0
0
0
0
0
0
0
DNC
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutschland GmbH & Co. KG.
C02-1224.01-.04
Page 6
Table 2
Panel #20020613
Individual Results
C-SAT 020093 (diluted 10% in vegetable oil)
Subject
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
DNC
=
Observations
48 Hours
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Did not complete study
CIR Panel Book Page 32
72 Hours
0
0
0
0
0
0
0
0
DNC
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutsehiand GmbH & Co. KG.
C02-1224.01-.04
Page 7
Table 3
Panel #20020613
Individual Results
C-SAT 020093 (diluted 30% in vegetable oil)
Subject
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
DNC
=
Observations
48 Hours
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Did not complete study
CIR Panel Book Page 33
72 Hours
0
0
0
0
0
0
0
0
DNC
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutschland GmbH & Co. KG.
C02-1224.0l-.04
Page 8
Table 4
Panel #200206 13
Individual Results
C-SAT 020093
Subject
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
DNC
=
Observations
48 Hours
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Did not complete study
CIR Panel Book Page 34
72 Hours
0
0
0
0
0
0
0
0
DNC
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutsohiand GmbH & Co. KG.
C02-1224.01-.04
Page 9
Table 5
Panel #20020613
Subject Data
Subject
Number
Initials
Age
Sex
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
RC
BA
JE
AR
WE
CV
DC
LE
CD
CO
AW
PR
LD
EV
TV
KS
SW
PM
KH
BW
LB
CW
23
46
57
31
63
35
43
47
20
34
73
55
38
34
36
64
36
43
39
63
56
56
F
F
F
M
M
F
F
F
F
F
F
M
F
F
M
F
F
M
F
F
F
F
CIR Panel Book Page 35
Substanz-Verwaltung CRT
aktuel?er User
IS
CNPeter Wierich/OU= DE/OUEMEAIO=Cognis
C-SAT-Nr
020093
Chemische
Bezeich nung
Trivial Name
S-GehaIt [%]
Losungsmittel
(Neben-)
Komponenten
Verunreinigungen
C12/15 Alkylbenzoate
Cetiol AB
100
Identiflzierung der Prufsubstanz
Herstelldatum
Verfallsdatum
01.09.2002
01.09.2003
Batch
RIS
S’s
CAS
CD22660003
51 Zi 200670000
lOslich
in/suspendierbar n
Farbe
Aggregatzustand bel
RT
pH-Wert
fluchtig bei RT
ParaffinOl, native Ole
68411 -27-8
farbios
Flussigkeit
Lagerung
CCC N.Mertscheit
Auftraggeber
ProduktbetreuerKleber
Gefah renhinweise (z. B. qiftiqlreizend/tzend/entzundIichIexpIosiv)
Weitere Informationen zur PrUfsubstanz
CIR Panel Book Page 36
C
(:92000
-
Consumer Product Testing Co.
EST, 1970
FINAL REPORT
CLIENT:
Cognis Deutschland GnibH & Co. KG.
Henkelstr. 67
D-4055 I
Duesseldorf, Germany
ATTENTION:
Mr. Peter Wierich
TEST:
Repeated Insult Patch Test
Protocol No.: 1.01
TEST MATERIAL:
C-SAT 020093
EXPERIMENT
REFERENCE NUMBER:
C02-1225.01
Richard R. Eisenberg, MIS.
Board Certified Dermatologist
Principal Investigator
J/Frk, R.N.
Study Director
This report is submitted for the exclusive use of the person. partnership, or corporation to whom it is addressed, and neither the report nor the
name of these Laboratories nor any member of its staff, may be used in connection with the advertising or sale of any product or process
without written authorization.
70 New Dutch Lane
•
Fairfield, New Jersey 07004-2514
•
(973) 808-71 11
CIR Panel Book Page 37
•
Fax (973) 808-7234
QUALITY ASSURANCE UNIT STATEMENT
Study No.: C02-1225.01
The objective of the Quality Assurance Unit (QAU) is to monitor the conduct and reporting of clinical
laboratory studies. These studies have been performed with adherence to ICH Guideline E6 for Good
Clinical Practice and requirements provided for in 21 CFR parts 50 and 56 and in accordance to standard
operating procedures and applicable protocols. The QAU maintains copies of study protocols and
standard operating procedures and has inspected this study on the date(s) listed below. The findings of
these inspections have been reported to management and the Study Director. All materials and data
pertinent to this study will be stored in the Archive Facility at 70 New Dutch Lane, Fairfield, New
Jersey, 07004, unless specified otherwise, in writing by the Sponsor.
Date(s) of inspection:
January 20, 2003
February 26, 2003
February 14, 2003
March 10,2003
March 11, 2003
Senior personnel involved:
Richard Hettenbach
Senior Director, Regulatory Affairs and Quality Assurance
Marie Terlizzese, M.S.
Quality Assurance Associate
The representative signature of the Quality Assurance Unit signifies that this study has been performed
in accordance with standard operating procedures and study protocol as well as government regulations
regarding such procedures and protocols.
CIR Panel Book Page 38
CognisDeutschland GmbH & Co. KG.
C02- 1225.01
Page 3
Objective:
To determine by repetitive epidermal contact the potential of a test material
to induce primary or cumulative irritation and/or allergic contact
sensitization.
Participants:
One hundered and twelve (112) qualified subjects, male and female, ranging
in age from 16 to 79 years, were selected for this evaluation. One hundered
and one (101) subjects completed this study. The remaining subjects
discontinued their participation for various reasons, none of which were
related to the application of the test material.
Inclusion Criteria:
a.
b.
c.
d.
e.
Exclusion Criteria:
a.
b.
c.
d.
a
Male and female subjects, age 16
and over.
Absence of any visible skin disease which might be confused with a skin
reaction from the test material.
Prohibition of use of topical or systemic steroids and/or antihistamines
for at least seven days prior to study initiation.
Completion of a Medical History form and the understanding and
signing of an Informed Consent form.
Considered reliable and capable of following directions.
Ill health.
Under a doctor’s care or taldng medication(s) which could influence the
outcome of the study.
Females who are pregnant or nursing.
A history of adverse reactions to cosmetics or other personal care
products.
Test Material:
C-SAT 020093
Study Schedule:
Panel #
Initiation Date
Completion Date
20030013
20030022
January 13, 2003
January 20, 2003
February 21, 2003
February 27, 2003
aWith parental or guardian consent
CIR Panel Book Page 39
Cognis Deutschland GmbH & Co. KG.
C02-1225.Ol
Page 4
Methodology
The upper back between the scapulae served as the treatment area.
Approximately 0.2 ml of the test material, or an amount sufficient to cover
the contact surface, was applied to the 3/4” x 3/4” absorbent pad portion of an
adhesive dressing*. This was then applied to the appropriate treatment site to
form an occluded patch.
Induction Phase:
Patches were applied three (3) times per week (e.g., Monday, Wednesday,
and Friday) for a total of nine (9) applications. The site was marked to ensure
the continuity of patch application. Following supervised removal and
scoring of the first Induction patch, participants were instructed to remove all
subsequent Induction patches at home, twenty-four hours after application.
The evaluation of this site was made again just prior to re-application. If a
participant was unable to report for an assigned test day, one (1) makeup day
was permitted. This day was added to the Induction period.
With the exception of the first supervised Induction Patch reading, if any test
site exhibited a moderate (2-level) reaction during the Induction Phase,
application was moved to an adjacent area. Applications are discontinued for
the remainder of this test phase, if a moderate (2-level) reaction was observed
on this new test site. Applications would also be discontinued if marked (3level) or severe (4-level) reactivity was noted.
Rest periods consisted of twenty-four hours following each Tuesday and
Thursday removal, and forty-eight hours following each Saturday removal.
Challenge Phase:
Approximately two (2) weeks after the final Induction patch application, a
Challenge patch was applied to a virgin test site adjacent to the original
Induction patch site, following the same procedure described for Induction.
The patch was removed and the site scored at the clinic twenty-four and
seventy-two hours post-application
*Manufactured by TruMed Technologies, Inc., Burnsville, MN
CIR Panel Book Page 40
Cognis Deutschland GmbH & Co. KG.
C02-1225.Ol
Page 5
Evaluation Key:
0
+
1
2
3
4
Results:
=
=
=
=
No visible skin reaction
Barely perceptible or spotty erythema
Mild erythema covering most of the test site
Moderate erythema, possible presence of mild edema
Marked erythema, possible edema
Severe erythema, possible edema, vesiculation, bullae and/or
ulceration
The results of each participant are appended (Table 1).
Observations remained within normal limits throughout the test interval.
Summary:
Under the conditions of this study, test material, C-SAT 020093, did not
indicate a potential for dermal irritation or allergic contact sensitization.
CIR Panel Book Page 41
Cognis Deutsehiand GmbH & Co. KG.
C02-1225.01
Page 6
Table 1
Panel #20030013
Individual Results
C-SAT 020093
Subject
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
24
-*
—
24*hr
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
=
=
1
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
—-----
0
0
0
0.
0
0
0
0
0
0
0
Induction Phase
6
4
5
—
7
8
9
Virgin Challenge
Site
24*hr 72br
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-DID NOT COMPLETE STUDY.
0
0
0
0
0
0
-DID NOT COMPLETE STUDY.
—-DID NOT COMPLETE STUDY
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
----—-----—---DID NOT COMPLETE STUDY
0
0
0
0
0
0
0
0
0
0
0
0
Supervised removal of l Induction and Challenge Patch
Subject unable to report as scheduled, instructed to remove patch and report on the next test day.
CIR Panel Book Page 42
-*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
---
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutschland GmbH & Co. KG.
C02-1225.01
Page 7
Table 1
(continued)
Panel #200300 13
Individual Results
C-SAT 020093
Subject
Number
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
24*
1
2
——---Induction Phase
3
4
6
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
24*hr
—
=
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
—
7
—--
8
9
Virgin Challenge
Site
24*hr 72 hr
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-----DID NOT COMPLETE STUDY---0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0’
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
DID NOT COMPLETE STUDY
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
DID NOT COMPLETE STUDY--0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Supervised removal of 1 Induction and Challenge Patch
CIR Panel Book Page 43
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Cogiiis Deutsohiand GmbH
C02-1225.01
Page 8
& Co. KG.
Table 1
(continued)
Panel #20030022
Individual Results
C-SAT 020093
Subject
Number
-
0
0
0
0
0
24*
*
W
24*hr
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
-
—-—-—----—
=
=
=
1
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Induction Phase--—--—
4
6
7
5
0
0
0
0
0
0
0
0
0
O
0
O
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
O
0
O
O
0
0
0
0
0
0
0
0
0
0
0
0
o
O
0
0
0
0
———
—-—--
8
Ow
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Ow
0
o
0
o
0
0
0
0
0
0
0
0
0
0
0
0
Ow
0
0
0
DID NOT COMPLETE STUDY
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ow
0
o
0
Ow
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ow
t1nduction and Challenge Patch
Supervised removal
Subject not present for supervised patch removal
Observation recorded 96 hours post challenge application. Subject
unable to report as scheduled.
Inclement weather. Subject unable to report as scheduled and was
instructed to report on the next scheduled test day.
CIR Panel Book Page 44
Virgin Challenge
Site
24*1w 721w
O
0
O
O
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
O
0
0
o
0
0
0
0
o
o
0
Cognis Deutsehiand GinbH & Co. KG.
C02-1 225.01
Page 9
Table 1
(continued)
Panel #20030022
Individual Results
C-SAT 020093
Virgin Challenge
Subject
Number
29
30
0
0
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
24*
DNC
W
24*hr
0
0
0
0
0
0
0
0
0
0
31
32
33
34
35
36
37
38
39
40
41
=
=
=
1
2
3
Induction Phase--—----—
7
6
5
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
—----—---—---—--—
-
o
o
o
o
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
o
------
0
0
0
+
0
—
8
—--
9
0
0
0
0
0
Ow
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
O
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NOT
COMPLETE
STUDY
DID
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
O
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
DID NOT COMPLETE STUDY--0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Supervised removal of l Induction and Challenge Patch
Did not complete study
Inclement weather. Subject unable to report as scheduled and was
instructed to report on the next scheduled test day.
CIR Panel Book Page 45
Site
24*hr 72 hr
O
0
0
O
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
O
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Cognis Deutschland GmbH & Co. KG.
C02-1225.01
Page 10
Table 2
Panel #20030013
Subject Data
Subject
Number
Initials
Age
Sex
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
SC
TJ
JS
WF
AL
DK
DW
CC
WM
CS
TM
AT
NP
RA
LS
MM
SV
EM
SM
MT
FP
ID
CT
DE
PR
PF
TM
AS
54
33
51
49
56
44
46
66
34
41
74
27
35
36
39
33
33
46
26
58
63
79
34
47
63
77
69
70
F
M
F
F
F
F
F
F
F
F
F
F
M
F
F
M
M
F
F
F
F
F
F
F
F
F
F
F
CIR Panel Book Page 46
Cognis Deutschland GmbH & Co. KG.
C02-1225.01
Page 11
Table 2
(continued)
Panel #20030013
Subiect Data
Subject
Number
Initials
Age
Sex
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
ES
SE
NE
PH
MR
DN
RG
AT
QA
RG
GG
MG
AA
DM
MM
KF
KP
PC
DK
DT
SK
SM
YL
KG
JS
JP
BC
JM
‘39
50
18
31
36
21
45
58
49
55
67
31
45
47
44
45
45
36
19
31
45
55
39
38
62
53
35
66
M
F
F
F
F
F
M
M
F
F
M
F
M
F
F
F
F
F
F
F
F
F
M
F
M
M
M
M
CIR Panel Book Page 47
Cognis Deutschland GmbH & Co. KG.
C02-1225.01
Page 12
Table 2
(continued)
Panel #20030022
Subject Data
Subject
Number
Initials
Age
Sex
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
TC
KF
AK
EH
KC
AC
DD
FD
BT
LC
GT
MW
LG
JB
JG
BH
PS
MV
JC
JS
PS
DM
DF
SB
VS
JD
LF
IC
18
45
72
51
23
27
47
53
46
32
40
48
40
16
44
25
28
47
45
44
17
22
69
52
28
42
36
59
F
F
F
M
F
M
F
M
F
F
F
F
F
F
M
M
F
F
F
F
M
M
F
F
F
M
F
F
CIR Panel Book Page 48
Cognis Deutschland GmbH & Co. KG.
C02-1225.0l
Page 13
Table 2
(continued)
Panel #20030022
Subject Data
Subject
Number
Initials
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
PH
RN
1V
GD
VA
ES
DS
RC
PS
ML
EA
GA
SM
SS
WB
JC
DR
LR
PD
PL
TB
CA
CS
EH
EA
CC
AS
SW
Age
Sex
58
74
52
47
48
48
39
63
26
.67
41
27
40
51
39
74
36
69
63
73
53
37
31
68
63
55
34
52
M
F
F
F
M
M
F
M
F
F
F
M
F
F
F
F
F
F
F
F
M
F
F
M
F
F
F
F
CIR Panel Book Page 49
Substanz-Verwaltung CRT
aktueller User
CN=Peter Wierich!OU=DE/OU=EMEAIO=Cognis
C-SAT-Nr
020093
Chemische
Bezeichnung
rrivial Name
S-Ge halt [%)
LOsungsmittel
(Neben-)
Komponenten
Verunreinigungen
C12115 Alkylbenzoate
Cetiol AB
100
Identifizierung der Prufsubstanz
Herstelldatum
Verfallsdatum
01.09.2002
01.09.2003
Batch
RIS
sLs
CAS
CD22660003
51 ZI 200670000
lslich
inlsuspendierbar in
Farbe
Aggregatzustand bei
RT
pH-Wert
flochtig bei RT
ParaffinOl, native Ole
68411 -27-8
farblos
FlOssigkeit
I
Lagerung
CCC N.Mertscheit
Auftraggeber
ProduktbetreuerKleber
Gefahrenhinweise (z.B. giftig/reizend!Lzend/enUndIich/expIosiv)
Weitere Informationen zur PrUfsubstanz
CIR Panel Book Page 50
Personal Care
Products Council
Committed to Safety,
Quality & Innovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDIENT REVIEW (CW)
-
FROM:
John Bailey, Ph.D.
Industry Liaison to the CIR Expert Panel
DATE:
July 9, 2010
SUBJECT:
Studies on a Body Lotion Containing 0.95% Isostearyl Benzoate
Consumer Product Testing Co. 2005. Repeated insult patch test of a body lotion containing 0.95%
Isostearyl Benzoate. Experiment Reference Number C05-0728 .01.
Skin Research Dpt. 2005. Assessment of the eye irritating potential of a cosmetic product (body lotion
containing 0.95% Isostearyl Benzoate) through alternative methods to the Draize test. Report
reference: CTOXJO5 161.
1101 17th Street, N.W., Suite 30O Washington, D.C. 20036-4702
202.331.1770
CIR Panel Book Page 51
I
202.331.1969 (fax)
www.personakarecouncil.org
Consumer Product Testing Co.
I-sr
FINAL RE PORT
CLIENT:
-
ew Jersey 07950-2451
ATTENTION:
James Flanagan
Sr. Cherr4ist Microbiology
TEST:
Repeated Insult Patch Test
ProtocoiNo.: 1.01
TEST MATERIAL:
i
Body Lotion TL45-.24-2
•
EXPERIMENT
REFERENCE NUMBER:
9ç
irJ
i3irnzc’c1-c
C05-072.01
Richard R. Eisenberg, M.D.
Board Cprtified Dermatologist
P-ank,
(‘xeLitive Vice President, Clinical Evaluations
Report Date:
‘O// 472
This report is submitted Jor the exetusive use of the person, parfnershi4, or corporation
to whom it ‘a addressed, and neither the report nor the
nrrrne of these Laboratories 001 any membor of its staff, may
be use in corinsciron with the edverltsin ot safe of any pcoduct or process
wittiest written authorization
() N(v IhItcIl Lant
•
Iiirfirki New ,Jers t45 14
t$73) O-7 II
CIR Panel Book Page 52
Iax (97;lf 8O-7234
Consumer Product Testing Co.
QUALITY ASSURANCE UNIT STATEMENT
Study No.: C05-0729.01
The objective of the Quality Assurance Unit (QkU) is to monitor the conduct and reporting of clinical
laboratory studies. These studies have been performed with adherence to the applicable ICH Guideline
E6 for Good Clinical Practice and requiremenuts provided for in 21 CFR parts 50 and 56 and in
accordance to standard operating procedures and applicable protocols. The QAU maintains copies of
study protocols and standard operating procedures and has inspected this study. All data pertinent to this
study will be stored in the Consumer Product Testing Company archive, unless speciiied otherwise, in
writing by the Sponsor.
Quality Assurance personnel involved:
)cdkô
Qu
ty Assurance
/o/i4
Date
The representative signature of the Quality Assurance Unit signifies that this study has been performed
in accordance with standard operating procedures and study protocol as well as government regulations
regarding such procedures and protocols.
70 New Duth Lane
FirfieIc1, New Jersw O704-2 14 973) 8Q-7 I 11 • Fax ($73) 8O87234
(linicI • Toxicology
\fliI\tiCI Chcmiry
Microbiology
CIR Panel Book Page 53
Page 3
Objective:
To determine by repetitive epidermal contact the potential of a test material
to induce primary or cumulative irritation and!or allergic contact
sensitization.
Participants:
One hundred fifteen (115) qualified subjects, male and female, ranging in age
from 16 to 78 years. rere selected for this evaluation. One hundred seven
(107) subjects completed this study. The remaining subjects discontinued
their participation for various reasons, none of which were related to the
application of the test material.
Inclusion Criteria:
a.
b.
d.
e.
Exclusion Criteria:
a.
b.
c.
d.
a
Male and female subjects, age 16
and over.
Absence of any viibIe skin disease which might be confused with a skin
reaction from the test material.
Prohibition of use of topical or systemic steroids andlor antihistamines
for at least seven dkys prior to study initiation.
Completion of a Medical History form and the understanding and
signing of an Informed Consent form.
Considered reliable and capable of following directions.
01 health.
Under a doctor’s care or taking medication(s) which could influence the
outcome of the study.
Females who are pregnant or nursing.
A history of advçrse reactions to cosmetics or other personal care
products.
Test Material:
Study Schethile:
Body Lotion TL45-24-2
Panel #
1nitiaton Date
Completion Date
20050393
20050401
Augut 15. 2005
August 17, 2005
September 23, 2005
September 29, 2005
‘With parental or guardian consent
CIR Panel Book Page 54
Page 4
Methodology:
The upper back between the scapulae served as the treatment area.
Approximately 0.2 nIl of the test material, or an amount sufficient to cover
the contact surface, vas applied to the 1’ x I’ absorbent pad portion of a
clear adhesive dressijg*. This was then applied to the appropriate treatment
site to form a semi-o4elusive patch.
Induction Phase:
Patches were applied three (3) times per week (e.g., Monday, Wednesday,
and Friday) for a totai of nine (9) applications. The site was marked to ensure
the continuity of patch application. Following supervised removal and
scoring of the first Induction patch, participants were instructed to remove all
subsequent lnductioa patches at home, twenty-thur hours after application.
The evaluation of this site was made again just prior to re-application. If a
participant was unabl to report for an assigned test day, one (1) makeup day
was permitted. This day was added to the Induction period. It was noted that
due to a holiday weekend, which occurred during the Induction Phase,
subjects who requited a makeup day experienced a delay between
applications.
With the exception of the first supervised Induction Patch reading, if any test
site exhibited a moerate (2-level) reaction during the Induction Phase,
application was moved to an adjacent area. Applications are discontinued for
the remainder of this test phase, if a moderate (2-level) reaction was observed
on this new test site. Applications would also be discontinued if marked (3level) or severe (4-level) reactivity was noted.
Rest periods consisted of twenty-four hours following each Tuesday and
Thursday removal, and forty-eight hours following each Saturday removal.
Chaliene Phase:
Approximately two (2) weeks after the final Induction patch application, a
Challenge patch was. applied to a virgin test site adjacent to the original
Induction patch site, following the same procedure described for Induction.
The patch was removed and the site scored at the clinic twenty-four and
seventy-two hours post-application.
*Manufacred by TruMed Technologies, Inc.,
Burnsville, MN
CIR Panel Book Page 55
Page 5
Evaluatkrn Key:
0
=
+
=
=
2
3
4
Results:
=
=
No visible skin eaction
Barely perceptible or spotty eiythema
Mild erythema covering most of the test site
Moderate erythena, possible presence of mild edema
Marked erythema, possible edema
Severe erythemà, possible edema, vesiculation, bullae and/or
ulceration
The results of each participant are appended (Table 1).
With one exception, observations remained negative throughout the test
interval.
it was noted that Subject #25, Panel #20050393, exhibited numerous
reactions during the Induction Phase and moderate (2) to barely perceptible
(+) responses post-challenge application. This subject also reacted to a
number of test materials on this shared panel. Although his medical profile
does not reflect a histoly of allergies, it is the Laboratory’s opinion that he is
a reactive individual. He will be prohibited from participation in future patch
tests.
Suimuary:
Under the conditions f this study, test material,
Body Lotion
TL45-24-2, did not irdicate a clinically significant potential for dermal
irritation or allergic contact sensitization.
CIR Panel Book Page 56
Page 6
Table 1
Panel #20050393
1ndiviIua1 Results
1.
Subject
Number
1
2
3
4
5
6
7
8
9
10
ii
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
24*
24*hr
0
(1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Body Lotion TL45-24-2
—-———-—------—-------hdueticPhase--—-—--—--—-—----—-—---1
2
3
4
5
6
7
8
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
Virgin Challenge
Site
24*hr 72br
0
0
0
0
Q
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
q
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
O.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ol
o
0
0
0
0
0
0
0
0
0
0
0
0
--—-.-—4———DID NOT COMPLETE STUDY-------—----——
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
+
1
÷**
0
0
0
2
1
0
0’
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
Supervised removal of l Induction and Challenge Patch
96 hour follow-up evaluation
CIR Panel Book Page 57
Page 7
Tb1e I
(cotinued)
Panel #20050393
individual Results
ody Lotion TL45-24-2
Subject
Number
-----——---------——-------Inductiot Phase
24*hr L
4
6
1_ 3
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
24*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
—-——----—--
7
8
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ol
0
0
0
0
0
0
Oj
0
0
0
0
0
0
0
0
0
01
0
0
o
0
0
0
01
0
0
0
0
0
0
0
---—-—-—---P NOT COMPLETE STUDY0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
01
0
0
0
0
0
0
0
0
o
0
0
0
0
0
ol
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
ol
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
ol
0
0
OI
o
0
0
0
0
0
01
0
0
0
0
Supervised removal of 1 Induction and Challenge Patch
CIR Panel Book Page 58
Virgin Challenge
Site
24*hr72 hr
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Page 8
Table I
(continued)
Panel #20050401
Individual Results
Body Lotion TL45-24-2
Subiect
Number
3
4
6
7
$
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
26
27
28
29
24*
1
2
3
Induction Phase—----—--—-—-—
4
5
6
7
o
0
0
0
o
o
o
o
o
o
o
o
o
o
o
o
o
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-—
24*hr
o
0
0
0
0
o
0
o
0
o
o
o
o
o
o
o
o
o
o
o
o
o
1
0
0
0
0
0
o
o
o
0
0
0
—
—
Virgin Challenge
Site
24*hr 72 hr
8
9
0
0
0
—DTD NOT COMPLETE STUDYo•
o
0
0
0
o
o
0
0
0
o
o
0
0
0
o
0
o
C)
0
o
o
C)
0
0
o
o
0
0
0
o
0
o
0
0
o
o
0
0
0
o
o
0
0
0
o
o
0
0
0
o
o
0
0
0
o
o
0
0
0
o
o
0
0
0
-DID NOT COMPLETE STUDY
•-DID NOT COMPLETE STUDYo
0
0
0
0
o
0
0
0
0
o
0
0
0
0
o
O
0
0
0
o
o
0
0
0
o
0
0
0
0
o
0
0
0
0
o
O
0
0
0
o
0.
0
0
0
o
0
0
0
0
o
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
o
o
o
o
o
o
o
o
o
o
o
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
o
o
o
o
o
—
o
e
Supervised removal of 1
Induction and Challengq Patch
CIR Panel Book Page 59
0
0
0
o
0
o
o
0
0
o
0
0
o
0
o
o
0
o
0
o
()
0
o
o
o
o
0
0
Page 9
Table 1
(cohtinued)
Panel #20050401
Individual Results
Body Lotion TL45-24-2
Subject
Number
24*hr
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
24
-----——---------------------Inductio Phase—
I
2
3
4
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
C)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
000
00
q
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
Ô
01
01
01
0!
o!
0
0
0
0!
01
0
0
o!
0!
0
0
0
0
0
0
—--—i
0
0
0
—-——
7
8
9
Virgin Challenge
Site
24*hr 72 hr
0
0
0
0
0
0
0
0
0
0
o
0
-1311) NOT COMPLETE TUDY
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
-DID NOT COMPLETE STUDY0
0
0
0
0
0
0
0
0
0
0
0
o
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
DID NOT COMPLETE STUDY-—-—-——
0
0
0
0
0
0
Supervised removal of 1 Induction and Chal1eng Patch
CIR Panel Book Page 60
Page 10
liable 2
Panel 20050393
Subject Data
Subject
Number
Initials
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
LG
AK
RM
VT
KL
ND
BA
SF
MM
EG
AG
VV
HG
MA
VA
GD
CD
JO
MV
JM
CN
AB
3M
IV
AD
RD
LZ
FS
AD
64
45
43
39
40
36
39
53
58
55
22
16
72
16
51
49
25
54
42
24
63
62
68
72
55
70
43
21
53
CIR Panel Book Page 61
M
F
F
F
F
M
F
F
M
F
M
M
M
F
M
F
F
F
F
F
F
M
F
F
M
M
F
M
F
Coty, Inc.
C05-0729.0 1
Page 11
Table 2
(coitinued)
Panel #20050393
Subject Data
Subject
Number
Initials
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
GS
JD
VD
AD
RW
AL
JM
RC
MG
LS
JD
LV
GN
EM
AM
RF
HP
iF
TM
DC
MC
DC
MC
AC
EF
EV
CW
—
76
21
73
69
49
77
44
20
66
40
59
72
36
29
60
35
75
70
52
64
30
18
39
57
76
65
75
CIR Panel Book Page 62
F
M
F
M
M
F
M
M
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Page 12
‘Fable 2
(continued)
Panel #20050401
Subjct Data
Subject
Number
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Initials
JV
LP
JP
LM
DD
CM
AS
PR
LM
CD
RT
LS
AP
JH
OS
SB
RB
CL
JC
RV
EM
BL
LD
JA
MA
MP
AL
AO
-
Age
Sex
66
38
34
48
49
53
58
66
35
65
21
42
57
47
26
30
57
54
31
66
42
43
35
37
52
51
42
46
46
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
F
F
F
F
F
M
M
F
F
F
F
CIR Panel Book Page 63
Page 13
Täbie2
(continued)
Panel #20050401
Subject Data
Subject
Number
Initials
Age
Sex
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
AS
JS
RM
SM
YV
RV
CB
RF
JR
RR
VR
PF
DB
TP
MW
JS
FF
ED
AL
LL
DS
AD
BR
JS
JY
YC
HM
JB
GZ
EH
57
55
35
65
51
26
21
40
30
55
60
37
28
64
71
54
51
45
78
76
67
41
34
39
47
41
77
36
41
58
M
F
F
M
F
F
F
M
F
F
M
M
F
F
F
M
M
F
M
F
F
M
F
M
M
F
F
F
F
F
CIR Panel Book Page 64
•
Assessment of the Eye Irritating Potential
of a Cosmetic Product through
Alternative Methods
to the Draize Test
Test Product:
Reference: TL45-24-2
Body Lotion
10
Report Date: 27 September 2005
Report Ref: CTOXJO5161
o
S° S
CONCLUSION:
Taking into account the responses of the 3 alternative methods used we
consider that the estimated Draize classification of the test product might be
slightly irritant with Draize score which might range from 0 to 15
According to our experience and with respect to the type of product tested (skin
care product), we consider that this product is as well tolerated as products
belonging to the same category.
According to the estimated Draize score, the following
be proposed:
warning may
“No statement”
Olivie OUCET Pharm.
Toxicotog
UROTOX)
Head of Skin esearch Opt
.,
Skin Research Opt
D.
Cell Toxicology lab.
CIR Panel Book Page 65
p. 1126
TABLE CONTENT
1- INTRODUCTION
2- TECHNICAL INFORMATIONS
21 Product characteristics
2-2 Testing facilities
2-3 Data storage
2-4 Authentication of the study
3- ALTERNATIVE METHODS USED
3-1 The Neutral Red Release (NRR) assay
3-2 The Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM)
3-3 The Reconstituted human Epithelial Culture (REC) assay
4- RESULTS AND CONCLUSIONS
4-1 NRR assay
4-2 HET-CAM
4-3 REC assay
5- FINAL ASSESSMENT
6- REFERENCES
Skin Research Opt
Cell Toxicology lab.
CIR Panel Book Page 66
p. 21 26
1-INTRODUCTION
There is a need to evaluate the eye irritation potential of cosmetic products for
purposes of consumer safety and reguatory requirements. For the time being, the
Draize rabbit eye test is practically the nly method for determining ocular irritation,
which is acceptable to various regulatory groups.
For the last few years, a clear desire, based on both ethical and scientific grounds.
has been arising to replace the use of nimals, in cosmetic product testing. In that
respect, a wide number of in vitro or ex vivo assays have been proposed worldwide
as alternatives to the Draize test. Despite the tremendous efforts concentrated either
by the Cosmetic Toiletries and Fragrances Association (CTFA) or the European
Community (EC) and British Home Office (BHO), none of these alternative methods
have been successfully validated.
However, in some particular fields suct as eye irritation, it is clear that under the
increasing pressure of consumer associations, regulatory agencies tend to be more
and more favorable to the use of these methods for safety assessment. For instance,
the French government recently registered the Hen’s Egg Test on Chorio-AllantoIc
Membrane (HET-CAM) and the Neutral Red Release (NRR) assay as official test
methods for determining the irritating potential of cosmetic products (Journal Officiel
de Ia République Française, 26112196, Annexe IV; 30/12199, Annexe VI).
The aim of this study was to predict the eye irritation potential of formulated products.
For that purpose, we developed a particular in vitro>> approach, which combines
several alternative methods. Indeed, many international studies have clearly
demonstrated the interest of combining at least 2 or 3 alternative methods when
assessing eye irritation through in vitro tests.
Skin Research Dpi
Cell Tox,cology lab.
CIR Panel Book Page 67
P. 3/26
Taking into account both the results obtained during the last international validation
studies (Balls et a!., 1995; Gettings et aL, 1990; Gettings et at. 1994; Gettings et aL,
1996) and the recent advances in te use of in vitro models we selected as
(<relevant>) alternative methods the 3 following in vitro tests:
-
•
-
the Neutral Red Release (NRR) assay
the Hens Egg Test on the ChorioAllaritoic Membrane (HET-CAM)
the Reconstituted Human Epithelial Culture (REC) assay
The combination of these different in vitro methods allows the assessment of
different end-points and thus explores various types of mechanisms (cytotoxicity,
acute vascular effect, toxicokinetic, transepithelial absorption,...) which are generally
considered as taking part in the eye irritation phenomena (Rougier et al., 1994).
The conclusion of the study results from a global assessment, systematically based
on the responses of the 3 methods used, since none single alternative method can
predict eye irritation with a sufficient level of safety.
Skin Research Dpt
—
Cell Toxicology lab.
CIR Panel Book Page 68
p4/26
2- TECHNICAL INFORMATIONS
2-1 Product characteristics
The product
Body Lotion, ref. TL45-24-2, was received from the sponsor
Morris Plains (USA) on the 16 August 2005.
The test product, identified as a skin care froduct, is a white milk, having a pH of 7.2
at 22.2°C.
Upon receipt, it was stored at room temperature in the Cell Toxicology Laboratory.
An aliquot of the test product was stored ml a specific room of the Skin Research Dpt.
According to the internal Skin Research. procedures, it will be kept there for a
minimum period of 3 years.
2-2 Testing facilities
The test were performed in the Cell Toxicology Laboratory of Skin Research Dpt
-
2-3 Data storage
All the data relative to the study will be stored in the premises of the Skin Research
Dpt for a period of 10 years.
Skin Research Opt
Cell Toxicølogy lab,
CIR Panel Book Page 69
5 1 26
2-4 Authentication of the study
I, the undersigned Olivier DOUCET, Director of the Study, certify that this study has
been carried out in the oremises of the
-
—
—
standard protocol under the responsibility of Myléne LANVIN and Technical
Investigators of the Cell Toxicology Laboratory.
Olivie OUCET Pharim
Toxicolog
UROTOX)
Head of Skin esearch Opt
D.
I, the undersigned Mylène LANVIN, Reponsible for the Study, certify that this study
has been performed under my supervising in accordance with our internal standard
protocol.
R
arch Assistant
Head of Cell Toxcoiogy Dpt
We, the undersigned Carine LINOSSIER, Connne THILLQU, Vincent COMTE and
Boris MERLIN, Technical Investigators, certify that all the observations and numerical
data presented in this document are an accurate reflection of the results obtained
during this study.
cr4ER
Tèehfàal Investigator
Cell Toxicology Dpt
Vincent COMZ
Technical Igvator
Cell
Skin Research Opt
CorirN1OU
Tec4&I)rrestigator
C5.e1foIogy Dpi
Boris MERLIN
Technical Inve
Cell
Cell Toxicology lab.
CIR Panel Book Page 70
tor
p. 61 26
3- ALTERNATPIE METHODS USED
3-1 The Neutral Red Release (NRR) assay
Principle of the method
The NRR assay with rabbit cornea cells (SIRC) is a short-term monolayer culture test
system in which cells are first exposed to Neutral Red dye (NR) then to the test
material. According to the toxicity of the product, the cells are damaged and release
their neutral red dye. The neutral red contained in surviving cells was extract with a
revelation solution and spectrophotometrically measured. The test product
concentration that gives rise to the release of 50% NR dye (NR
) is used as
50
endpoint to reflect the cytotoxicity of the test product.
Two stages can be necessary to asses$ the NR
50 of a test product. The first Stage
allows the estimation of the NR
50 wheéas the second stage permit to accurately
assess the final score.
Materials
Chemicals: Sodium Dodecyl Sulfate (SDS) and Sodium Chloride (NaCI) were
purchased from Sigma Chemical Co. (St Louis, MO, USA). Neutral red dye was
supplied by Fluka AG (Buchs, CH). Modified Eagle’s Medium (MEM), fcetal calf
serum, antibiotics (penicillin/streptomycin 5000Ul/5000jgIml and fungizon
amphotericin B 250jglmI), MEM Non Essential Amino Acids (NEAA) and PhosphateBuffered Saline (PBS) were supplied by lnvitrogen (Cergy-Pontoise, France). Before
using, ftal calf serum was maintained in a bain-marie at 56°C during 30 minutes in
order to obtain a modified” fcatal calf serUm.
Rabbit cornea SIRC cells: Rabbit cornea fibroblasts SIRC (ATCC n°CCL6O) were
bought in the United States at ATCC (American Type Culture Collection Rockville,
Maryland, USA) through a French supplier (CERDIC, Sophia Antipolis, France). Cells
were cultured according to the internal procedures of our laboratory for freezing,
unfreezing and subculturing. Briefly, cells were maintained in medium MEM
supplemented with 2% antibiotics and 1% MEM Non Essential Amino Acids. This
completed medium was extemporaneously supplemented with 10% of ‘rnodified”
fcatal calf serum. Cells were incubated in humidified atmosphere at 37°C, 5% CO
.
2
Experimental procedure
Cell seeding: For treatment, cells were seeded in all the wells of 24-well plates. The
plates were incubated for 24 hours at 37°C, 5% CO
.
2
Skin Research Dpt
Cell Toxicology b
CIR Panel Book Page 71
p. 7126
Application of the neutral red dye: The NRR assay was carried out according to the
principle of the colorimetric test described by Borenfreund and Puerner (1984, 1985).
After incubation, I ml of neutral red solution test, centrifuged before using, was
added to each well of the plates. The plates were incubated for 3 hours at 37°C, 5%
.
2
Co
Test product dilutions: According to the physico-chemical characteristics of the test
product, the dilutions ware performed extemporaneously in an hydrophilic (wtv) or a
lipophilic substance (w/w). During the first stage, the product was tested diluted at
0%; 5%; 15%; 25%; 35% and 50%.
According to this preliminary results, some dilutions were selected for the second
stage. The principle is given in the following Table 1.
Table 1: Dilutions to be selected for the second stage
1
Stage n°1
NR5O (%)
<4
4and6
0.1
1
>6and<13
—
>17 and <23
23arid27
>27and<33
33and37
>37anci<46
46 and 50
>
50
Stagen°2
[iIutions to be tested
1
5
%)
5
10
.
5
10
15
5
20
20
25
25
30
30
35
4
10
351
15
25
—
30
35
—____
50
40
50
60
Slight cytbtoxIcity I Unnecessary Stag2
-
Test product application: After incubation for 3 hours, the neutral red solution was
removed and I ml of complete culture medium was added in each well of the plate.
The plate was maintained at room temperature for 30 minutes. Then each well was
rinsed with 2 ml of PBS and 500 p1 of ech test product dilution were added in the
same time in two wells of the plate. Aftera 55-second contact (or 25 seconds for the
positive control), each well was rinsed with PBS. The plate was gently stirring
throughout contact time.
Skin Research Dpt
Cell Toxicology lab.
CIR Panel Book Page 72
p. 8126
Revelation of the cytotoxicity: An ethanol/acetic acid/distilled water solution was then
added to each well. The plates were gently stirring during about 15 minutes until
having an homogeneous coloration. 200 .iI of the resulting solutions were put, in
dupilcate, in the wells of a 96-well plate.
Reading: The optical densities (O.D.) were read at 540 nm by using a multi-well
spectrophotometer. The ethanol/acetic acid/distilled water solution served as ‘blank’,
Control solution application: The positive control (Sodium Dodecyl Sulfate
=
SDS)
was tested diluted at 0%; 0.01%: 0.05%; 0.2% and 0.25%. The dilutions were
performed in 0.9% sodium chloride sokition. The dilution 0% which represents the
negative control was applied to the cells during 55 seconds whereas the dilutions
0.01%; 0.05%; 0.2% and 0.25% were applied during only 25 seconds.
Test scoring: Data were expressed as a percentage of cytotoxicity, compared to the
negative control (dilution 0%). The NR
50 was calculated by interpolation from the
curve representing the percentage of viability versus the concentration of test
product.
The cytotoxicity of the product was obtained from the 50
NR according to the scale
presented in Table 2.
Table 2: Cytotoxicity scale from the NR endpoint
% of death
obSerVed at the
dilution_50%
50 (%)
NR
>
20
50
>
‘
25 and
20 and
50
25
Clssiflcation
Negligible cytotoxicity (PNI)
<
50
Not very important cytotoxicity (SI)
COTY Conclusion
Slightly cytotoxic (SI)
Moderate cytotoxicity (MI)
Moderately cytotoxic (MI)
Important Cytotoxicity (I)
Cytotoxic (I)
The conforrrnty of the study was checked bY using a positive control. According to the
internal procedure, this study complied if the NR
50 of the positive control ranged from
0.127% to 0.185%.
Skin Research Opt
Cell Toxicology lab.
CIR Panel Book Page 73
p. 9126
3-2 The Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM)
Principle of the method
The Het-Cam is an in vitro method used to evaluate the irritant potential of a test
material (J.O.R.F., 26/12196, Annexe IV). The test procedure is based on the
assessment by a trained person of the immediate effects following application of test
product to the chorioallantoic membraneof 10-day-old fertile eggs. The determination
of the Net-Cam score, according to the scale described by Luepke (1985, 1986)
allows the assessment of the irritating potential of the test product.
Materials
Chemicals: Sodium Dodecyl Sulfate (SDS) and Sodium Chloride (NaCl) were
purchased from Sigma Chemical Co. :(St Louis, MO, USA). Sterilized water for
injections (Wi) was purchased from Aguettant Laboratory (Lyon, France).
Hen’s eggs: Fresh fertile White Legom hen’s eggs, weighing 50 -65 g, were supplied
by INRA (Tours, France).
Experimental procedure
Upon their arrival, all the defective eggs and eggs which weight is not ranged from 50
to 65 g, were eliminated. The hen’s eggs were incubated at 15°C during at least 48
hours. Then, they were placed, on their long axis, in a rotating incubator under a
temperature of 37.5°C ± 1°C; 60% ± 5% relative humidity (Union FrancoSuisse,
Evreux, France) for 10 days. The eggshell was removed around the airspace. After a
5 ml saline solution (NaCl 0.9% with distilled water) application and the removal of
the inner shell membrane, the vascular chorioallantoic membrane (CAM) was
exposed to the air.
Test product application: Four eggs are treated with 0.3 ml of the product, tested neat
or diluted according to the type of product. Previously maintained at a temperature of
37.5°C, the test product was applied onto the surface of the CAM. After a 20-second
contact, the membrane was gently rinsed off by using 5 ml (10 ml or more if
necessary) of saline solution kept at 37.5°C.
Investigator observations: Observations were achieved by using a specific lamp
KL1 500 electronic (SCHOTT, France) emitting a cold and white light. Blood vessels
and albumen were continuously observed by a trained person for a 5-minute period.
Irritant effects, such as hyperhaemia, haemorrhage and coagulation (opacity and/or
thrombosis), were scored according to their occurrence within the test period.
Skin Research Dpi
Cell Toxicology lab.
CIR Panel Book Page 74
p. 10126
Control solutions application: Two eggs treated with a sodium dodecyl sulfate
solution in sterilized water for injections (SDS solution) served as positive control
while at least 2 eggs treated with saline solution were used as negative controL
If the test product was diluted in mineral oil, 2 eggs were treated with this lipophilic
diluant to check its Het-Cam score.
The irritating effect of the test product (if any) was quantified according to the scoring
system described in the French regulaticn (J.O.R.F., 26(12196, Annexe IV) presented
in Table 3.
Table 3: Hat-Cam scoring syøtem according to Luepke’s scale
Vascular
effect
Q<t3O.
Time (t)
30s.<t2min.
2min.<t5min
Hyperhaemia
5
3
1
Haemorrhage
7
5
3
Coagulation
9
7
5
For each parameter (Hyperhaemia, Haemorrhage, Coagulation) the individual scores
obtained from the 4 eggs were averaged. The sum of these 3 values gave the so
called “Het-Cam score” of the test product on a scale ranging from 0 to 21.
The magnitude of the eye irritating potential of the test product was then calculated
according to the classification developed by Luepke (1985, 1986) and described in
the French regulations (J.O.R.F., 26/12/90, Annexe IV), see Table 4.
Table 4: Test product classification
Hot-Cam score
CIasslIcatIofl
Score < 1
1 Score < 5
5 Score < 9
Score 9
Practicallynon irritant
I
SlIghtly imtant
Moderately irritant
Irritant
.
COW Conclusion
Slightly irntant (SI)
Moderately irritant (Ml)
Irritant (I)
The conformity of the study is checked by using controls. According to the internal
procedure, the study complied if the Het-Cam score for the positive control ranged
from 15 to 18 and the Hat-Cam score for the negative control ranged from 0 to 1.
Skin Research Dpi
Cell Toxology lab.
CIR Panel Book Page 75
.
11/ 26
3-3 The Reconstituted human Epithellal Culture (REC) assay
Principle of the method
The REC assay is a cytotoxicity test based on a time course approach. The
formulated product is applied onto three-dimensional reconstituted human epithelial
cultures, having the feature of the epithelial part of the cornea. The quantification of
the test product cytotoxicity is performed through a colorimetric assay: the MIT test
(Mosmann, 1983). The determination of a simplified mean cytotoxicity index (SMCI)
is used to quantify the time course toxicity for the applied substance, according to the
procedure described by Doucet et al. (198).
Materials
Chemicals: Sodium Dodecyl Sulfate (SDS) and 3-(4, 5-dimethylthiazol-2-yl) 2, 5diphenyltetrazolium bromide (MiT) was purchased from Sigma Chemical Co. (St
Louis, MO, USA). Isopropanol was supplied by Carlo Erba (Milan, Italy) and
Phosphate—Buffered Saline (PBS) by Invitrogen (Cergy-Pontoise, France). Saline
solution and modified culture medium (MCDB 153) were supplied by SkinEthic
Laboratories (Nice, France).
Reconstituted human Epithelial Cultures (REC): Reconstituted human epithelial
cultures were supplied by SkinEthic Laboratories (Nice, France). They were obtained
by culturing transformed human keratinocytes (TR146 cell line) derived from
squamous carcinoma (Regnier at al., 1987; Rupniak et al., 1985).
Experimental procedure
Test product application: The product wa tested neat or diluted according to the type
of product. Test sample was directly applied onto the apical surface of the epithelial
culture. Product was gently spread with a brush. Cultures were treated with the test
product in duplicate. The cultures were transferred to a 24-well culture dish, each
well containing fresh medium MCDB 15, They were incubated at 37°C, 5%C0
2 /
95% air atmosphere for 1 hour, 3 hours and 24 hours. After each exposure time,
cultures were washed with PBS, and the MiT assay (Mosmann, 1983) was
performed. After incubation in MiT reagent, the formazari crystals were extracted by
isopropariol. Optical densities were read at 540 nm, by using a spectrophotometer
(isopropanol served as blank”).
Skin Research Dpt
-
Cell Toxicology lab.
CIR Panel Book Page 76
—
p. 12126
Control solutions application: For each time, 2 cultures treated with a sodium dodecyl
sulfate solution in saline (SDS solution) served as positive control while 2 cultures
treated with saline solution were used as negative control.
If the test product was diluted in mineral oil, 2 cultures treated with this lipophilic
diluant were used also as negative control.
The results were expressed as a percentage of cytotoxicity compared with the
negative control. The time course of toxicity for the applied product was expressed as
a cumulative simplified mean cytetoxicity index (SMCI) calculated over 24 hours, as
follows:
(%cyt. lh)
SMCI
+
(%cyt. 3h/3)
+
(%cyt. 24hl24)
=
3
with:
%cyt. lh = % of cytDtoxicity of the test product after 1 hour.
%cyt. 3h % of cytotoxicity of the test product after 3 hours.
%cyt. 24h = % of cytotoxicity of the test product after 24 hours.
The cytotoxicity of the product was determined according to the classification
presented in Table 5.
Table 5: Cytotoxicity scale from the SMCI endpoint
The conformity of the study is checked y using a positive control, According to the
internal procedure, the study compIle only when the SMCI of the positive control lies
within the confidence internal range.
The product classification in terms of eye irritation results from a global assessment
based on the responses of the 3 in vitro itiethods used. The following Table 6 reflects
this multi-technical approach and gives information about the proposed safety
classification of the test product. This latter is extrapolated from the results of the 3
alternative methods and presented as an estimated Draize classification. Based on
this, an attempt is made for issuing somespecific US warnings,
Skin Research Dpt
Cell Tocology lab.
CIR Panel Book Page 77
p. 13/26
CIR Panel Book Page 78
8
ox
3
REC Assay
Hot-Cam
NRR Assay
Score < 24.5
Score >= 9
Score <= 25
Score >= 24.5
Score > 9
Score <= 25
<
REC Assay
Hot-Cam
NRR Assay
15
NRR Assay
<=
Score < 15
Score < 9
25< Score <= 50
REC Assay
Hot-Cam
7.5
Score <7.5
Score <9
Score >50
REC Assay
Hot-Cam
NRR Assay
-____________
In Vitro Threshold
Test Method
-
50)
Strongly Irritant
(Draize score : 50.1 110)
Irritant
(Draize score : 30.1
-
Moderately Irritant
(Draize score: 15.1 30)
Avoid contact with eyes.
If contact occurs, flush with water.
No statement
US Warning
Determination of appropriate warning statements.
Suitability to market the product has to be considered.
This product may cause irritation. Avoid contact with eyes.
If contact occurs, flush with plenty of water.
It irritation persists, consult a physician.
—.-----__-—.------_---------—--—-
Slightly lrritant
(Draize score :0- 15)
Estimated European Draize
Classification
Table 6 : Table of concordance between the In vitro scores and the estimated Draize classification
for skin care products, sun care products, alcoholic products and make-up products.
4- RESULTS AND CONCLUSIONØ
4-1 NRR assay
4-1-1 Summary
The Neutral Red Release (NRR) assab’ conducted on rabbit cornea fibrobiasts
SiRC is an in vitro method currently used to assess the cytotoxicfty of a test product
after a short contact time of the test sibstance with the cells by measuring the
neutral red release from pre-loaded cells (Brantom at aL, 1997; Reader et aL, 1989).
The cytotocity is revealed by the cncentration of test product (NR) which
inhibited of 50% the cell survival and grov.th.
n this study, the procedure used was adpted from the protocol described in French
regulation as official method for the ssessment of the irritating potential of
formulated cosmetic products (JO.RR. 30112199, Annexe VI).
Under the experimental conditions used,an NR
5 supehor to 50% was obtained fo
the product
Body Lotion, ref. TL45-24-2. From this result. the test
product was considered slightly cytotoic.
4-1-2 Results
Stage 1
Th firct
f thic iirv wc inititd in
the oremises of the
19 September
2005 and was completed on the 20 Septernber2uu.
The test product
Body Lotiorli was tested diluted at 0%: 5%: 5%: 25%:
35% and 50% in saline solution.
The optical densities and the percentags of viability obtained for the test product
and the positive control are presented in Tables 7 and 8 respectie’. The graphic
assessment of the NR for the test product and the positive control were presented
in Fig. 1 and 2 respectively.
Skin Research Dp
Cell Toxipoogy lab.
CIR Panel Book Page 79
p. 15126
Table 7: Optical densities (O.D.) and cytotoxicity
(%)
obtained for the test product
and the negative control (representing the 0% dilution) during the stage n°1
(O.D.) and cytotoxicity (%) obtained for the test product
and the negative control
—
Product dilution
(%)
0
5
15
25
35
50
Well 1
0.951
0.962
0.964
0.962
0,913
0.744
Well 2
0.944
0.99
1.012
0.992
0.931
0.743
We113
Well 4
0.951
-
-
-
-
0.962
Mean
SD
0.952
0.007
Cytotoxicity (%)
0.00
-
I
-
-
-
-
-
0,97
0.021
0.988
0.034
0.977
0.021
0.922
0.744
0.013
0.001
0.00
0.00
0.00
3.15
21.90
-
100
80
>60,
:x
40:
>
C.)
20
0
10
-
•‘._
.
0
20
30
Product dilutions
40
50
(%)
Fig. 1: Assessment of the NR
50 for the test product
NR>50%
Skin Research Dpt
Cell Toxicology lab.
CIR Panel Book Page 80
p. 16/26
Table 8: Optical densities (O.D.) and cytotoxicity (%) obtained for the positive control
1
9
and the negative control (representing the 0% dilution) during the stage n
(O.D.) and cytotoxicity (%) obtained for the positive control
and the negative control
(%)
0
O.01
0.05
0.2
0.25
Well 1
Wel12
We113
Well 4
Mean
SD
1.058
1.075
1.059
1.052
1.061
0.010
i.083
1.082
0.861
0.770
0.449
0.246
0.229
-
-
-
-
-
-
-
1.083
0.001
0.816
0.064
0.430
0.028
0.238
0.012
Cytotoxicity (%)
0.00
0.00
23.14
59.52
77.62
Product dilution
100
0.410
1
80 i
60
C.)
x
0
40
0
20
0
0
0.05
0.1
0.15
Product dilutions
0,2
0.25
(%)
Fig. 2: Assessment of the NR
50 for the positive control
I
NRO.160%
Skin Research Opt
Cell Toxicology lab.
CIR Panel Book Page 81
p. 17/26
Stage 2
According to table 2 and to the results obtained during the first stage of this study, it
was not necessary to perform the stage 2
4-1-3 Conclusion
Under the experimental conditions use, the NR
50 of the product
Body Lotion was superior to 50%. Frm this result, the test product may be
considered slightly cytotoxic.
According to our experience and with respect to the type of product tested (skin care
product), we consider that this product is as well tolerated as products belonging to
the same category.
Skin Research Dpi
cell Toxióology lab.
CIR Panel Book Page 82
p. 18126
4-2 HET-CAM
4-2-1 Summary
The Hen’s Egg Test-Chorioallantoic Melnbrane (Het-Cam) is an in vitro method
currently used to assess the eye irritating potential of a test product (Balls et aL,
1995; Gettings et al., 1994). The test procedure is based on the evaluation of
immediate effects following application of:the test substance onto the surface of the
chorloallantoic membrane of 10-day-old feitile hen’s eggs.
In this study, the protocol used was adapted from Luepke (Luepke, 1985; Luepke
and Kemper, 1986) and was performed according to the method described by the
French regulation (JD.R.F., 26112196, Annexe IV). The Het-Cam score of the product
Body Lotion, ref. TL45-24-2, was determined after application to the
chorioallantoic membrane of 0.3 ml of neat test material.
Under the experimental conditions used, the Het-Cam score of the test product was
7.0. Consequently, this product may be classified as moderately irritant when applied
neat to the hen’s egg chorioallantoIc membrane.
4-2-2 Results
This study was initiated in the oremises of the
completed
on the 13 September 2005.
Date of arrival of the eggs:
Date of incubation at 15°C:
Date of incubation at 37,5°C:
30 August 2005
30 August 2005
01-02 September 2005
The individual results obtained for the test product, tested neat, the positive and the
negative controls are respectively presented in Tables 9, 10 and 11.
Skin Research Opt
Cet TxcØlogy lab,
CIR Panel Book Page 83
p 19126
Body Lotion ref. TL4S-24-2
Test product:
Table 9: Individual egg scores obtained for the test product
0
0
o
0
Mean Score
=
Classification: Moderate’y irritant
7.0
Remarks: A very slight haemorrhage re ction was observed to tre second treated
egg whereas slight ones were observed t the others treated eggs
Table 10: Individual egg
Mean Score
=
obtained for the positive control
16.0
Negative cor,troi: saline solution
Table 11: Individual egg score obtained for the negative control
Mean
Mean Score
0
0
a
0
Skin Research Opt
Cell To cology lab,
CIR Panel Book Page 84
p. 20126
4-23 Conclusion
Under the experimental conditions usd, the Het-Cam score of the product
Body Lotion was 7.0. Frcn this result, the test product may be
considered moderately irritant when app!ied neat to the hen’s egg CAM.
According to our experience and with respect to the type of product tested (skin care
product), we consider that this product is as well tolerated as products belonging to
the same category.
Skin Research Opt
C&I Thxiogy lab
CIR Panel Book Page 85
p21/26
4-3 REC assay
4-3-1 Summary
The Reconstituted human Epitheliat Culture (REC) assay is an in vitro method used
to assess the cytotoxicity of a test product through a three-dimensional epithelial
modal. After application of the test product, the cytotoxicity is evaluated by a rapid
colorimetric test: the MU test, according to the protocol described by Mosmann
(1983).
In this study, the protocol used was adapted from the test procedure described by
Doucet at al. (1998). The time course of toxicity for the applied product, expressed as
a cumulative simplified mean cytotoxicity index (SMCI) was used as endpoint.
Under the experimental conditions used, a SMCI of 1.87 was calculated. From this
result, the product
ody Lotion. ref. TL45-24-2, was considered slightly
cytotoxic when applied neat onto the reconstituted epithelial cultures.
4-3-2 Results
This study w inititd in th nrmiscs nf the I
on the 15 September 2005.
Cell cultures
Date of arrival:
Batch n°:
14 September 2005
05022B0902
Date of expiry:
Age of the culture:
23 September 2005
7 days
The REC score, expressed as a simplified mean cytotoxicity index (SMCI), obtained
for the product, tested neat is presented irilTable 12.
Table 12: REC score (SMCI) and cytotoxic potential obtained for the test product
CIR Panel Book Page 86
4-33 Conclusion
Under the experimental conditions used, he SMCI of the test product
Body Lotion was 1.87. From this result, the test product may be considered slightly
cytotoxic when applied neat onto reconstiti ted human epithelial cultures.
According to our experience and with respect to the type of product tested (skin care
product), we consider that this product is as well tolerated as products belonging to
the same category.
Skin Research Opt
Cell ToxiIogy lab.
CIR Panel Book Page 87
p. 231 2
5- FINAL ASSESSMENT
The aim of this study was to predict the ee irritation potential of formulated products
by using a particular <<in vitro>> approach, which combines several alternative
methods:
-
the Neutral Red Release (NRR) assay
the Hen’s Egg Test on the Chorio.AUantoic Membrane (HET-CAM)
the Reconstituted Human Epithelial Culture (REC) assay
For the tested product,
and classifications were obtained:
NRR assay:
HET-CAM:
REC assay:
Body Lotion, ref. TL45-24-2, the following results
50%
Score = 7.0
SMCI 1.87
50
NR
>
slightly cytotoxic
moderately irritant
slightly cytotoxic
Taking into account the responses of these 3 methods, we consider that the
estimated Draize classification of the test product might be slightly irritant with
Draize score, which might range from 0 to 15.
According to our experience and with respect to the type of product tested (skin care
product), we consider that this product is as well tolerated as products belonging to
the same category.
According to the estimated Draize score, the following
proposed:
No sta1ement”
Skin Research Dpt
CeKThxicØogy lab.
CIR Panel Book Page 88
iS warning may be
6- REFERENCES
BALLS, M, BOTHAM, P.A., BRUNER, L.H, and SPIELMANN, H. (1995). The
ECIHO International Validation Study on Alternatives to the Draize Eye Irritation Test.
Toxicology In Vitro. 9, 871-929.
-
BORENFRE1JND, E. and PUERNER, J.A. (1984). A simple quantitative procedure
using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult.
Methods. 9, 7-9.
-
BORENFREUND, E. and PUERNER, J.A. (1985). Toxicity determined in vitro by
morphological alteration and neutral red absorption. Toxicol Letters. 24, 119-124.
-
BRANTOM P.G., BRUNER L.H., CH4\MBERLAIN M. et a), (1997). A summary
report of the COLIPA international validation study on alternatives to the Draize rabbit
eye irritation test (1997). Toxicology in vilo 11, 141-179.
-
DOUCET, 0., LANVIN, M. and ZASIROW, L. (1998). A new in vitro human
epithelial model for assessing the eye. irritating potential of formulated cosmetic
products. In Vitro & Molecular Toxicology 11, 273-283.
-
GETTINGS, S. D, DIPASQUALE, L. C., BAGLEY, 0. M., et al. (1990). The CTFA
evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize
primary eye irritation test (phase I) hdro-alcoholic formulations: a preliminary
communication. In Vitro Toxicology. 3, 293-302.
-
GETTINGS, S. D., DIPASQUALE, L. Ci., BAGLEY, D. M., et al. (1994). The CTFA
evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize
primary eye irritation test (phase II) p11/water emulsions. Food and Chemical
Toxicology. 32, 943-976
-
GETTINGS, S. D., LORDO R. A., HINTZE K. L., et al. (1996). The CTFA evaluation
of alternatives program: an evaluation df in vitro alternatives to the Draize primary
eye irritation test (phase ill) surfactant-based formulations. Food and Chemical
Toxicology. 34, 79-117.
-
Skin Research Dpt
Cell Thxiology lab.
*
CIR Panel Book Page 89
p. 25! 26
JOURNAL OFFICIEL DE LA REPUBLIQUE FRANcAISE. Arrêté du 29 Novembre
1996, J.O. du 26 Décembre 1996. Annexe IV. Méthode Officielle d’Evaluation dii
Potential Irritant par Application sur Ia Membrane Chorio-AllantoIdienne de l’oeuf de
poule.
-
JOURNAL OFFICIEL DE LA REPUBL1UE FRANçAIsE. Arrêté dii 27 Décembre
1999. J.O.R.F. du 30 Décembre 1999. Annexe VI. Méthode officielle devaluation dii
potentiel irritant par application directe sur monocouche do fibroblastes de cornée de
lapin par Ia méthode do relargage dii rouge neutre.
-
LUEPKE, NP. (1985). Hen’s egg chorioallantoic membrane test for irritation
potential. Food and Chemical Toxicology 23, 287-291.
-
LUEPKE, N.P. and KEMPER, RH. (1986). Het-Cam test: an alternative to the
Draize eye test. Food and Chemical Toxicology. 24, 495-496.
-
MOSMANN, T. (1983). Rapid colorirnetric assay for cellular growth survival:
Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, 55-63.
-
READER S.J., BLACKWELL V., OHARA R., CLOTHIER R., GRIFFIN G. and
BALLS M. (1989). A vital dye release method for assaying the short-term cytotoxic
effects of chemicals and formulations. ATLA 17. 28-33.
-
REGNIER, M., DESBAS, C., BAILLY, C., and DARMON, M. (1987). Differentiation
of normal and tumoral human keratinocytes cultured on dermis. Reconstruction of
either normal or tumoral architecture. In Vitro Cell Dev, Bio. 24, 625-632.
-
ROUGIER, A., COTTIN, M., DE SILVA, 0., et al. (1994). The use of in vitro
methods in the ocular irritation assessment of cosmetic products. Toxicology In Vitro.
8, 893-905.
-
RUPNIAK, H.T., ROWLATT, C., LANE, E.B., et at. (1985). Characteristics of four
new human cell lines derived from squamous cell carcinomas of the head and neck.
J. NatI. Cancer Inst. 75, 621-635.
-
Skin Research Dpt
Cell Toxicology lab,
CIR Panel Book Page 90
p. 26)26
VCRP Data for Alkyl Benzoates
Ingredient
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
C12-15 ALKYL BENZOATE
Use Category
01B - Baby Lotions, Oils, Powders, and Creams
03A - Eyebrow Pencil
03C - Eye Shadow
03D - Eye Lotion
03E - Eye Makeup Remover
03F - Mascara
03G - Other Eye Makeup Preparations
04B - Perfumes
04E - Other Fragrance Preparation
05A - Hair Conditioner
05B - Hair Spray (aerosol fixatives)
05C - Hair Straighteners
05F - Shampoos (non-coloring)
05G - Tonics, Dressings, and Other Hair Grooming Aids
05I - Other Hair Preparations
07A - Blushers (all types)
07B - Face Powders
07C - Foundations
07D - Leg and Body Paints
07E - Lipstick
07F - Makeup Bases
07G - Rouges
07I - Other Makeup Preparations
08B - Cuticle
Cuticle Softeners
Softeners
08C - Nail Creams and Lotions
10A - Bath Soaps and Detergents
10B - Deodorants (underarm)
10E - Other Personal Cleanliness Products
11A - Aftershave Lotion
11D - Preshave Lotions (all types)
11E - Shaving Cream
11G - Other Shaving Preparation Products
12A - Cleansing
12C - Face and Neck (exc shave)
12D - Body and Hand (exc shave)
12E - Foot Powders and Sprays
12F - Moisturizing
12G - Night
12H - Paste Masks (mud packs)
12I - Skin Fresheners
12J - Other Skin Care Preps
13A - Suntan Gels, Creams, and Liquids
13B - Indoor Tanning Preparations
13C - Other Suntan Preparations
CIR Panel Book Page 91
No. of Uses
9
1
28
20
1
1
18
1
13
12
6
3
2
42
33
16
26
42
1
66
7
1
17
1
1
11
6
1
15
1
5
3
30
91
102
5
174
35
11
3
48
17
36
9
C16-17 ALKYL BENZOATE
10A - Bath Soaps and Detergents
2
ISOSTEARYL BENZOATE
12C - Face and Neck (exc shave)
1
STEARYL BENZOATE
STEARYL BENZOATE
01B - Baby Lotions, Oils, Powders, and Creams
07C - Foundations
2
1
CIR Panel Book Page 92
Personal Care’
Products Council
Committed to Safety,
Quality & Innovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDIENT REVIEW (CIR)
-
FROM:
John Bailey, Ph.D.
Industry Liaison to the CIR Expert Panel
DATE:
July 26, 2010
SUBJECT:
Comments on the Scientific Literature Review on Alkyl Benzoates
The following 10 substances were included in an HPV submission to EPA.. It is not clear if this
submission (found at http ://www .epa. gov!chemrtklpubs/summaries/benzylde/c 1 3450tc.htm)
was reviewed for relevant information.
Benzaldehyde
p-Methoxybenzaldehyde
m-Methoxy-p-hydroxybenzaldehyde
Benzyl acetate
Benzyl benzoate
Methyl benzoate
Methyl p-methylbenzoate
Methyl 2-hydroxybenzoate
Pentyl 2-hydroxybenzoate
Benzyl 2-hydroxybenzoate
p.2 As reference 12 was published in 2004, the meaning of “began a couple of years ago” is not clear,
and will become less clear after the CIR report is published.
p.2 It would be helpful if the product categories in which use was reported in the Council survey were
also included in the text. Rather than stating that no uses were reported for the rest of the
ingredients, it would be helpful if the names of the ingredients for which no uses were reported
were stated.
p.2 The statement about the permitted uses of methanol in the EU suggests that the EU regulations
were checked for all the ingredients in the report. Please see EU Annex VI entry la for
permitted uses of Methyl Benzoate, Ethyl Benzoate, Propyl Benzoate and Butyl Benzoate as
preservatives.
p.2 As the alcohols are not included in the report. The information about alcohols can be deleted
from the Non-Cosmetic Use section. If this information is left in the report, a reference should
be added for the use of isopropyl alcohol as a topical antimicrobial.
p.3 It is not clear why the discussion regarding metabolism of acetates is included in this report on
benzoates.
-
-
-
-
-
11011 7th Street, N.W., Suite 3O0 Washington, D.C. 20036-4702
202.331.1770
CIR Panel Book Page 93
202.331.1969 (fax)
www.personalcarecouncil.org
p.3 If Kirk-Othmer (reference 23) cited any primary references regarding the dermal penetration of
benzoic esters, the primary references should be obtained and cited.
p.3 Please define NI
.
50
p.3 How many rabbits were used in the dermal LD
50 study of Methyl Benzoate.
p.3 Please provide the species used in the Benzoic Acid and Sodium Benzoate studies.
p.3 How many rats were used in the chronic study of Methyl Benzoate. Although this study is under
the Methyl Benzoate subheading, and the text says “Methyl benzoate”, the title of the study
(reference 33) in the reference section indicates the study was about Sodium Benzoate. Please
state whether or not histopathological examinations of major organs were completed in this
study.
p.4 What was the concentration of Methyl Benzoate used in the dermal irritation study in rabbits.
p.4 Please correct the spelling of “strume”, “hyperkeatosis”, “straatmu”, “hyperkertosis”
p.4 The information on Benzyl Benzoate, needs to be removed from this report and added to Benzoic
Acid, Benzyl Benzoate et al. report.
p.4 Did reference 35 report any maternal effects?
p.4 Please provide the number of rats per treatment group used in reference 37.
pA Please provide the number of mice, hamsters and rabbits per group used in reference 37. On what
gestation days were these species treated?
p.5 Please provide the gestation days on which the hamsters were treated with Benzoic Acid.
p.5 It is not clear what 50.5% vs 12.5% represents, mortality of the parental and Fl cohorts combined
of controls compared to treated mice? What is meant by a “100% food restriction test”? How
long was this test?
p.5 Reference 33 is a carcinogenesis bioassay. Please provide the results concerning carcinogenicity
in the Carcinogenicity section.
p.S Please define ADI. What is the value of the ADI for Benzoic Acid?
p.S Were the studies of Benzoic Acid really clinical studies, or were they case reports?
p.5 What concentrations of Benzoic Acid were negative for sensitization, phototoxicity and
photosensitivity?
Where is the Summary for this report?
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
CIR Panel Book Page 94