Memorandum
To:
CIR Expert Panel Members and Liaisons
From:
Monice M. Fiume MMF
Senior Scientific Analyst/Writer
Date:
November 18, 2010
Subject:
Re-Review of Triethanolamine, Diethanolamine, and Ethanolamine
In 1983, the Expert Panel published a safety assessment on triethanolamine (TEA), diethanolamine
(DEA), and monoethanolamine (MEA; now named ethanolamine) with the conclusion that TEA, DEA,
and MEA are safe for use in cosmetic formulations designed for discontinuous, brief use followed by
thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin,
the concentration of ethanolamines should not exceed 5%. MEA should only be used in products that do
not contain N-nitrosating agents.
Since that 1983 assessment, new data have been published, including an NTP report on TEA that shows
some evidence of carcinogenic activity in female mice and on DEA that shows clear evidence of
carcinogenic activity in male and female mice. Subsequent to these studies, several follow-up studies to
identify a mode of action have appeared in the literature. While DEA, carcinogenicity in mice, and
mode of action in mice have been discussed at a number of Panel meetings, and industry has made
presentations on this subject, the question of a re-review was never formally raised. Therefore, this rereview package t was prepared for the Panel as a procedural follow-through.
While these three ingredients were grouped together in the original assessment, continuing to keep them
together may not be the best approach. So, three separate re-review document are being presented. If
the Panel decides that any of these ingredients should be re-opened, you have the option to re-open one,
two, or all three of the ingredients, and the report can be split or kept as one document.
As a reminder of the Panel’s discussions at past meetings, the minutes from the June 1999, June 2008,
and June 2009 meetings are being provided, as well as a synopsis of the presentation given by Dr.
William Stott, who represented the Alkanolamines Panel of the American Chemistry Council, regarding
a proposed mechanism of action for carcinogenicity and its relevance to humans. In addition to those
minutes, information/correspondence previously submitted to the CIR, as well as Dr.Andersen’s previous memos to the Panel regarding issues involving DEA, also are being provided.
Dialkyl- and dialkanolamines (including DEA) and their salts are on the European Union (EU) list of
substances which must not form part of the composition of cosmetic products, due to concern over their
readiness for nitrosamine formation. Dialkanolamines are also completely prohibited for use in
cosmetic formulations in Canada.
Monoalkylamines, monoalkanolamines (including MEA) and their salts are allowed for use with
restrictions by the EU, as are trialkylamine, trialkanolamines (including TEA), and their salts.
The CIR has issued a number of reports on TEA-, DEA-, and MEA-containing ingredients. A list of
these ingredients is attached, with the conclusion reached by the CIR, to give you an indication of the
impact this report has. (The number of TEA-, DEA-, and MEA-containing ingredients not yet reviewed
is quite extensive.)
As a final note, you will notice that the format used for these re-review documents looks different than
what you are accustomed to. The CIR is in the process of updating the presentation of data. For
example, rather than breaking Animal Toxicology section into Acute, Short-Term, Subchronic, and
Chronic studies, this information will be presented under Acute (Single Dose) Toxicity and Repeated
Dose Toxicity. Feedback on the new format is appreciated.
-2-
TEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR
TEA Dodecylbenzenesulfonate
safe as used when formulated to be non-irritating (DiscusTEA Tridecylbenzenesulfonate
sion stated that TEA was previously reviewed and found
safe as used)
TEA-EDTA
safe as used (TEA was not mentioned in Discussion)
TEA Lactate
safe for use in cosmetic products at concentrations ≤10%,
at final formulation pH > 3.5, when formulated to avoid
increasing sun sensitivity or when directions for use
include daily use of sun protection; safe for use in salon
products at concentrations ≤30%, at final formulation pH 2
3.0, in products designed for brief, discontinuous use
followed by thorough rinsing from the skin, when applied
by trained professionals, and when application is accompanied by directions for daily use of sun protection (TEA
was not mentioned in Discussion)
TEA Lauryl Sulfate
can be used without significant irritation at a final concentration thereof not exceeding 10.5%; greater concentrations may cause irritation, especially if allowed to remain
in contact with the skin for significant periods of time
(TEA was not mentioned in Discussion)
TEA Salicylate
safe as used when formulated to avoid skin irritation and
when formulated to avoid increasing the skin's sun sensitivity, or, when increased sun sensitivity would be expected,
directions for use include the daily use of sun protection
(TEA was not mentioned in Discussion)
TEA Stearate
safe as used in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing; in
products intended for prolonged contact with the skin, the
concentration of TEA Stearate should not exceed 15% in
formulation; should not be used in products under
conditions resulting in N-nitrosation reactions (Discussion
states TEA conclusion)
TEA Cocoyl Hydrolyzed Collagen
safe as used (TEA was not mentioned in Discussion)
Final Report, 2008
JACT 12(3) 1993 (orig.)
IJT 21(S2) 2002
IJT 17(S1) 1998
JACT 1(4) 1982
IJT 22 (S3) 2003
JACT 14(3) 1995
JACT 2(7) 1983
(not reopened in 2005)
DEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR
safe as used in rinse-off products and safe at concentraJACT 15(6) 1996
tions ≤10% in leave-on cosmetic products.; should not
JACT 5(5) 1986 (orig.)
be used as an ingredient in cosmetic products in which
N-nitroso compounds are formed (original Discussion
addressed nitrosation, but not levels of free DEA)
DEA Dodecylbenzenesulfonate
safe as used when formulated to be non-irritating
Final Report, 2008
(Discussion stated that DEA was previously reviewed
JACT 12(3) 1993 (orig.)
and found safe as used)
Final Report, 1995
Isostearamide DEA
safe for use in rinse-off products; in leave-on products,
Myristamide DEA
these ingredients are safe for use at concentrations that
Stearamide DEA
will limit the release of free ethanolamines to 5%, but
with a maximum use concentration of 40%; these ingredients should not be used in cosmetic products in which
N-nitroso compounds may be formed (Discussion
addressed DEA)
JACT 5(5) 1986
Lauramide DEA
safe as used; should not be used as ingredients in cosLinoleamide DEA
metic products containing nitrosating agents (original
Oleamide DEA
Discussion addressed nitrosation, but not levels of free
(Cocamide DEA. originally)
DEA)
Cocamide DEA
-3-
Acetamide MEA
Cocamide MEA
Isostearamide MEA
Myristamide MEA
Stearamide MEA
MEA Salicylate
MEA-CONTAINING INGREDIENTS REVIEWED BY THE CIR
safe at ≤7.5% in leave-on products and safe as used in
JACT 12(3) 1983
rinse-off products; cosmetic formulations containing
(not reopened in 2008)
Acetamide MEA should not contain nitrosating agents
or significant amounts of free acetamide (Discussion
addressed nitrosation)
IJT 18(S2) 1999
safe as used in rinse-off cosmetic products and safe at
concentrations up to 10% in leave-on products; should
not be used as an ingredient in cosmetic products containing N-nitrosating agents, or in product formulations
intended to be aerosolized (Discussion refers to
cocamide MEA)
safe for use in rinse-off products; in leave-on products,
Final Report, 1995
these ingredients are safe for use at concentrations that
will limit the release of free ethanolamines to 5%, but
with a maximum use concentration of 17%; these ingredients should not be used in cosmetic products in which
N-nitroso compounds may be formed (Discussion
addressed MEA)
safe as used when formulated to avoid skin irritation and IJT 22(S3) 2003
when formulated to avoid increasing the skin's sun sensitivity, or, when increased sun sensitivity would be
expected, directions for use include the daily use of sun
protection (MEA was not mentioned in Discussion)
-4-
TEA, DEA, MEA HISTORY
Original Report: In 1983, the Expert Panel determined that these ingredients were safe for use
in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing
from the surface of the skin. In products intended for prolonged contact with the skin, the
concentration of ethanolamines should not exceed 5%. Ethanolamine (MEA) should be used
only in rinse-off products. Triethanolamine (TEA) and diethanolamine (DEA) should not be used
in products containing N-nitrosating agents.
June 1999: discussed NTP carcinogenicity results; presentations were made by Dr. LehmanMcKeeman and Dr. Stott
June 2008: presentation was made by Dr. Stott; Acetamide MEA was discussed, with reference
to the MEA, DEA, TEA report
June 2009: discussed DEA carcinogenicity; mentioned that the DEA report was not be reopened
December 2010: formal rereview package was presented to the Panel
SEARCH STRATEGY – TEA/DEA/MEA
TOXLINE
PUBMED
13
83
15
21
Jan 17. 2010
DEA
111-42-2 & choline
111-42-2 & carcinogen*
choline & deficiency &
human & cancer
TEA
102-71-6 & carcinogen*
102-71-6 & choline
MEA
Jan 25, 2010
102-71-6 OR 111-42-2
(1980-current)
EU
not to be used
38
restrictions
55
5
11
2
restrictions
1003 (downloaded 58)
UPDATED SEARCH May 31, 2010
(102-71-6 OR 111-42-2 OR 141-43-5) AND (REPRODUCTI* OR TERATOGEN*) – 142 (Toxline); 41
(DART)
(102-71-6 OR 111-42-2 OR 141-43-5) AND (DEVELOPMENT* OR FETOTOX*) – 378 (Toxline); 47
(DART)
(102-71-6 OR 111-42-2 OR 141-43-5) AND TOX*
(102-71-6 OR 111-42-2 OR 141-43-5) AND (GENOTOX* OR MUTAGEN* OR CLASTOGEN*) – 286
Toxline); 7 (DART); 9 (CCRIS)
(102-71-6 OR 111-42-2 OR 141-43-5) AND (SENSITIZA* OR SENSITIZE* OR SENSITIS* OR
IRRIT*) – 306 (Toxline); 6 (DART)
(102-71-6 OR 111-42-2 OR 141-43-5) AND (METBOLI* OR ABSORB* OR ABSORP* OR
DISTRIBUT* OR EXCRET*) – 403 (Toxline); 18 (DART)
141-43-5 AND CARCINOGEN* – 193
141-43-4 AND CHOLINE - 0
Total Download (most duplicates removed): 1218
UPDATED SEARCH – Sept 21, 2010 – last 12 mos
102-71-8 OR 111-42-2 OR 141-43-5
128 hits/1 useful
Re-Review of Triethanolamine
Introduction ................................................................................................................................................................................. 1 Chemistry..................................................................................................................................................................................... 1 N-Nitrosodiethanolamine Formation ...................................................................................................................................... 1 Use ............................................................................................................................................................................................... 2 Cosmetic ................................................................................................................................................................................. 2 Non-Cosmetic ......................................................................................................................................................................... 2 Toxicokinetics.............................................................................................................................................................................. 3 Absorption, Distribution, Metabolism, Excretion ................................................................................................................... 3 Dermal ................................................................................................................................................................................. 3 Oral ...................................................................................................................................................................................... 4 Other .................................................................................................................................................................................... 4 Toxicological Studies .................................................................................................................................................................. 4 Acute Toxicity ........................................................................................................................................................................ 4 Dermal ................................................................................................................................................................................. 4 Oral ...................................................................................................................................................................................... 4 Other .................................................................................................................................................................................... 5 Repeated Dose Toxicity .......................................................................................................................................................... 5 Dermal ................................................................................................................................................................................. 5 Oral ...................................................................................................................................................................................... 5 Inhalation ............................................................................................................................................................................. 6 Reproductive and Developmental Studies ................................................................................................................................... 6 Dermal ................................................................................................................................................................................. 6 Oral ...................................................................................................................................................................................... 6 Genotoxicity ................................................................................................................................................................................ 7 In Vitro.................................................................................................................................................................................... 7 In Vivo .................................................................................................................................................................................... 7 Carcinogenicity ............................................................................................................................................................................ 7 Dermal ................................................................................................................................................................................. 7 Oral ...................................................................................................................................................................................... 8 Irritation and Sensitization ........................................................................................................................................................... 8 Irritation .................................................................................................................................................................................. 8 Skin ...................................................................................................................................................................................... 8 Mucosal ............................................................................................................................................................................... 9 Sensitization ............................................................................................................................................................................ 9 Provocative Testing ....................................................................................................................................................... 10 Phototoxicity/Photoallergenicity ........................................................................................................................................... 10 Clinical Use ............................................................................................................................................................................... 11 Case Studies .......................................................................................................................................................................... 11 Miscellaneous Studies ............................................................................................................................................................... 11 N-Nitrosodiethanolamine Formation .................................................................................................................................... 11 Possible Mode of Action for Carcinogenic Effects ............................................................................................................... 11 Risk Assessment ........................................................................................................................................................................ 12 Exposure Assessment............................................................................................................................................................ 12 Tables......................................................................................................................................................................................... 13 Table 1. Definition and Structure ......................................................................................................................................... 13 Table 2. Physical and Chemical Properties .......................................................................................................................... 13 Table 3. Current and historical frequency and concentration of use of TEA according to duration and type of exposure ... 13 References ................................................................................................................................................................................. 14 INTRODUCTION
Triethanolamine (TEA), an ingredient that functions as a fragrance ingredient, surfactant – emulsifying agent, and
pH adjuster in cosmetic products, has previously been reviewed by the CIR Expert Panel. In 1983, the Expert Panel concluded that TEA is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from
the surface of the skin. In products intended for prolonged contact with the skin, the concentration of TEA should not exceed
5%. TEA should not be used with products containing N-nitrosating agents. In the 1983 assessment, data demonstrated that
TEA was a mild skin and eye irritant, and that irritation increased with increasing ingredient concentration.
This current document contains summaries of data obtained from a search of published literature, as well as summary information from the original safety assessment
CHEMISTRY
TEA is an amino alcohol. The structure and synonyms of TEA are given in Table 1, and chemical and physical
properties are given in Table 2. TEA is produced commercially by aminating ethylene oxide with ammonia. The
replacement of three hydrogens of ammonia with ethanol groups produces TEA. TEA contains small amounts of
diethanolamine (DEA) and ethanolamine (MEA).
TEA is reactive and bifunctional, combining the properties of alcohols and amines. The reaction of ethanolamines
and sulfuric acid produces sulfates. TEA can act as an antioxidant against the autoxidation of fats of both animal
and vegetable origin. TEA can react with nitrite or oxides of nitrogen to form N-nitrosodiethanolamine (NDELA).
The optimum pH for nitrosamine formation is between 1 and 6; however the rate of NDELA formation in the pH
range of 4-9 is four to six times greater in the presence of formaldehyde.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine1
TEA is produced by reacting 3 moles of ethylene oxide with 1 mole of ammonia; additional ethylene oxide will continue to react to produce higher ethylene oxide adducts of TEA.2 Typically, ethylene oxide is reacted with ammonia in a
batch process to produce a crude mixture of approximately one-third each MEA, DEA, and TEA. The crude is later
separated by distillation.
Based on unpublished survey data collected by the Food and Drug Administration (FDA), a DEA impurity level of
0.3% was found in TEA samples.3
N-Nitrosodiethanolamine Formation
Nitrosamines are compounds containing the R1R2N-NO functional group. Nitrosation is the process of converting
organic compounds (e.g., alkyl amines) into nitroso derivatives (e.g., nitrosamines) by reaction with nitrosating agents.
These agents include nitrous acid (HNO2), oxides of nitrogen (e.g., nitrites), and other compounds capable of generating a
nitrosonium ion, NO+.
The formation of a specific nitrosamine, N-nitrosodiethanolamine (NDELA), from reaction of TEA with nitrite was
examined in vitro and in vivo.4 The TEA used in these studies had an impurity content of 0.4% DEA. In an aqueous (aq.)
matrix, approximately 3% TEA converted to NDELA at a pH of 4.0 in the presence of acetic acid. At the same pH, in the
presence of sulfuric or hydrochloric acid, only about 1% of the TEA was nitrosated. At pH 7, the greatest nitrosation to
NDELA, 0.5%, occurred in the presence of sulfuric acid. No conversion of TEA to NDELA was detected at pH 2 or 10. In
nutrient broth cultures (neutral pH), 0.08% and 0.68% of the TEA was nitrosated to NDELA in a diluted (high cecal
inoculum) and full-strength (low cecal inoculum) media. (The percent nitrosation was determined using values that were
corrected for DEA impurity-related NDELA formation.)
1
USE
Cosmetic
TEA functions in cosmetics as a fragrance ingredient, surfactant – emulsifying agent, and pH adjuster.5 In 1981,
according to data provided through the Food and Drug Administration (FDA) Voluntary Cosmetic Registration Program
(VCRP), TEA was used in 2757 formulations; 2235 of those products were leave-on formulations, and 2419 involved dermal
exposure. Products containing TEA were used at concentrations of ≤0.1% to >50%, with most formulations containing ≤5%
TEA.1 VCRP data obtained in 2010 report that TEA is used in 4015 formulations; 3208 of those products are leave-on formulations, and 3304 involve dermal exposure.6 According to data submitted by industry in response to a survey conducted
by the Personal Care Products Council (Council), TEA is used at concentrations of 0.0002-19%.7 In leave-on products, the
reported use concentrations range from 0.0002-6%; the 6% leave-on use is reported for face and neck creams, lotion, and
powders. The complete current and historical use data for TEA are shown in Table 3. The number of leave-on and dermal
contact uses have increased substantially since the original review, and the maximum concentration of use has decreased.
Also, the number of non-coloring hair formulations and formulations involving mucous membrane exposure increased
greatly since 1981.
TEA is used in aerosolized products, and effects on the lungs that may be induced by aerosolized products containing this ingredient are of concern.
The aerosol properties that determine deposition in the respiratory system are particle size and density. The parameter most closely associated with deposition is the aerodynamic diameter, da, defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic
diameter of ≤ 10µm are inhalable. Particles with a da from 0.1 - 10µm settle in the upper respiratory tract and particles with a
da < 0.1 µm settle in the lower respiratory tract.8,9
Particle diameters of 60-80 µm and ≥80 µm have been reported for anhydrous hair sprays and pump hairsprays,
respectively.10 In practice, aerosols should have at least 99% of their particle diameters in the 10 – 110 µm range, and the
mean particle diameter in a typical aerosol spray has been reported as ~38 µm.11 Therefore, most aerosol particles are
deposited in the nasopharyngeal region and are not inhalable.
The melting point of TEA is very close to room temperature. Accordingly, depending on storage and application
conditions, aerosolized TEA may be a liquid/vapor, instead of a particle.
TEA is listed by the European Commission (EC) in Annex III Part 1: the list of substances which cosmetic products
must not contain, except subject to the restrictions and conditions laid down.12 Trialkylamine, trialkanolamines, and their
salts, including TEA, are allowed at a maximum concentration of 2.5% in non rinse-off products. In non-rinse-off and other
products, the following limitations apply for TEA: do not use with nitrosating systems; minimum allowable purity is 99%;
maximum allowable secondary amine content is 0.5% in raw material; maximum allowable nitrosamine content is 50 µg/kg;
and must be kept in nitrite-free containers.
According to data obtained from Health Canada, some leave-on type products reportedly use TEA as high 10 and
30%, with some reporting 100% TEA. 13
Non-Cosmetic
TEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes, mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate for
resins, plasticizers, and rubber chemicals. It is used as a lubricant in the textile industry, as a humectant and softening agent for
hides, as an alkalizing agent and surfactant in pharmaceuticals, as an absorbent for acid gases, and in organic syntheses.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
2
TEA has uses as an indirect food additive (21 CFR 175.105).14 TEA is also used as a rust inhibitor in water-based
metalworking fluids.15
TOXICOKINETICS
Absorption, Distribution, Metabolism, Excretion
Dermal
[14C]TEA (purity not specified), 2000 mg/kg, was applied to a 2.0 cm2 area of skin on the back of mice, and the dosing area was not protected.16 Peak radioactivity occurred in the blood 2 h after application, with a half-life of absorption and
elimination of 10.2 min and 18.6 h, respectively. When compared to a dose of 1000 mg/kg [14C]TEA in acetone, which was
applied to a 1.0 cm2 area, the concentrations of radioactivity in the blood were 5-fold greater with the 2000 mg/kg dose.
(Additionally, dermal application of 1000 mg/kg in acetone resulted in approximately 1000-fold higher 14C blood radioactivity levels than found in mice dosed intravenously (i.v.) with 1.0 mg/kg.) Another group of mice was also dosed dermally
with 2000 mg/kg [14C]TEA in water; 200 µl, pH 7.0, was applied using a glass ring to protect the dosing site. The blood
concentrations of radioactivity of mice dosed with aq. TEA were then compared to those dosed with neat TEA, and similar
results were found.
The blood kinetics and absorption, distribution, metabolism, and excretion (ADME) of [14C]TEA were determined
following dermal application of 2000 mg/kg neat [14C]TEA without occlusion to male C3H/HeJ mice.17 (Non-radiolabeled
TEA was 99.6% pure; radiochemical purity was 98.6%.) TEA was extensively and rapidly absorbed following a single open
application of 2000 ml/kg neat [14C]TEA. The majority of the radioactivity, 49-62% of the total dose (~58-72% of the absorbed dose), was excreted in the urine, primarily as unmetabolized TEA. DEA and MEA were not detected in the urine. Approximately 18-28% of the total dose (~20-32% of the absorbed dose) was excreted in the feces. The amount of radioactivity
remaining in the body after 48 h ranged from 3.3-6.1%, and the amount recovered at the application site ranged from 1.22.1% for the open applications and 6-11% for the occluded applications.
A study was performed to compare the toxicokinetics of the dermal absorption of [14C]TEA in C3H/HeJ mice to
F344 rats.18 At 48 h after dermal application of 1000 mg/kg TEA to mice, approximately 60% of the radioactivity was
recovered in the urine and 20% in the feces. Less than 10% of the radioactivity was recovered at the application site. In the
urine, 95% of the recovered radioactivity was as unchanged TEA. [14C]TEA was absorbed more slowly and less extensively
in rats. (Additional details, including purity and detailed results for rats, were not provided.)
The National Toxicology Program (NTP) examined the ADME of TEA following dermal administration to B6C3F1
mice and F344 rats.19 With mice, groups of 4 females were given a single dose of 79 or 1120 mg/kg [14C]TEA in acetone;
the dose contained 12-15 µCi, with the appropriate amount of non-labeled TEA in a volume of 190 µl/dose. (Radiochemical
purity of [14C]TEA was 97%; the purity of non-labeled TEA was confirmed, but the purity was not stated.) The dose was
applied to a 1.44 cm2 area of clipped skin, and a non-occlusive cover was used. Approximately 60-80% of the dose was abssorbed, and absorption increased with increasing dose. In the urine 22.5-27.5% and 48-56% of the dose was recovered after
24 and 72 h, respectively, and TEA was excreted mostly unchanged. Approximately 5-9 and 8-13% of the dose was recovered in the feces at the same time periods.
With rats, groups of 4 females were given a single dermal dose of 68 or 276 mg/kg [14C]TEA in acetone; the dose
contained 65 µCi, with the appropriate amount of non-labeled TEA in a volume of 190 µl/dose. The dose was applied to a 12
cm2 area of clipped skin, and a non-occlusive cover was used. Only 19-28% of the dose was absorbed over 72 h; absorption
increased with increasing dose, but not significantly. In the urine, 13-24% of the dose was recovered in 72 h as mostly un3
changed TEA. The amount recovered in the feces after 72 h was <0.25%. Very little radioactivity, <1%, was present in the
tissues; a number of tissues had elevated concentrations of radiolabel relative to blood.
Oral
TEA (purity not specified) was administered orally as a single dose or as a repeated dose for 5-6 days.20 (Dosing
details were not described.) At 24 h after administration of the single dose, the excretion ratio of unchanged dose in the urine
and feces was 53 and 20% of the dose, respectively. With repeated administration, the excretion ratio per day remained constant. Gender did not affect the ratios. TEA glucuronide was detected, but in a very small amount. (Actual concentration not
specified.) TEA was rapidly absorbed in the gastrointestinal tract, and excreted mostly in the urine in unchanged form.
Other
A group of 27 male C3H/HeJ mice was given an i.v. injection of 1 mg/kg [14C]TEA as an aq. solution (0.5 mg/ml),
and the dose volume was 2 ml/kg.17 (Non-radiolabeled TEA was 99.6% pure; radiochemical purity was 98.6%.) Radioactivity in the blood declined in a biphasic, exponential manner for 24 h, with a relatively rapid initial phase of [14C] elimination,
followed by a slower terminal phase. The majority of the radioactivity, approximately 69%, was excreted in the urine,
primarily as unmetabolized TEA. TEA and MEA were not detected in the urine. Some of the radioactivity, ~17%, was excreted in the feces. The average amount of radioactivity recovered in the tissues was 3.1%.
The NTP examined the ADME of TEA following i.v. administration to B6C3F1 mice and F344 rats.19 Groups of 4
female mice and 4 female rats were given a single i.v. dose of 3 mg/kg [14C]TEA in isotonic saline. For mice, the dose contained 6 µCi, with the appropriate amount of non-labeled TEA, for a dosing volume of 2 ml/kg. (Radiochemical purity of
[14C]TEA was 97%; the purity of non-labeled TEA was confirmed, but the purity was not stated.) At 24 h, 26 and 14% of the
dose was excreted in the urine and feces, respectively, while at 72 h, these values were 62 and 28%, respectively. TEA was
excreted mostly unchanged. Less than 0.5%, was detected in expired carbon dioxide. A number of tissues contained higher
concentrations of TEA equivalents relative to blood. For rats, the dose contained 47 µCi, with the appropriate amount of
non-labeled TEA, for a dosing volume of 1 ml/kg. Much more of the radioactivity was excreted in the urine for rats compared to mice, and excretion was more rapid. Approximately 90% of the dose was recovered in the urine in 24 h, and 98% in
72 h, mostly as unchanged TEA. Like mice, <0.5%, was detected in expired carbon dioxide. Only 0.9% of the radioactivity
was detected in the tissues after 72 h.
TOXICOLOGICAL STUDIES
Acute Toxicity
Dermal
The acute dermal toxicity of TEA was examined using groups of 6 rabbits. Undiluted TEA, 91.8 and 88.1%
active, was applied to the intact and abraded skin of 3 rabbits under a 24 h occlusive patch. The exposure to actual
TEA was 2 g/kg. None of the animals died. Mild erythema and edema were reported at 24 h.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Oral
The acute oral toxicity of TEA was determined using guinea pigs and rats. In guinea pigs, undiluted TEA has an
LD50 of 8 g/kg, and the LD50 of TEA in a gum arabic solution was between 1.4 and 7.0 g/kg. Using rats, the oral
LD50 of undiluted TEA ranged from 4.19 g/kg- 11.26 g/kg. The purity ranged from 78.6% TEA (with 8.6% DEA
and 1.7% MEA) to unspecified high purity.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
4
Other
The i.p. LD50 of TEA was 1.45 g/kg for mice.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Repeated Dose Toxicity
Dermal
A closed patch continuous exposure test was performed using 10 guinea pigs in which commercial and high purity
TEA, 8 g/kg, was applied daily 5 days/wk. All guinea pigs died by the 17th application; adrenal, pulmonary,
hepatic, and renal damage were observed. In a 13-wk study, 1 mg/kg of a hair dye formulation containing 0.10.15% or 1.5% TEA was applied to the backs of 12 rabbits for 1 h, twice weekly. The test site skin was abraded
for half of the animals. No systemic toxicity was observed, and there was no histomorphologic evidence of toxicity. In a 6-mos study in which TEA was applied caudally to rats for 1 h/day, 5 days/wk, no toxic effects were
observed with a 6.5% solution. However, using a 13% solution, changes (not specified) were seen in liver and
central nervous system function. The addition of 1.4 mg/l TEA to the drinking water of the rats dosed dermally
with 13% TEA did not increase the toxic effects.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
In a 2-wk study, undiluted TEA (purity not specified) was applied dermally to B6C3F1 mice at doses of 0-3.37 g/kg
and to F344 rats at doses of 0-2.25 g/kg, 5 days/wk.21 Chronic active necrotizing inflammation of the skin at the application
site occurred at a greater frequency and severity in rats than in mice. No renal or hepatic lesions were detected with either
species.
In a 2 wk and a 13-wk dermal study with up to 100% TEA (99.3% pure) in acetone using male and female C3H/HeJ
mice, no toxicity or irritation was observed. 22 The only treatment-related effect was a mild thickening of the epidermis. In a
13-wk NTP dermal study using male and female B6C3F1 mice, application of 250-2000 mg/kg bw TEA in acetone or 4000
mg/kg neat resulted in decreased mean body weights and body weight gains for some male mice.23 (Purity of TEA was 99%.
Functional group titration indicated <0.4% MEA or DEA.) Clinically, irritation was observed for the highest dose group,
while microscopically, inflammation was observed for this dose group and acanthosis was noted for all dose groups, with
severity increasing with dose. Absolute kidney and liver weights of males and females of the 4000 mg/kg group and relative
kidney to body weights of males dosed with ≥1000 mg/kg were increased compared to controls. Absolute and relative spleen
weights were also significantly increased in high dose females compared to controls.
In a 13-wk dermal study using male and female F344/N rats, application of 125-1000 mg/kg bw TEA (99% pure) in
acetone or 2000 mg/kg neat, resulted in significant decreases of mean body weights and body weight gains in the high dose
animals. 23 Functional group titration indicated <0.4% MEA or DEA present.) Clinically, irritation was observed at the
application site, and microscopic lesions included acanthosis and inflammation. Kidney weights of males and females dosed
with ≥500 mg/kg were increased compared to controls, and dosed females, but not males, had greater incidences of
nephropathy, as compared to controls.
Oral
Oral studies were conducted in which groups of 8-20 rats were dosed with 0.2-2.61 g/kg/day TEA for 60 days to 6
mos, and groups of 8 guinea pigs were dosed with 0.2-1.6 g/kg/day TEA for 60 or 120 doses. Repeated oral
ingestion of TEA produced evidence of hepatic and renal damage. Some deaths occurred in groups of rats fed
≥0.17 g/kg/day TEA.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Male and female B6C3F1 mice and F344 rats were given drinking water containing 0 -8% TEA (purity not specified) for 14 days.24,25 Male and female dose high dose mice, and male and female rats given ≥4% TEA, had decreased body
5
weights. All but one of the high dose rats were euthanized early due to severe dehydration. No treatment-related changes
were observed in mice given 4% TEA or rats given 2% TEA in the drinking water.
Inhalation
In a 5-day dose-range finding inhalation study, 5 male and 5 female Wistar rats were exposed, nose only, to target
concentrations of 0-400 mg/m3 TEA (98.9% pure) for 6 h.26 Concentration-dependent laryngeal inflammation and edema
were observed at microscopic examination, and the no observed adverse effect concentration (NOAEC) was 100 mg/m3. The
full, 28 day/20 exposure study used target concentrations of 0, 20, 100, and 500 mg/m3, and the mass median aerodynamic
diameter (MMAD) was 0.7-1.1 µm. A functional observational battery was conducted using 7 rats/sex/group. Minimal to
moderate focal inflammation in the submucosa of the larynx was observed; effects were concentration-dependent. No systemic toxicity was observed, and there were no effects on organ weights. There were no indications of neurotoxicological
effects. Based on the results of this study, the 90-day NOAEC for local irritation was calculated to be 4.7 mg/m3 .
In a 14-day inhalation study, B6C3F1 mice and F344 rats were exposed to 0-2000 mg/m3 TEA (purity not specified)
6h/day, 5 days/wk, for 2 wks.27,28 Female mice and male and female rats of the high dose group had decreased body weights,
and male mice of the high dose group had increased kidney weights. Increased kidney weights in rats dosed with ≥500
mg/m3, and decreased thymus and heart weights in mice at all doses, were not clearly associated with TEA. The only
histopathologic observation was a minimal acute inflammation of the laryngeal submucosa in both mice and rats; however,
this occurred sporadically and there was no dose-response associated with this lesion.
In a 28-day inhalation study, groups of 10 male and 10 female Wistar rats were exposed to 0.02, 0.1, and 0.5 mg/l
TEA (purity not specified) as an aerosol, 6 h/day, 5 days/wk, according to the Organisation for Economic Co-operation and
Development (OECD) guideline 412.29 Exposure was head and nose, and he particle size was 0.6-1.1 µm. The only clinically significant observation was a slight irritation of the upper respiratory tract in the high dose group. The no-observed effect
level (NOEL) for systemic toxicity was >0.5 mg/l, and the NOEL for upper respiratory tract irritation was 0.02 mg/l.
REPRODUCTIVE AND DEVELOPMENTAL STUDIES
Dermal
Hair dyes containing 0.1-0.15% or 1.5% TEA were applied topically to the shaved skin of groups of 20 gravid rats
on days, 1, 4, 7, 10, 13, 16, and 19 of gestation, and the rats were killed on day 20 of gestation. No developmental
or reproductive effects were observed.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
TEA, 0.5 g/kg in acetone (purity not stated), was applied dermally to clipped skin on the back of male and female
F344 rats.30 A volume of 1.8 ml/kg was applied daily for 10 wks prior to mating, during mating, and through gestation and
lactation. No effect on mating or fertility or offspring growth or survival was observed. A similar study was performed in
which Swiss CD-1 mice were given daily applications of 2 g/kg TEA at a volume of 3.6 ml/kg.31 No adverse reproductive
effects were observed.
Oral
A Chernoff-Kavlock teratogenicity screening test was performed using mated female CD-1 mice, in which the animals were dosed orally by gavage with 1125 mg/kg TEA on days 6-15 of gestation.32 (It was stated that the TEA was the
“purest grade commercially available”.) No adverse reproductive effects were observed.
6
GENOTOXICITY
In Vitro
Undiluted TEA, at concentrations of ≤100 mg/plate, was not mutagenic in Salmonella typhimurium with or without metabolic activation. TEA with sodium nitrite, but not TEA alone, was mutagenic in Bacillus subtilis without
metabolic activation. NDELA, which is not mutagenic in B. subtilis without metabolic activation, was found in
the mixture. In an unscheduled DNA synthesis test in which primary rat hepatocyte cultures were exposed to 10-8
to 10-1 M TEA and [3H]thymidine, simultaneously, TEA did not appear to cause DNA-damage-inducible repair.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
TEA, in distilled water or dimethylsulfoxide, was not mutagenic to Escherichia coli33 or S. typhimurium, with or
without metabolic activation, at doses of 0-20,000 µg/plate.33-35 TEA (88.2% purity) did not cause gene conversion in
Saccharomyces cerevisiae.34 TEA was negative in a rec assay at doses of 0-4000 µg/disk. No induction of sister chromatid
exchanges occurred in Chinese hamster ovary (CHO) cells at 0-1010 µg/ml without metabolic activation and 0-10,100 µg/ml
with metabolic activation,36 and chromosomal aberrations were not induced in cultured rat liver cells34 or at doses of 5-100
µg/ml in cultured Chinese hamster cells.33,36 TEA, 25-500 µg/ml, was negative in a cell transformation assay using hamster
embryo cells.33
In Vivo
A mouse peripheral blood micronucleus test was performed using samples collected from mice that were dosed
dermally for 90-days with 0-4 g/kg TEA in an NTP study.37 Results were negative in both male and female mice.
CARCINOGENICITY
Dermal
In a series of 3 experiments using a total of 560 CBA x C57Bl6 male mice, the carcinogenic effects of 99%+ pure
TEA and 80%+ industrial grade TEA and the cocarcinogenic effect of TEA and syntanol DC-10 (not defined)
were examined over a 14-18 mo timeframe. TEA was not carcinogenic or cocarcinogenic. .
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
The first carcinogenicity study of TEA using B6C3F1 mice performed by the NTP was deemed inadequate due to a
Helicobacter hepaticus infection.23 Therefore, a second 2-yr study was performed in which TEA in acetone was applied dermally at doses of 200-2000 mg/kg to male B6C3F1 mice and at doses of 100-1000 mg/kg to female B6C3F1 mice.38 (Purity
of TEA was 99+%. Using high-performance liquid chromatography/mass spectrometry, 0.491% DEA was detected as an impurity. A slight increase in DEA was seen in acetone and ethanol solutions after 11 days of storage; the dose formulations
were prepared approximately every 2 wks.) The body weights of high dose males were decreased compared to controls during wks 17-37 and at the end of the study. Dermal irritation increased with increasing dose, and was more severe in males
than in females. At necropsy, treatment-related epidermal hyperplasia, suppurative inflammation, and ulceration and dermal
chronic inflammation occurred at the application site in most test groups, and the incidence and severity increased with increasing dose. Lesions were found, and it was concluded that there was equivocal evidence of carcinogenic activity of TEA
in male mice, based on the occurrence of liver hemangiosarcoma, and some evidence of carcinogenic activity in female mice,
based on increased incidences of hepatocellular adenoma.
In a 2-yr NTP dermal carcinogenicity study using F344/N rats, TEA in acetone was applied at doses of 32- 125
mg/kg in acetone to males and at doses of 63-250 mg/kg to females.23 (Purity of TEA was 99%. Functional group titration
indicated <0.4% MEA or DEA.) Irritation was observed at the application site, and frequency increased with increasing
dose. At the interim necropsy, the absolute and relative kidney weights of high dose females were significantly greater than
the controls. Microscopically, at the site of application, dermal lesions, including acanthosis, inflammation, and/or ulcera7
tion, were observed. It was concluded that there was equivocal evidence of carcinogenic activity in male rats, based on a
marginal increase in the incidences of renal tubule cell adenoma, and there was no evidence of carcinogenic activity in female
rats.
The carcinogenic potential of TEA was evaluated using a Tg·AC transgenic mouse model.39 Groups of 10-15 female
homozygous mice were dosed dermally with 3-30 mg TEA/mouse in acetone, 5x/wk for 20 wks. TEA was inactive in
Tg·AC mice.
Oral
Groups of 40 male and 40 female ICR-JCL mice were fed a diet containing 0.01, 0.03, or 0.3% TEA throughout
their lifetime. The malignant tumor incidence was 2.8, 27, and 36% for females, respectively, and 2.9, 9.1, and
3.6% for males, respectively. Treated females had a much higher incidence of thymic and non-thymic tumors in
lymphoid tissues than treated males. Survival was similar for treated and control animals.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
The oral carcinogenic potential of TEA was examined by administering 1 and 2% TEA in drinking water to male
and female B6C3F1 mice for 82 wks.40 (DEA was present as an impurity at 1.9 %.) Body weights of male mice of the 2%
group were decreased during wks 1-20 when compared to controls. No significant changes in organ weights were observed.
No dose-related increase of the incidence of any tumors was observed in the treated groups, and there was no evidence of
carcinogenic potential of TEA upon oral administration.
Drinking water containing 1 or 2% TEA was given to male and female F344 rats for 2 yrs.41 (DEA was present as
an impurity at 1.9%.) From wk 69 on, the dose concentrations for females were reduced by half because of associated nephrotoxicity. A dose-related decrease in body weight gain was reported for male and female test group rats, and a dose-dependent increase in mortality, starting at wk 60, was observed. Absolute and relative kidney-to-body weights were significantly
increased in males and females, and the increase was dose-related. Severe chronic nephropathy was statistically significantly
increased in males of the high dose group and females of both dose groups. No treatment-related effects were found in the
liver. There was no increase in the incidence of any tumors in the treated groups compared to controls when using the Chisquare test. Since increased nephrotoxicity appeared to affect the lifespan of the treated animals, especially the females, an
age-adjusted statistical analysis was performed on the incidences of main tumors or tumor groups for males and females, and
a positive trend was noted in the occurrence of hepatic tumors (neoplastic nodule/hepatocellular carcinoma) in males and of
uterine endometrial sarcomas and renal-cell adenomas in females. The researchers stated that, because these tumors have
been observed spontaneously in F344 rats, and since their incidences in the control group was lower than that of historical
controls, the occurrence of the tumors may not be attributable to TEA. Instead, increased incidence of renal tumors in the
high-dose group may have been associated with renal damage. The researchers concluded that TEA was toxic to the kidneys,
especially in females, but it was not carcinogenic to F344 rats.
IRRITATION AND SENSITIZATION
Irritation
Skin
Non-Human
The primary skin irritation potential of undiluted TEA was determined using rabbits. After 10 open applications
of 0.1 ml to rabbit ears and 10 unoccluded applications to the intact skin of the abdomen, and 3 semi-occluded 24h applications to abraded skin, it was concluded that TEA was slightly to moderately irritating, and prolonged or
repeated exposure may be irritating. Twenty-four h occluded patch tests using groups of 8 male rabbits were
performed in 22 laboratories; the primary irritation score ranged from 0-5.5/24, and the total score for all 22
laboratories was 27.3/400. In a preliminary study, occlusive dermal applications of 50-100% aq. TEA to pairs of
8
guinea pigs resulted in one erythematic reaction to undiluted TEA, and in another preliminary study, no irritation
was observed when 5, 10, or 25% TEA was applied to the backs of guinea pigs.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
The irritancy potential of TEA (purity not specified) was evaluated in an ear swelling test using female BALB/c
42
mice.
A significant increase in irritancy was observed with 25 and 50% TEA compared to the vehicle (4:1 acetone/olive
oil) group.
Human
Clinical studies were performed with formulations containing TEA. In a few studies on formulations containing
0.45-2.4% TEA, the researchers concluded that no irritation was observed, while short-lived acute irritation was
reported for formulations containing 1.9-2.6% TEA. However, according to the Expert Panel’s interpretation of
the results of a number of other studies, formulations containing 0.83-20.04% TEA were irritating.
In clinical provocative testing using 5-10 “hyper reactors,” 100% TEA produced an irritant reaction on nonscarified skin, 10% TEA in ethanol was a marked irritant on scarified skin, while 5% in ethanol was a slight
irritant on scarified skin.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
A patch test with TEA (purity not specified) was performed on 20 subjects, and erythema and transepidermal water
loss (TEWL) were measured, and the contents of suction blister fluids (SBF) were evaluated for primary proinflammatory
mediators.43 Aq. TEA, 0-100%, was applied occlusively for 24 h; 100-200 µl, concentrated to 20 µl with drying, were applied. The percent of non-responders to 100% TEA was 80%; those that did respond had weak and non-uniform erythema.
The incidence was below or about that found with the solvent controls. For the challenge phase, 765 µmol/cm2 TEA was
applied occlusively to 12 subjects for 6-24 h. No increase in TEWL or change in eicosanoid profile of the SBF was observed. TEA was a non-irritant.
Mucosal
Non-Human
The ocular irritation potential of 0.005-0.1 ml undiluted TEA was evaluated in a number of studies using rabbits.
With high concentrations and long contact time, TEA may be irritating to rabbit eyes. Using rabbits, 10% aq. TEA
produced essentially no irritation with or without rinsing. A formulation containing 12.6% TEA, 0.1 ml, was evaluated in a study using 6 rhesus monkeys. Slit lamp examination revealed some corneal effects in 2 monkeys at 24
h and slight positive fluorescein staining in one monkey at 72 h.
The vaginal irritation potential of a spermicidal formulation containing 1.92% TEA was evaluated by placing 0.5
ml of the ointment inside the vaginas of 6 rats for 3 days. The formulation was a non-irritant.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Sensitization
In Vitro
The sensitization potential of TEA was evaluated in a local lymph node assay (LLNA) performed with groups of 5
BALB/c mice.42 This study was performed in conjunction with the ear-swelling test described previously. Lymphocyte proliferation increased with dose, but the increases were not statistically significant. TEA was not identified as a sensitizer in the
LLNA.
Non-Human
TEA was not a sensitizer to guinea pigs when 20 guinea pigs were given dermal applications of undiluted TEA
1x/wk for 3 wks, followed by challenge applications 14 and 21 days after dosing. No sensitization was seen when
four lots of TEA were evaluated using groups of 20 guinea pigs; induction applications were applied for up to 6 h,
1x/wk, for 3 wks, and the challenge was performed after 14 days. One of the studies used undiluted TEA during
induction, while the other 3 studies used 50% TEA at induction. All four studies used challenge patches with 90%
TEA. No sensitization was observed in a similar study in which induction patches contained a 25% active TEA
solution, and a challenge patch with the 25% solution was applied after 1 wk of non-treatment.
9
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
The hypersensitivity of mice to TEA (99+% pure) was determined.44 TEA, in an acetone:olive oil mixture (4:1) at
concentrations of 3%, 10%, or 30%, was applied daily for 5 consecutive days to groups of 8 female B6C3F1 mice, and the
animals were challenged 7 days later with a 30% solution. For some animals, dermabrasion, as well as intradermal injections
of Freund’s complete adjuvant (FCA), was used. There were no treatment-related effects on survival or body weights. There
were no statistically significant or dose-related hypersensitivity responses to TEA observed with a radioisotopic method or in
an ear swelling test, with or without FCA.
Results were negative in three maximization studies examining the sensitization potential of TEA.15 In the first test,
performed using Pirbright-White guinea pigs, induction consisted of intradermal injections of 2% TEA (98.9% pure) in isotonic saline and epicutaneous application of undiluted TEA, and challenge used 10% TEA in isotonic saline. In the second
test using 20 Dunkin-Hartley guinea pigs, intradermal and epicutaneous inductions used 1.5% technical grade TEA and 25%
technical grade TEA with 10% sodium lauryl sulfate pre-treatment, respectively, and challenge doses consisted of 1, 5, and
10% technical and analytical grade TEA. The third study used 15 animals and the same induction protocol (grade of TEA
not specified); 2/15 reacted to 10% TEA after 1, but not 3, days.
Human
In patch tests in which TEA was tested at 5%, 2% aq, or 5% in petrolatum, 9/479, 23/500, and 2/100 subjects had
positive reactions for contact dermatitis. The Expert Panel interpreted these findings as sensitizing. In a patch test
with 64 subjects in which 0.5 ml of 1% TEA (containing 88.6% TEA and 6% DEA) was used, the test solution
was not sensitizing.
The majority of formulations containing 0.83-4.2% TEA were not sensitizing, and a formulation containing
20.04% TEA, tested on 26 subjects, was not considered sensitizing when it produced 2 slight reactions upon challenge. However, according to the interpretation of the Expert Panel, there were a few cosmetic formulations
containing 2.1 and 2.4% TEA that the Panel determined to be sensitizing. In other studies with cosmetic formulations containing 2.1% TEA, the researchers concluded that reactions observed at challenge were probably due to
skin fatigue.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Provocative Testing
A group of 737 patients was patch tested with 6 different emulsifiers, including 2.5% TEA (purity not specified) in
petrolatum.45 The patch tests were performed according to International Contact Dermatitis Research Group (ICDRG)
recommendations. A total of 39 patients had positive reactions to the emulsifiers, and 20 of those patients, 5 males and 15
females, had positive reactions to TEA. There were 106 (0.1%) irritant reactions reported. The results were clinically relevant in 7 patients. Many of the patients allergic to TEA were also allergic to other ingredients.
Over a 15-yr period, provocative patch testing using TEA was performed on 85,098 subjects.15 There were 323
(0.3%) positive reactions to TEA, and most of the reactions (289; 0.3%) were weak positives. The researchers stated that
occupational exposure was not a risk factor for TEA contact allergy.
Phototoxicity/Photoallergenicity
Non-Human
A formulation containing 1% TEA was applied to the stripped skin of 6 guinea pigs, and each animal was then
exposed to ultraviolet A (UVA) light for 2 h. No erythema or edema was observed.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Human
There were no phototoxicity or photosensitization reactions with a number of formulations containing 0.45-4.2%
TEA, nor were there any reactions with a formulation containing 20.04% TEA. However, in one study with a
formulation containing 4.2% TEA, the Expert Panel felt that the formulation was either mildly phototoxic or there
10
was UV enhancement of an irritant response.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
CLINICAL USE
Case Studies
Eczema of the face of 2 female patients was exacerbated by a cream that contained TEA.46 Patch testing was performed using the ICDRG standard series, a cosmetic battery, the TEA-containing cream, and TEA at 5%, 2%, and 1% in
petrolatum (pet.). Both patients reacted to the TEA-containing cream (+ reaction) and to 5% TEA pet. (++ reaction). One
patient reacted to 2% TEA (+ reaction), and neither reacted to 1% TEA pet. Patch tests were negative for all other compounds. In a control group of 50 subjects, patch testing with 5% TEA pet. was negative.
Over a 4-yr period, the incidences of positive patch test reactions to the same TEA-containing cream were 69/171
patients in one clinic and 49/191 in another.47 It was hypothesized that the difference between the clinics was due to differences in sampling methods; the first clinic tested only those patients that had recently used the TEA-containing cream or who
had suspected reactions. In follow-up patch testing with a total of 54 subjects from the 2 clinics, 15 of which were controls,
19 subjects had a positive allergic response to the TEA-containing cream, 40 had a positive irritant response, and 13 had negative responses. With 1.45-5% TEA, 6 subjects had a positive response. However, with 5-20% TEA stearate, 8/8 patients
and 15/15 controls had a positive irritant response. (TEA stearate was tested because it was demonstrated that TEA stearate
was formed from the combination of TEA and stearic acid in formulation.) The researchers postulated the reactions were
irritant reactions to TEA stearate. (The amount of TEA stearate present in formulation was estimated to be 4.8%, and 6/23
subjects patch tested with 5% TEA stearate had irritant reactions.)
Two cases of occupational asthma in metal workers exposed to cutting fluid containing TEA were reported.48
Exposure to TEA at temperatures higher than that of ambient air was a common feature.
MISCELLANEOUS STUDIES
N-Nitrosodiethanolamine Formation
In vivo, female B6C3F1 mice were dosed dermally or orally with 1000 mg/kg TEA, in conjunction with oral exposure to sodium nitrite.4 Following 7 days of dermal dosing, no NDELA was detected in the blood, ingesta, or urine of test,
vehicle control, or sodium nitrite control mice. (The limits of detection for the blood, ingesta, and urine were 0.001, 0.006,
and 0.47 µg/ml, respectively.) With a single oral dose, the concentrations of NDELA found in the blood and ingesta of mice
2 h post-dosing were 0.001 ± 0.0005 µg/g and 0.044 ± 0.059 µg/g, respectively.
Possible Mode of Action for Carcinogenic Effects
It has been reported that choline deficiency induces liver cancer in rodents; therefore, the potential of TEA to cause
choline deficiency in the liver of female B6C3F1 mice was investigated as a mode of tumorigenesis.49 Female mice were
dosed dermally with unoccluded applications of 10-1000 mg/kg/day TEA in acetone, 5 days/wk for 3 wks, and female CDF
rats were dosed in a similar manner with 250 mg/kg/day TEA. (Purity of TEA was 99+%; DEA impurity levels were 0.04
and 0.45%.) No clinical signs of toxicity were noted for mice or rats. Phosphocholine and betaine levels were statistically
significantly decreased in the high dose mice, and choline levels were decreased in these mice. The decrease in phosphocholine levels was variable, but dose-related. (More pronounced effects were observed when the TEA having 0.45% DEA
impurity was used.) In rats, no changes in choline or its metabolites were noted. The potential of TEA to inhibit the uptake
of [3H]choline by CHO cells was also investigated, and a dose-related decrease was observed. The researchers concluded
that TEA may cause liver tumors in mice via a choline-depletion mode of action, and this effect is likely caused by the inhibi11
tion of choline uptake by the cells. The researchers stated that, while DEA impurity may contribute to choline depletion, a
choline-deficiency mode of tumorigenesis appears to be a property of TEA, exclusive of any DEA impurity.
RISK ASSESSMENT
Exposure Assessment
The dermal exposure of consumers to TEA was estimated assuming a presence of 2.5% TEA in cosmetic products
(based on the limit set by the EC) and that all TEA is unreacted.50 Using an EC algorithm method, the dermal exposure of
consumers to an eye make-up powder is 0.0125 mg/kg bw/day and to a body lotion is 6.25 mg/kg bw/day. Using a
DERMAL program method, the dermal potential dose rate for a bar soap containing 2.5% TEA is 5.182 mg/day.
According to an evaluation of TEA by the International Agency for Research on Cancer (IARC) Working Group,
there is inadequate evidence in humans, as well as in animals, for the carcinogenicity of TEA.51 The overall evaluation of the
IARC is that TEA is not classifiable as to its carcinogenicity to humans (Group 3).
12
TABLES
Table 1. Definition and Structure
Ingredient
Definition
CAS No.
Triethanolamine The product of the ammonolysis of
102-71-6
three stoichiometric equivalents of
ethylene oxide
Synonyms
Function(s) in Cosmetics
2,2’,2”-nitrilotris[ethanol]
trolamine
TEA
fragrance ingredient
surfactant-emulsifying agent
pH adjuster
Formula/Structure
HO
OH
N
HO
Table 2. Physical and Chemical Properties
Property
Physical Form
Color
Odor
Molecular Weight
Density/Specific Gravity
Viscosity
Vapor Pressure
Vapor Density
Melting Point
Boiling Point
Water Solubility
Other Solubility
log Kow
Disassociation Constant
pKa
Value
clear viscous liquid
colorless to pale yellow
Ammonical
149.19
1.124 (20°C)
590.5 cP @25°C
<0.01 mm Hg @ 20°C
5.14 mmHg
21.6°C
335.4°C @ 760 mm Hg
miscible in water
insoluble in benzene, ether, and petroleum distillates
miscible with methanol or acetone; sparingly soluble in hydrocarbon solvents; readily
forms salts with organic and inorganic acids
-1.59 @ 20°C
Reference
1
19
1
1
1
18
19
18
19
19
19
1
18
50
26
7.76 @ 25°C
Table 3. Current and historical frequency and concentration of use of TEA according to duration and type of exposure
# of Uses
Conc. of Use (%)
data year
19811
20106
19811
20107,52
Totals
2757
4015
≤0.1 - >50*
0.0002-19
Duration of Use
Leave-On
Rinse Off
2235
522
3208
807
≤0.1 - >50*
≤50
0.0002-6
0.0003-19
Exposure Type
Eye Area
Possible Ingestion
Inhalation
Dermal Contact
Deodorant (underarm)
Hair - Non-Coloring
Hair-Coloring
Nail
Mucous Membrane
Bath Products
Baby Products
404
17
101
2419
2
129
49
18
15
11
6
428
30
84
3304
12
413
6
14
215
24
22
≤25
≤1
≤5
≤0.1 - >50*
0.1-1
≤50
0.1-25
≤10
1-50
≤10
0.1-50
0.2-4
0.2-1
0.001-2
0.0002-19
0.1-0.4
0.0003-6
1-13
0.2-3
0.1-19
0.4-2
0.2-2
* - included unreported concentration of use
13
REFERENCES
1.
Elder RE (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll
Toxicol. 1983;2:(7).
2.
Dow Chemical Company. The alkanolamines handbook. 1988. Midland, MI: The Dow Chemical Company.Secondary reference
in Knaak et al. 1997.
3.
Kraeling MEK, Yourick JJ, and Bronaugh RL. In vitro human skin penetration of diethanolamine. Food Chem Toxicol.
2004;42:1553-1561.
4.
Saghir SA, Brzak A, Markham DA, Bartels MJ, and Stott WT. Investigation of the formation of N-nitrosodiethanolamine in
B6C3F1 mice following topical administration of triethanolamine. REg Toxicol Pharmacol. 2005;43:10-18.
5. Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal
Care Products Council, 2010.
6. Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database. 2010. Washington, DC:
FDA.Updated May 4.
7.
Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey. 10-13-2010.
Unpublished data submitted by the Council.
8.
James AC, Stahlhofen W, Rudolf G, and et al. Annex D. Deposition of inhaled particles. Annals of the ICRP. 1994;24:(1-3):231232.
9.
Oberdorster G, Oberdorster E, and Oberdorster J. An emerging discipline evolving from studies of ultrafine particles. Environ
Health Perspect. 2005;113:(7):823-839.
10.
Bowers D. Unpublished information on hair spray particle sizes provided at the September 9, 1999 CIR Expert Panel meeting.
1999.
11.
Johnson MA. The influence of particle size. Spray Technology and Marketing. 2004;Nov 24-27.
12.
European Commission.European Commission Enterprise and Industry. Cosmetics - Cosing. Annex III/1,62 - Trialkylamines,
trialkanolamines and their salts. 2010.
http://ec.europa.eu/enterprise/cosmetics/cosing/index.cfm?fuseaction=search.details&id=28313&back=1. Accessed 67-2010.
13.
Heatlh Canada.Cosmetic Ingredient Hotlist. 2010. http://www.hc-sc.gc.ca/cps-spc/person/cosmet/info-ind-prof/_hot-listcritique/hotlist-liste_1-eng.php. Accessed 10-29-2010.
14.
Food and Drug Administration.Everything Added to Food in the United States (EAFUS). 5-27-0010.
http://www.fda.gov/Food/FoodIngredientsPackaging/ucm115326.htm. Accessed 6-8-2010.
15.
Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine.
Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60:243-255.
16.
Waechter JM and Rick DL. Triethanolamine: Pharmacokinetics in C3H/HeJ mice and Fischer-344 rats following dermal
administration. 1988. Midland. MI: Dow Chemical Co.Secondary reference in Knaak et al. 1997.
17.
Stott WT, Waechter JM, Rick DL, and Mendrala AL. Absorption, distribution, metabolism and excretion of intravenously and
dermally administered triethanolamine in mice. Food Chem Toxicol. 2000;38:1043-1051.
18.
Triethanolamine. 1990. Chapter: 7.6. Ethel Browning's Toxicity and Metabolism of Industrial Solvents. Buhler DR and Reed DJ.
Amsterdam/New York/Oxford: Elsevier.
19.
National Toxicology Program. NTP Technical report on the toxicology and carcinogenesis studies of triethanolamine (CAS No.
102-71-6) in B6C3F1 mice. (Dermal study). NTP TR 518. 2004.
20. Kohri N, Matsuda T, Umeniwa K, Miyazaki K, and Arita T. Development of assay method in biological fluids and biological fate
of triethanolamine. Yakuzaigaku. 1982;42:(4):342348.
14
21.
Melnick R and Hejtmancki M Mezza L Ryan M persing R Peters A. Comparative effects of triethanolamine (TEA) and
diethanolamine (DEA) in short-term dermal studies. (Secondary reference in Melnick and Tomaszewski 1990). The
Toxiologist. 1988;8:127.
22.
DePass LR, Fowler EH, and Leung H-W. Subchronic dermal toxicity study of triethanolamine in C3H/HeJ mice. Fd Chem
Toxicol. 1995;33:(8):675-680.
23.
National Toxicology Program. Toxicology and carcinogenesis studies of triethanolamine (CAS No. 102-71-6) in F344/N rats and
B6C3F1 mice. (Dermal studies.) TR No. 449. 1999.
24.
Hejtmancik M, Mezza L, Peters AC, and Athey PM. The repeated dose dosed water study of triethanolamine (CAS No. 102-716) in Fischer-344 rats. 1985. Columbus OH: Columbus Division Laboratories.Unpublished data summarized in Knaak
et al. 1997.
25.
Hejtmancik M, Mezza L, Peters A, and Athey PM. The repeated dose dosed water study of triethanolamine (CAS No. 102-71-6)
in B6C3F1 mice. 1985. Unpublished data summarized in Knaak et al. 1997: Columbus Division
Laboratories.Unpublished data summarized in Knaak et al. 1997.
26.
Gamer AO, Rossbacher R, Kaufmann W, and van Ravenzwaay B. The inhalation toxicity of di- and triethanolamine upon
repeated exposure. Food Chem Toxicol. 2008;46:2173-2183.
27.
Mosberg A, McNEill D, Hejtmancik M, Persing RL, and Peters A. The repeated dsoe inhalation study of triethanolamine (CAS
No. 102-71-6) in Fischer-344 rats. 1985. Columbus OH: Battelle Columbus Division Laboratories.Unpublished data
summarized in Knaak et al. 1997.
28.
Mosberg A, McNEill D, Hejtmancik M, Persing RL, and Peters A. The repeated dose inhalation study of triethanolamine (CAS
No. 102-71-6) in B6C3F1 mice. 1985. Columbus OH: Battelle Columbus Division Laboratories.Unpublished data
summarized in Knaak et al. 1997.
29.
BASF AG. Twenty-eight day inhalation study of TEA using Wistar rats. Unpublished report, Project No. 40I0711/89098. OECD
SIDS Initial Assessment Report for SIAM 3 (Triethanolamine). 1993. Secondary reference in OECD 1995 - SIDS Initial
Assessment Report for SIAM 3.
30.
Battelle Columbus Laboratories. Mating trial dermal study of triethanolamine (CAS No. 102-71-6) in Fischer 344 rats. Final
report (NIH Cotnract No. N01-ES-45068). Secondary reference in NTP 2004. 1988.
31.
Battelle Columbus Laboratories. Mating trial dermal study of triethanolamine (CAS No. 102-71-6) in Swiss CD-1 mice. Final
report (NIH Cotnract No. N01-ES-45068). Secondary reference in NTP 2004. 1988. Secondary reference in NTP
2004.- TR 518.
32.
Environmental Protection Agency.High Production Volume (HPV) Challenge Program Revised Test Plan and Robust Summaries
for Polyphosphoric Acid Esters of Triethanolamine, Sodium Salts. 11-30-2004.
http://www.epa.gov/oppt/chemrtk/pubs/summaries/plyacdts/c14950rr.pdf.
33.
Inoue K, Sunakawa T, Okamoto K, and Tanaka Y. Mutagenicity tests and in vitro transformation assays on triethanolamine.
Mutat Res. 1982;101:305-313.
34. Dean BJ, Brooks TM, Hodson-Walker G, and Hutson DH. Genetic toxicology testing of 41 industrial chemicals. Mutat Res.
1985;153:57-77.
35.
Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, and Zeiger E. Salmonella mutagenicity tests: II. Results from the
testing of 270 chemicals. Environ Mutagen. 1986;8:(Suppl 7):1-119.
36.
Galloway SM, Armstrong MJ, Reuben, Colman, Brown B, Cannon C, Bloom AD, Nakamura F, Ahmed M, Duk S, Rimpo J,
Margolin BH, Resnick MA, Anderson B, and Zeiger E. Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: Evaluation of 108 chemicals. Environ Mol Mutagen. 1987;10:(Suppl 10):1-175.
37.
Witt KL, Knapton A, Wehr CM, Hook GJ, Mirsalis J, Shelby MD, and MacGregor JT. Micronucleated erythrocyte frequency in
peripheral blood of B6C3F1mice from short-term. prechronic, and chronic studies of the NTP carcinogenesis bioassay
program. Environ Mol Mutagen. 2000;36:163-194.
38.
National Toxicology Program. Toxicology and carcinogenesis studies of triethanolamine (CAS NO. 102-71-6) in B6C3F1 mice.
(Dermal study.) NTP TR 518. 2004.
15
39.
Tennant RW, French JE, and Spalding JW. Identifying chemical carcinogens and assessing potential risk in short-term bioassays
using transgenic mouse models. Environ Health Perspect. 1995;103:942-950.
40.
Konishi, Y., Denda, A., Uchida, K., Emi, Y., Ura, H., Yokose, Y., Shiraiwa, K., and Tsutsumi, M. Chronic toxicity
carcinogenicity studies of triethanolamine in B6C3F1, mice. Fundam.Appl Toxicol. 1992;18:(1):25-29.
41.
Maekawa, A., Onodera, H., Tanigawa, H., Furuta, K., Kanno, J., Matsuoka, C., Ogiu, T., and Hayashi, Y. Lack of carcinogenicity
of triethanolamine in F344 rats. J Toxicol Environ Health. 1986;19:(3):345-357.
42.
Anderson SE, Brown KK, Butterworth LF, Fedorowicz A, Jackson LG, Frasch HF, Beezhold D, Munson AE, and Meade BJ.
Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components. J Immunotoxicol.
2009;6:(1):19-29.
43.
Müller-Decker K, Heinzelmann T, Fürstenberger G, Kecskes A, Lehmann W-D, and Marks F. Arachidonic acid metabolism in
primary irritant dermatitis produced by patch testing of human skin with surfactants. Toxicol Appl Pharmacol.
1998;153:59-67.
44.
National Toxicology Program.Abstract for IMM90005. The immunotoxicity of triethanolamine (CAS No. 102-71-6). 2-21-2005.
http://ntp.niehs.nih.gov/?objectid=03E847AC-AC78-4065-BE8E52A8BB9CFBA7. Accessed 10-14-2010.
45.
Tosti A, Morelli R, and Bardazzi F. Prevalence and sources of sensitization to emulsifiers: a clinical study. Contact Derm.
1990;23:68-72.
46. Jones SK and Kennedy TC. Contact dermatitis from triethanolamine in E45 cream. Contact Derm. 1988;19:(3):230.
47.
Batten TL, Wakeel RA, Douglas WS, Evans C, White MI, Moody R, and Ormerod AD. Contact dermatitis from the old formula
E45 cream. Contact Derm. 1994;30:159-161.
48.
Savonius B, Keskinen H, Tupperainen M, and Kanerva L. Occupational asthma caused by ethanolamines. Allergy. 1994;49:877881.
49. Stott WT, Radtke BJ, LinscombeVA, Mar MH, and Zeisel SH. Evaluation of the potential of triethanolamine to alter hepatic
choline levels in female B6C3F1 mice. Toxicol Sci. 2004;79:(2):242-247.
50.
Organisation for Economic Co-operation and Development. SIDS Initial Assessment Report for SIAM 3 (Triethanolamine).
http://webnet.oecd.org/Hpv/UI/SIDS_Details.aspx?id=CEF02D50-2294-462B-8027-FB4EC67D5D81. 2-16-1995.
51.
International Agency for Research on Cancer (IARC). Triethanolamine. IARC Monogr Eval.Carcinog Risks Hum. 2000;77:381401.
52.
Personal Care Products Council. Updated concentration of use of triethanolamine. 11-8-2010. Unpublished data submitted by the
Council (3 pp).
16
Re-Review of Diethanolamine
Introduction ................................................................................................................................................................................. 1 Chemistry..................................................................................................................................................................................... 1 Use ............................................................................................................................................................................................... 1 Cosmetic ................................................................................................................................................................................. 1 Non-Cosmetic ......................................................................................................................................................................... 2 Toxicokinetics.............................................................................................................................................................................. 2 Absorption, Distribution, Metabolism and Excretion ............................................................................................................. 2 In-Vitro ............................................................................................................................................................................... 2 Dermal ................................................................................................................................................................................ 3 Non-Human ................................................................................................................................................................... 3 Human ........................................................................................................................................................................... 4 Oral..................................................................................................................................................................................... 4 Non-Human ................................................................................................................................................................... 4 Other................................................................................................................................................................................... 5 Non-Human ................................................................................................................................................................... 5 Percutaneous Absorption ........................................................................................................................................................ 6 Non-Human ................................................................................................................................................................... 6 Human ........................................................................................................................................................................... 6 Toxicological Studies .................................................................................................................................................................. 7 Acute (Single Dose) Toxicity ................................................................................................................................................. 7 Oral..................................................................................................................................................................................... 7 Other................................................................................................................................................................................... 7 Repeated Dose Toxicity .......................................................................................................................................................... 7 Dermal ................................................................................................................................................................................ 7 Oral..................................................................................................................................................................................... 8 Inhalation............................................................................................................................................................................ 9 Reproductive and developmental studies................................................................................................................................... 10 Dermal .............................................................................................................................................................................. 10 Oral................................................................................................................................................................................... 11 Inhalation.......................................................................................................................................................................... 11 Genotoxicity .............................................................................................................................................................................. 12 In Vitro ................................................................................................................................................................................. 12 Carcinogenicity .......................................................................................................................................................................... 12 Dermal .............................................................................................................................................................................. 12 Irritation and Sensitization ......................................................................................................................................................... 13 Irritation ................................................................................................................................................................................ 13 Skin .................................................................................................................................................................................. 13 Mucosal ............................................................................................................................................................................ 13 Sensitization .......................................................................................................................................................................... 13 Provocative Testing ..................................................................................................................................................... 14 Miscellaneous Studies ............................................................................................................................................................... 14 N-Nitrosodiethanolamine Formation .................................................................................................................................... 14 Immunotoxicity..................................................................................................................................................................... 14 Inhibition of Choline Uptake ................................................................................................................................................ 15 Species Selective Effect on DNA Synthesis ......................................................................................................................... 15 Effect on Hippocampal Neurogenesis and Apoptosis ........................................................................................................... 15 Effect on Cell Proliferation and Apoptosis in the Liver........................................................................................................ 16 Possible Mode of Action for Carcinogenic Effects............................................................................................................... 16 Risk Assessment ........................................................................................................................................................................ 17 Tables......................................................................................................................................................................................... 18 Table 1. Definition and Structure......................................................................................................................................... 18 Table 2. Physical and Chemical Properties .......................................................................................................................... 18 Table 3. Current and historical frequency and concentration of use according to duration and type of exposure ............... 18 Table 4. Percutaneous penetration study details (Study 1) .................................................................................................. 19 Table 5. Percutaneous penetration study details (Study 2) ................................................................................................. 19 References ................................................................................................................................................................................. 20 INTRODUCTION
Diethanolamine (DEA), an ingredient that functions in cosmetics as a pH adjuster, has previously been reviewed by
the CIR Expert Panel.1 In 1983, the Expert Panel concluded that DEA is safe for use in cosmetic formulations designed for
discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of DEA should not exceed 5%. DEA should not be used with products containing Nnitrosating agents. In that review, it was shown that DEA was a mild skin and eye irritant, and that irritation increased with
increasing ingredient concentration.
Since that time, DEA has been discussed at a number of Panel meetings. It was first discussed at the June 1999
meeting, and then at the meetings in June 2008 and 2009. The Panel has also heard industry presentations on DEA.
However, no formal action was taken regarding a re-review of DEA. A substantial number of published documents have
become available since the 1983 report was issued. The Panel should now formally decide if the new information, which
included a carcinogenicity study performed by the National Toxicology Program (NTP) warrants a re-review DEA, or does it
confirm the original conclusion. To help with your decision, this document contains summaries of data obtained from a
search of published literature, as well as summary information from the original safety assessment.
CHEMISTRY
DEA is an amino alcohol. DEA is produced commercially by aminating ethylene oxide with ammonia.
The replacement of two hydrogens of ammonia with ethanol groups produces DEA. DEA contains small
amounts of triethanolamine (TEA) and ethanolamine (MEA).
DEA is reactive and bifunctional, combining the properties of alcohols and amines. At temperatures of
140°-160°C, DEA will react with fatty acids to form ethanolamides. The reaction of ethanolamines and
sulfuric acid produces sulfates and, under anhydrous conditions, DEA may react with carbon dioxide to
form carbamates. DEA can act as an antioxidant in the autoxidation of fats of both animal and vegetable
origin. DEA can react with nitrite or oxides of nitrogen to form N-nitrosodiethanolamine (NDELA). The
optimum pH for nitrosamine formation is between 1 and 6; however the rate of NDELA formation in the
pH range of 4-9 is four to six times greater in the presence of formaldehyde.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine1
The structure and synonyms of DEA are given in Table 1, and chemical and physical properties are given in Table 2.
DEA is produced by reacting 2 moles of ethylene oxide with 1 mole of ammonia.2 Typically, ethylene oxide is
reacted with ammonia in a batch process to produce a crude mixture of approximately one-third each MEA, DEA, and TEA.
The crude is later separated by distillation. Dow Chemical Company reports that DEA is commercially available with a
minimum purity of 99.3%, containing 0.45% max. DEA and 0.25% max TEA.3
DEA is structurally similar to choline and ethanolamine.4
USE
Cosmetic
5
DEA functions in cosmetics as a pH adjuster. In 1981, according to data provided through the Food and Drug
Administration (FDA) Voluntary Cosmetic Registration Program (VCRP), DEA was used in 18 formulations, and all but one
of those products were rinse-off formulations.1 Twelve uses were in hair coloring products. Products containing DEA were
used at concentrations of ≤5%. VCRP data obtained recently indicate that DEA is used in 30 formulations; 15 are leave-on
formulations and 15 are rinse-off, and 13 uses are in non-coloring hair formulations.6 According to data submitted by
1
industry in response to a survey conducted by the Personal Care Products Council (Council), DEA is used at concentrations
of 0.0008-0.3%.7 The highest concentration used in leave-on products is 0.06% in moisturizing products. The complete
current and historical use data for DEA are shown in Table 3.
Reportedly, “DEA per se is rarely if ever used in personal care products.”8 The potential for exposure to DEA exists
from the use of alkanolamides of DEA (which are condensation products of DEA and fatty acids).
DEA is present in aerosolized products, and potential effects on the lungs of aerosolized products containing this
ingredient are of concern.
The aerosol properties that determine deposition in the respiratory system are particle size and density. The parameter most closely associated with deposition is the aerodynamic diameter, da, defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic diameter of ≤ 10µm are inhalable. Particles with a da from 0.1 - 10µm settle in the upper respiratory tract and particles with a
da < 0.1 µm settle in the lower respiratory tract.9,10
Particle diameters of 60-80 µm and ≥80 µm have been reported for anhydrous hair sprays and pump hairsprays,
respectively.11 In practice, aerosols should have at least 99% of their particle diameters in the 10 – 110 µm range and the
mean particle diameter in a typical aerosol spray has been reported as ~38 µm.12 Therefore, most aerosol particles are
deposited in the nasopharyngeal region and are not inhalable.
According to the opinion of the Scientific Committee on Consumer Safety of the European Commission, dialkyland dialkanolamines and their salts are on the list of substances which must not form part of the composition of cosmetic
products.13 According to the Commission’s opinion paper, all amines occur only in the form of their salts in all cosmetic
products. This is because all amines are alkaline compounds which are always neutralized by an acid component to produce
their salts. There is concern about the potential for nitrosamine formation; in principle, secondary amines are potential
precursors of nitrosamines.
In Canada, DEA is completely prohibited as per the Cosmetic Hotlist; Health Canada prohibits the use of
dialkanolamines, including DEA.14 This prohibition is based on the EU prohibition in the Cosmetics Regulation, Annex II.
The use of DEA in product formulations in Canada is being investigated because of reported use at concentrations of ≤3%.
(Health Canada, personal communication).
Non-Cosmetic
DEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes,
mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate
for resins, plasticizers, and rubber chemicals. DEA is used as lubricants in the textile industry, as humectants and softening
agents for hides, as alkalizing agents and surfactants in pharmaceuticals, as absorbents for acid gases, and in organic syntheses.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
DEA has uses as an indirect food additive (21 CFR 175.105).15
TOXICOKINETICS
Absorption, Distribution, Metabolism and Excretion
In-Vitro
Three full thickness skin preparations from CD rats, CD-1 mice, New Zealand White (NZW) rabbits, and 6 from
female mammoplasty patients were used to compare the dermal penetration of DEA through the skin of different species.16
[14C]DEA (96.5% purity; sp. act. 15.0 mCi/mmol) was applied to the skin sample undiluted, or as an aq. solution, at a dose of
20 mg/cm2. Dose volumes of 35 µl undiluted [14C]MEA or 95 µl of the aq. solution (37% w/w) were applied to the exposed
surface of the skin (1.77 cm2). These volumes maintained an infinite dose during the 6 h exposure period. Skin sample
2
integrity was confirmed with the use of a reference chemical, [14C]ethanol. With undiluted DEA, the cumulative dose
absorbed was 0.04% in rat skin, 1.30% in mouse skin, 0.02% in rabbit skin, and 0.08% in human skin. With aq. DEA, an
increase in the cumulative dose absorbed was seen in all species: 0.56% in rat skin, 6.68% in mouse skin, 2.81% in rabbit
skin, and 0.23% in human skin.
Penetration of undiluted and aq. DEA was greater through mouse skin than any other skin sample. With undiluted
DEA, penetration was similar for rat, rabbit, and human skin samples. Penetration of DEA was greater for an aqueous
solution than for undiluted DEA. The researchers hypothesized that this may be attributable to elevated skin hydration
caused by the application of the aqueous solution to the skin.
Human liver slices were incubated with 1 mM [14C]DEA (>97% purity).17 After 4 and 12 h, 11 and 29% of the
DEA, respectively, absorbed into the liver slices. The radioactivity was comprised mostly of DEA (85-97%); four other
metabolites were present at low concentrations. The liver slices were fractionated into aqueous, organic, and pellet fractions.
The aqueous-extractable radioactivity was comprised primarily of DEA, with up to four other components. DEA-derived
radioactivity in the organic extracts was predominately (>90%) comprised of phospholipids containing non-methylated
headgroups. DEA was readily absorbed and incorporated into ceramides, forming mostly ceramide-phosphDEA, and it was
slowly methylated therein.
Dermal
Non-Human
A single dose of 81.1 mg/kg DEA in 15 µl ethanol was applied to a 1 cm2 area of the intrascapular region of male
B6C3F1 mice.18 After 48 h, 58.1% of the dose was recovered in tissues or excreta; 28.3% of the dose was excreted in the
urine, 4.4% in the feces, and 2.2% was present in the skin at the site of application.
A single occluded dose of [14C]DEA was applied to a 19.5 cm2 area on the back of rats for 6 h, and the treated site of
50% of the animals was rinsed.19 In the animals that were not rinsed, 80% of the dose was found in the wrappings, and 3.6%
found in the skin. In rinsed animals, rinsates from the wrappings contained 58% of the dose, and from the skin contained
26% of the dose. Unrinsed animals absorbed 1.4% of the dose, while those that were rinsed absorbed 0.64%. The majority
of the radioactivity was found in the carcass, liver, and kidneys.
The researchers also conducted a repeated-dose percutaneous-absorption study in which as a pre-exposure, 1500
mg/kg/day non-radiolabeled DEA was applied occlusively under a 2 in x 2 in gauze square to the backs of rats 6 h/day for 3
or 6 days. [14C]DEA, 1500 mg/kg/day, was then applied for 3-6 consecutive days under occlusion; each dose was left in
contact with the skin for 48 h. Totals of 21 and 41% of the dose were absorbed by the animals dosed for 3 and 6 days,
respectively. The majority of the recovered radioactivity was in the wrapping. The carcass, liver, and kidneys contained
most of the radioactivity in the animals. Totals of 4.3 and 13% of the radioactivity were recovered in the urine of animals
dosed with non-radiolabeled DEA for 3 and 6 days, respectively. Pre-exposure to DEA resulted in a 1.5- to 3.0-fold increase
in the absorption rate.
Groups of 4-5 male Fischer 344 rats were used to evaluate the absorption, distribution, metabolism, and excretion
(ADME) of DEA (99% purity) following dermal administration.20 The dose, which contained 6-20 µCi radiolabel, unlabeled
DEA, and ethanol, for a total dose volume of 25 µl per dose, was applied to a 2 cm2 area of skin under a non-occlusive
covering. Absorption increased significantly with increasing dose. After 48 h, only 2.9% of the radioactivity was absorbed
following dermal application of 2.1 mg/kg, while 10.5 and 16.2% of the radioactivity was absorbed with doses of 7.6 and
27.5 mg/kg, respectively. Of the amount absorbed, 1.2, 4.3, and 4.5% of the radioactivity was recovered at the dose site
3
following application of 2.1, 7.6, and 27.5 mg/kg [14C]DEA, respectively. The greatest tissue/blood ratios were found in the
liver and lung.
Groups of 4-5 male B6C3F1 mice were used in dermal studies of the ADME of DEA. The total volume for mice
was 15 µl/dose, and the dose was applied to a 1 cm2 area of skin using a non-occlusive covering. Absorption through mouse
skin was greater than through rat skin. At 48 h after dermal application of 8 and 23 mg/kg [14C]DEA, 26.8 and 33.8% of the
dose was absorbed in the mice. A statistically significant increase in the amount absorbed after dosing with 81 mg/kg,
58.1%, was observed. The amount of radioactivity found at the site of application was only 4.0, 3.1,and 2.2% of the dose
following application of 8, 23, and 81 mg/kg [14C]DEA, respectively, and the amount excreted in the urine 48 h after application was 7.5%, 10.4, and 16.4%, respectively. Again, the tissue/blood ratio was greatest in the liver and kidneys.
DEA, 80 mg/kg bw in acetone, was applied to a 2 cm2 area on the backs 7 female C57BL/6 mice for 11 days.21
DEA and its methylated metabolites accumulated in the liver and plasma of mice. Also, a statistically significantly decrease
in hepatic concentrations of choline and its metabolites were reported.
Human
Three female subjects applied a lotion containing 1.8 mg DEA/g lotion to their entire body 2x/day for 1 mo.21
Blood samples were collected 1 day prior to the start of dosing, at 1 wk, and at 1 mo. Two of the subjects completed 1 mo of
application, while the third completed 3 wks. Application of the DEA-containing lotion for 1 mo resulted in increased
concentrations of DEA and dimethylDEA in plasma. However, when compared to mice that were dosed with 80 mg/kg
bw/day DEA for 11 days, the human absorption rates were 100- to 200- times less than those found in mice.
Oral
Non-Human
Four male Fischer 344 rats were dosed orally by gavage with 7 mg/kg aq. [14C]DEA to examine the ADME of DEA
(>97% purity).17 The amount excreted in the urine at 24 and 48 h was 9 and 22%, respectively, and the amount in the feces
was 1.6 and 2.4%, respectively. At 48 h after dosing, 27% of the dose accumulated in the liver, 5% in the kidneys, and 0.32,
0.27, 0.19, and 0.18% in the spleen, brain, heart, and blood, respectively. As measured in the liver and the brain, 87-89% of
the radioactivity distributed into the aq. phase, and 70-80% of that total radioactivity was as unchanged DEA. In the liver,
the remaining radioactivity was distributed between three methylated metabolite fractions, while in the brain, only one minor,
non-methylated metabolite was present.
With repeat oral dosing, DEA continued to accumulate in tissues, reaching steady states at 4-8 wks. The percent of
the dose measured in the blood, brain, and liver decreased during 8 weeks of repeated oral dose of 7 mg/kg/day when compared to the single dose post-48-h values. Again, DEA was the major radioactive component. Using high-performance
liquid chromatography (HPLC), almost all of the organic-extractable hepatic radioactivity eluted with the PC fraction, with
greater than 95% of the material in the form of phospholipids containing an N,N-dimethyl-DEA headgroup. In the brain, the
entire organic-extractable radioactivity eluted in the PE fraction, and it was almost entirely comprised of DEA-containing
headgroups.
According to the researchers, the results of this study demonstrated that DEA is O-phosphorylated and N-methylated, and that these metabolites are incorporated as the polar headgroups in aberrant phospholipids. The researchers felt this
was evidence that DEA and MEA, a naturally-occurring alkanolamine, share common biochemical pathways of transformation. Retention and bioaccumulation of DEA-derived radioactivity was attributed partly to aberrant phospholipids being
incorporated into tissues, most probably in cell membranes.
4
Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME of a single oral administration of [14C]DEA
(99% purity).20 The dose administered contained from 2-200 µCi radiolabeled, unlabeled DEA, and water to achieve a target
dose of 5 ml/kg bw. Gastrointestinal absorption was nearly complete after ≤200 mg/kg DEA. After 48 h, 22% of the dose
was excreted in the urine and 2.4% in the feces, primarily as unchanged DEA. Radioactivity was not detected in carbon
dioxide. Excretion was mostly unchanged DEA. A total of 57% of the radioactivity was found in the body 48 h after dosing
with 7 mg/kg [14C]DEA. Disposition of radioactivity was greatest in the liver (27.3%), muscle (16.3%), skin (5.1%), and
kidneys (5%); only 0.2% of the radioactivity was found in the blood after 48 h. Dose did not affect distribution in the tissues
The researchers then evaluated the ADME of DEA upon repeat oral exposure. Four rats were dosed orally with 7
mg/kg/day [14C]DEA for 5 days. Approximately 40% of the total dose was excreted during dosing. At 48 h after the last
dose, a total of 42% of the radioactivity was found in the tissues, with the greatest distribution in the liver (18.2%), muscle
(12.4%), kidneys (5.56%), and skin (4.18%). To further investigate DEA bioaccumulation, rats were dosed with 7 mg/kg/day
DEA, 5 days/wk, for 2, 4, or 8 wks. During the last week of each dosing period, 69, 79, and 92% of the dose was excreted,
respectively. After 8 wks of dosing, most of the radioactivity was recovered as unchanged DEA; however there were also
significant amounts of poorly retained metabolites, N-methylDEA, and another metabolite that was tentatively identified as a
quaternized lactone. The % dose recovered in the liver after 2, 4, and 8 wks of dosing was 12.3, 7.9, and 4.12%, respectively.
It was estimated that steady-state for bioaccumulation occurred after 4 wks of repeat dosing, except for the blood, which
continued to bioaccumulate DEA throughout dosing. Clearance of bioaccumulated DEA appears to be a first-order process,
with a whole body elimination half-life of ~6 days.
Other
Non-Human
Rats (number not specified) were given a single i.v. dose of 7.5 mg/kg [14C]DEA.22 After 48 h, 28 and 1% of the
dose was excreted in the urine and feces, respectively. The remainder was found in the tissues, with the highest concentrations in the liver and kidneys. (Additional details not provided.)
Groups of Sprague-Dawley rats (number per group not specified) were given an i.v. dose of 10 or 100 mg/kg DEA
in physiological saline.19 Animals were killed 96 h after dosing. Peak blood concentrations appeared 5 min after dosing.
Elimination from the blood was biphasic. Totals of 25 and 36% of the dose were excreted in the urine with 10 and 100
mg/kg DEA, respectively.
Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME [14C]DEA (99% purity) in phosphate-buffered
saline after intravenous (i.v.) administration.20 After 48 h, 28% of the dose was excreted in the urine, 0.6% in the feces, and
only 0.2% in carbon dioxide. Using HPLC, it was determined that most of the radioactivity in the urine was present as
unchanged DEA. A total of 54% of the dose was found in the body 48 h after dosing with 7 mg/kg [14C]DEA, and as with
oral dosing, the greatest disposition was found in the liver (27%), muscle (15%), skin (4.5%), and kidneys (4%). Only 0.2%
of the radioactivity was found in the blood after 48 h.
Groups of 5 female Sprague-Dawley rats were dosed i.v., via the cannulated jugular vein, with 10 or 100 mg/kg
14
[ C]DEA (97.4% purity); the dose volume was 2 ml/kg, and each rat received ~4.2 µCi 14C.23 Blood samples were taken at
various intervals up to 84 h after dosing. The peak concentrations of radioactivity in both the plasma and the red blood cells
were observed 5 min after dosing. Clearance of radioactivity from the plasma was calculated to be approximately 50 and 93
ml/h/kg for the low and high dose, respectively. In blood, these values were 84 and 242 ml/h/kg, respectively
Urine and feces were collected for 96 h after dosing. During this time, the major route of excretion was urinary; 25
and 36% of the dose was recovered in the urine. Urinary excretion was rapid at the high dose level, with 23% of the dose
5
recovered in the first 12 h. Only 8.5% of the dose was recovered during this time frame with the low dose. The majority of
the radioactivity was recovered in the carcass; 35 and 28% for the low and high dose, respectively. In the tissues at 96 h after
dosing with 10 and 100 mg/kg DEA, approximately 21 and 17% of the dose was recovered in the liver, 7 and 5% in the kidneys, and 5 and 5% in the skin, respectively. The researchers stated that because the majority of the administered radioactivity was recovered in the tissues, particularly in the liver and kidneys, this indicated a propensity for bioaccumulation.
There was some evidence that the bioaccumulation was dose-dependent.
Percutaneous Absorption
Non-Human
[14C]DEA was applied to a 2 cm2 area of the intrascapular region of male F344/N rats at a dose of 2.1, 7.6, or 27.5
mg/kg bw.18,22 After 48 h, 2.9, 10.5, or 16.2% of the dose, respectively, was absorbed. It was shown that a 10-fold increase
in the concentration of DEA resulted in a 450-fold increase in the rate of absorption.
[14C]DEA was applied to a 1.0 cm2 area of the back of 4 B6C3F1 mice, and the site was protected.22 Approximately
59% of the dose was absorbed, and the absorption rate was 19.9 µg/cm2/h.
The in vitro absorption of 2 mg/cm2 [14C]DEA was determined using fuzzy rat skin.24 A total of 1.4% of the
applied dose was applied over 24 h, with 1.9% of the dose remaining in both the stratum corneum and viable dermis/epidermis. Values were similar at 72 h.
Human
The percutaneous absorption of cosmetic formulations spiked with [14C]DEA (95-99% purity) was examined using
viable and non-viable human skin.25 Details regarding the product types used, the relevant ingredient compositions, the doses
applied, absorption of DEA, and retention in the skin are given in Table 4. Very little DEA was found in the receptor fluid,
with only 0.1% of the applied dose recovered in the receptor fluid of the shampoo and hair dye formulations. While the
amount absorbed was similar for these two product types, the distribution and localization were different, with most of the
DEA penetrating from the shampoos being localized in the stratum corneum, and that from the hair dyes being found in
deeper epidermal and dermal layers. With the lotion, 0.6-1.2% of the dose recovered in the receptor fluid. Approximately
65% of the penetrated DEA from the TEA lotion formulation was found in the lower layers of the skin. DEA did not appear
to be covalently bound to the skin, and extended times before analysis did not result in any statistically significant difference
compared to the values obtained after 24 h. Repeat application studies with viable skin did not produce a significant change
of dose absorbed into receptor fluid. However, with non-viable skin, absorption into the receptor fluid from the lotion
increased each day, from 0.6% on the first day to 2.6% on the third day; testing showed that the skin barrier did not remain
intact for the entire 72 h. Using a shampoo and a lotion formulation, non-viable skin gave penetration values similar to
viable skin. The researchers concluded that most of the DEA that penetrated was not available for systemic absorption.
Another study examining the percutaneous penetration of cosmetic formulations spiked with radiolabeled and
unlabeled DEA was performed mimicking simulated use conditions using fresh and frozen human skin samples.26 Details
regarding the product types used, the relevant ingredient compositions, the doses applied, absorption of DEA, and retention
in the skin are given in Table 5. Very little DEA penetrated the skin; 0.011-0.034% of the applied dose of the shampoo
formulations penetrated, 0.024-0.063% of the applied dose of the hair dye formulations, and 0.508% of the bubble bath
formulation penetrated the skin. With a moisturizer formulation, 0.605% of the applied dose penetrated fresh skin, while
0.456% penetrated frozen skin.
The researchers also examined the penetration of a simple aq. 1% solution of DEA through fresh and frozen human
skin samples. The cumulative 24 h percutaneous absorption was approximately 5-fold greater in frozen skin compared to
6
fresh skin, i.e., 8.87 µg/cm2 compared to 1.73 µg/cm2. The 24-h cumulative penetration values represented 0.433 and
0.086% of the dose for frozen and fresh skin, respectively. The amounts of DEA remaining on and in the skin were 1.68 and
1.14% of the applied dose recovered from the frozen and fresh skin samples, respectively. The researchers further investigated the distribution of [14C]DEA between aqueous and lipid fractions of viable skin strata. The radioactivity on the skin
samples from 2 donors (n=12) was determined after a 24 h exposure. At 24 h, the cumulative permeation value was 0.405%
of the applied dose for one donor (abdominal skin) and 0.067% for the other (breast skin). (The researchers stated that it may
be significant that one sample was abdominal skin and the other was breast skin.) Tape-strip profiles for both donors
appeared to indicate that the DEA had become evenly distributed throughout the stratum corneum. The majority of DEA was
recovered in aqueous extract, as opposed to organic extract, of the epidermal and dermal tissue, which suggested that the
material was in the free state and not associated with the lipid fraction.
TOXICOLOGICAL STUDIES
Acute (Single Dose) Toxicity
Oral
The acute oral toxicity of DEA was determined using guinea pigs and rats. Using groups of 2-3 guinea
pigs, all guinea pigs survived with dosing of 1.0 g/kg, but none survived dosing with 3.0 g/kg DEA gum
arabic solution. Using groups of 5 rats, the oral LD50 of undiluted DEA 0.71-0.80 ml/kg, and for a group of
6 rats, the LD50 of DEA in water was 1.82 g/kg. Using 90-120 rats, 20% aq. DEA had an acute LD50 of
1.41-2.83 g/kg, based on results of testing performed over a 10 yr time period. With groups of 10 rats, a
hair preparation containing 1.6% DEA had an LD50 of 14.1 g/kg when diluted and 12.9 ml/kg when
undiluted.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Other
The intraperitoneal (i.p.) LD50 of DEA was 2.3 g/kg for mice.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Repeated Dose Toxicity
Dermal
In a 13-wk study, 1 mg/kg of a hair dye formulation containing 2.0% DEA was applied to the backs of 12
rabbits for 1 h, twice weekly. The test site skin was abraded for half of the animals. No systemic toxicity
was observed, and there was no histomorphologic evidence of toxicity
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
DEA was applied dermally to groups of 5 male and 5 female B6C3F1 mice 5x/wk for 2 wks at doses of 0-2500
mg/kg bw in 95% ethanol.27 All of the male and 3 of the female high-dose test animals died during the study. Ulceration,
irritation, and crusting were observed at the application site of male mice of the 1250 and 2500 mg/kg groups and females of
the 2500 mg/kg group. Microscopically, moderate to marked epidermal ulceration and inflammation were observed in these
animals. Ulcerative necrosis extended into the underlying dermis. Minimally severe acanthosis, without inflammation, was
seen in the 160, 320, and 630 mg/kg dose groups. Absolute and relative liver weights increased in a dose-dependent manner
in males and females. The LOAEL was 160 mg/kg bw.
Repeated dermal exposure of DEA (99.6% purity) in 96% ethanol was applied to the shaved backs of the following
groups of B6C3F1 mice for the following time periods: 8 males and 8 females were dosed daily with 0 or 160 mg/kg bw
(dose volume 2.13 ml/kg bw) for 1 wk, followed by a 3-wk recovery period; groups of 10 males were dosed 5 days/wk with 0
7
or 160 mg/kg bw for 1, 4, or 13 wks; and groups of 8 males were dosed 5 days/wk with 0-1250 mg/kg for 1 or 13 wks.28
With the last dosing scheme, application of 630 and 1250 mg/kg DEA was discontinued, and the animals killed, after 1 wk
due to severe skin lesions; the high dose was then 160 mg/kg. In the other animals, including controls, some erythema and/or
focal crust formation was observed and attributed to the procedure and/or the vehicle, but not to DEA. No DEA-related
deaths were observed. Body weights were not affected by dosing. Statistically significant increases in liver weights were
seen in mice dosed with ≥10 mg/kg bw/day DEA for 1 or more weeks. Microscopically in the liver, eosinophilia was found
in animals dosed with ≥40 mg/kg DEA for 1 or more weeks, and hepatocellular giant cells were seen in animals dosed with
160 mg/kg for 13 wks.
Unoccluded dermal applications of 0-600 mg/ml DEA (purity >99%) in acetone were applied to the backs of 10
male and 10 female B6C3F1 mice, 5 days/wk for 13 wks, at doses of 0-1250 mg/kg bw.29 (The area of the dose site was not
provided.) Two male mice and 4 female mice dosed with 1250 mg/kg DEA were killed in moribund condition. Final mean
body weights of male mice dosed with 1250 mg/kg were statistically significantly decreased compared to controls. Clinical
signs of toxicity, observed in males and females dosed with 630 and 1250 mg/kg DEA, were irritation, crust formation, and
thickening at the application site. Ulceration and inflammation were observed in the 630 and 1250 mg/kg dose groups.
Dose-dependent increases in absolute and relative liver weights, associated with hepatocellular cytological changes, were
observed, and hepatocellular necrosis was seen in males dosed with 320-1250 mg/kg DEA. Absolute kidney weights were
statistically significantly increased in males and females of all test groups and relative kidney weights were increased in
males of all test groups and females dosed with 630 or 1250 mg/kg DEA; nephropathy was not found. Heart weights were
increased in high dose males and females, and degeneration was reported.
Unoccluded dermal applications of 0-500 mg/ml DEA (purity >99%) in 95% ethanol were applied to the backs of 10
male and 10 female F344/N rats, 5 days/wk for 13 wks, at doses of 0-500 mg/kg bw.30 (The area of the dose site was not
provided.) One male and 2 females given 500 mg/kg died or was killed in moribund condition during the study. Clinical
signs of toxicity, observed in males and females given 125-500 ppm DEA, were irritation and crusting at the application site.
Final mean body weights were statistically significantly decreased in males dosed with 250 and 500 mg/kg and females
dosed with 125-500 mg/kg DEA. Increases in absolute and relative kidney weights were observed with increased incidence
of renal lesions. Increases in absolute and relative liver weights were not accompanied by an increase in hepatic lesions.
Demyelination in the medulla oblongata was seen in all high dose animals and 7 females given 250 mg/kg DEA; this lesion
was minimal in severity.
Oral
Oral studies were conducted in which neonatal rats were dosed with 1-3 mM/kg/day DEA, as a neutralized
salt, on days 5-15 after birth, male rats were dosed with 4 mg/ml neutralized DEA in drinking water for 7
wks, and rats were fed 0-0.68 g/kg/day DEA in feed for 90 days. Repeated oral ingestion of DEA produced
evidence of hepatic and renal damage. Deaths occurred in the 7 wk and 90 day studies.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Female CD-1 mice, 3 per group, were dosed orally, by gavage, with 10-1000 mg/kg bw DEA in distilled water for 7
days.
31
One animal of the 100 mg/kg group died, and the death was considered test article-related. (Two deaths in the 1000
mg/kg group were attributed to gavage error.)
In a 13-wk study, 10 male and 10 female B6C3F1 mice were dosed orally, by gavage, 5 times/wk with 0-800 mg/kg
DEA in deionized water.32 Two males of the high dose group died during the study. Body weights and body weight gains of
8
high dose males were reduced. No clinical signs of toxicity were noted. Treatment-related renal lesions were found, but
details were not provided.
Groups of 10 male albino rats were given feed containing 0-1000 mg/kg bw/day DEA (99% purity) for 32 days.33
Nine of the 10 high dose animals died during the study. All test animals had decreased hemoglobin and hematocrits, with an
increased white blood cell count. The relative liver weights of animals fed 0.01 and 0.1% DEA, and the absolute liver
weights of animals of the 0.1% group, were increased. In a repeat 30-day study using the same procedure and dose levels, 7
of the 10 high dose animals died, and the remaining 3 high-dose animals killed, prior to study termination; body weights and
feed consumption were significantly reduced in this group. Again, hemoglobin and hematocrit were reduced in the 0.1%
group, and the hemoglobin value was reduced in the 0.1% group.
Groups of 10 male and 10 female B6C3F1 mice were given 0-10,000 ppm DEA (>99% purity) in the drinking water
for 13 wks.29 The pH of the solution was adjusted to 7.4. All males and females of the 5000 and 10,000 ppm groups and 3
females of the 2500 ppm group died during the study. Body weight gains were decreased in males of the 2500 ppm group
and females of the 1250 and 2500 ppm groups. No significant gross effects were noted at necropsy. Statistically significant,
dose-dependent increases in absolute and relative liver weights were observed, with hepatocellular cytological alterations and
necrosis in animals given ≥2500 pm DEA. Absolute and relative kidney weights also increased dose-dependently, with
statistically significant increases in mice given 1250 or 2500 ppm DEA, and neuropathy in all male test groups and female
test groups given 2500 or 5000 ppm DEA. Heart weights were increased in female mice given 2500 ppm DEA, and relative
heart weights were increased in males of the 2500 ppm group and females of the 1250 and 2500 ppm groups.
Groups of 10 male F344/N rats were given 0-5000 ppm and 10 female F344/N rats were given 0-2500 ppm DEA in
the drinking water for 13 wks.30 Two males of the high dose group died during the study. The following dose-related effects
were observed: decreases in body weight gains in males and females; hematological effects; increases in kidney weights
accompanied by renal lesions; and increases in relative liver weights in males and females. The brain and spinal cord were
also identified as targets of DEA toxicity.
Inhalation
Short-term inhalation of 200 ppm DEA vapor or 1400 ppm DEA aerosols produced respiratory difficulties
and some deaths in male rats. Inhalation of 25 ppm for 216 continuous hours resulted in increased liver
and kidney weights, while exposure of male rats to 6 ppm DEA following a “workday” schedule for 13 wks
caused decreased growth rate, increased lung and kidney weights, and some deaths.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
In a 2-wk dose-range finding inhalation study, 10 male and 10 female Wistar rats were exposed, nose only, to target
concentrations of 0-400 mg/m3 DEA (>99% pure) for 6 h/day, 5 days/wk.34 Mass median aerodynamic diameter (MMAD)
was 0.6-1.9 µm. Test article-related effects were not seen with 100 or 200 mg/m3 DEA. With 400 mg/m3 DEA, decreased
body weights and body weigh gains were seen in males and relative and absolute liver weights were seen in females. Microscopically, no effects were seen in the respiratory tract; the larynx was not examined.
Based on the results of the dose-range finding study, target concentrations of 0, 15, 150, and 400 mg/m3 DEA were
used in a 90-day study, in which groups of 13 male and 13 female rats were exposed via inhalation to 65 exposures, 6-h/day 5
days/wk. The MMAD was 0.6-0.7 µm. A functional observational battery was conducted using 10 rats/gender/group. Body
weight gains were reduced in males of the high dose group. Some statistically significant effects on clinical chemistry values
were seen in the mid and high dose group. Males and females of the high dose group had statistically significant increases in
blood content in the urine, and males of the mid and high dose groups excreted significantly elevated amounts of renal tubu9
lar epithelial cells. Relative liver weights were statistically significantly increased in males and females of the high dose
group and females of the mid-dose group, and relative kidney weights were statistically significantly increased in males and
females of the mid and high dose groups. Focal squamous metaplasia of the ventral laryngeal epithelium was observed in
test animals of all groups, and a concentration-dependent increase in laryngeal squamous hyperplasia, and in the incidence
and severity of local inflammation of the larynx and trachea, were observed. There were no indications of neurotoxicological
effects.
In a third study, test groups of 10 male and 10 female Wistar rats were exposed to target concentrations of 0, 1.5, 3,
and 8 mg/m3 DEA using the same dosing schedule as above, and recovery groups of 10 females were exposed to 3 or 8
mg/m3, with a post-exposure period of 3 mos. No dose-related clinical signs were observed. Liver weights of test, but not
recovery, females dosed with 8 mg/m3 were statistically significantly increased. Laryngeal effects were similar to those
described above. No microscopic changes were observed in the upper respiratory tract of recovery animals. The 90-day no
observed effect concentration (NOAEC) was determined to be 1.5 mg/m3 DEA.
In inhalation studies, Sprague-Dawley rats, Hartley guinea pigs and Beagle dogs (number per species and sex not
specified) were each exposed to 0.5 ppm DEA for 6 h/day, 5days/wk, for a total of 45 exposures.33 All animals survived
until study termination. There were no clinical signs of toxicity, and no evidence of irritation. No gross or microscopic
lesions were observed at necropsy.
REPRODUCTIVE AND DEVELOPMENTAL STUDIES
Dermal
Hair dyes containing up to 2% DEA were applied topically to the shaved skin of groups of 20 gravid rats
on days, 1, 4, 7, 10, 13, 16, and 19 of gestation, and the rats were killed on day 20 of gestation. No
developmental or reproductive effects were observed.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
A reproductive and developmental toxicity study was performed in which 0-320 mg/kg DEA (98.5% purity) in
ethanol was applied to a 2 cm2 area on the back of 15 male C57BL/6 mice, 15 per group, for 4 wks.35 (It was not stated
whether the test area was covered.) These males were then mated with untreated females. In the parental male mice, sperm
motility was significantly decreased in a dose-dependent manner. In male pups, a significant decrease in epididymis weight
was seen in the 80 mg/kg group at postnatal day (PND) 21, and reductions in male reproductive weights of high-dose pups
was seen at PND 70. There were no significant differences in skeletal formation, and differences in growth and development
parameters were not significant.
Doses of 0-320 mg/kg DEA (98.5% purity) in ethanol were applied to a 2 cm2 area on the backs of groups of 10
gravid female C57BL/6 mice on day 6 of gestation through PND 21. The body weights of male and female pups of the high
dose group were statistically significantly decreased compared to controls. No specific differences in organ weights were observed in pups of the test groups as compared to controls. There were no significant differences in skeletal formation. Some
dose-dependent effects on growth and developmental parameters were noted. Sperm motility was decreased in male pups,
but this result was not statistically significant.
CD rats and NZW rabbits were used to evaluate the potential of DEA (≥99.4% purity) to produce developmental
toxicity with dermal exposure.36 Groups of 25 gravid CD rats were dosed dermally with 150-1500 mg/kg/day DEA in
deionized water, 6 h/day, on days 6-15 of gestation, under an occlusive covering. Dosing volume was 4 ml/kg/day. Control
animals were dosed with vehicle only. Teratogenic effects were not seen at any dose. Dermal application of 500 mg/kg DEA
10
resulted in alterations of maternal hematological parameters, but did not affect embryonal/fetal development. Other signs of
maternal toxicity were seen at this dose and with 1500 mg/kg/day. The NOEL for embryonal/fetal toxicity was estimated to
be 380 mg/kg/day, which incorporates an adjustment for the 10-24% deficit in expected dose that occurred on days 12-15 of
gestation.
Groups of 15 mated rabbits were dosed dermally with 35-350 mg/kg/day DEA in deionized water, 6 h/day, on days
6-18 of gestation, under an occlusive covering. Dosing volume was 2 ml/kg/day, and controls were dosed with vehicle only.
Dermal administration of 350 mg/kg/day DEA produced severe skin irritation in rabbits, and signs of maternal toxicity were
observed at this dose. No developmental toxicity was observed, and there was no evidence of teratogenicity at any dose. The
NOEL for maternal toxicity of DEA in rabbits was 35 mg/kg/day, and the embryonal/fetal NOEL was 350 mg/kg/day DEA.
Oral
In a Chernoff-Kavlock screening test, groups of 4 gravid CD-1 mice were dosed orally, by gavage, with 0-2605
mg/kg bw DEA in distilled water on days 6-15 of gestation.31 Two, 3, and 4/4 animals of the 720, 1370, and 2605 mg/kg
dose groups died during the study. Rough hair coats were observed at all dose levels. Group of 50 female gravid CD-1
female mice were dosed orally, by gavage, with 0 or 450 mg/kg bw DEA in distilled water on days 6-15 of gestation. No
animals died during the study. The reproductive index and average number of live litters on day 0 were not affected by
dosing, but the average number of live litters on day 3 was reduced. Mean body weights and body weight gains of pups were
also decreased on PND 3.
Gravid Sprague-Dawley rats were dosed orally, by gavage, with 50-1200 mg/kg/day DEA on days 6-15 of gestation.
37
Maternal mortality was observed at doses of 50-1200 mg/kg/day. At doses of 50 and 200 mg/kg/day, no differences
in gross developmental endpoints were found between test and control animals. The NOEL for embryonal/fetal toxicity was
200 mg/kg/day DEA. However, maternal weight gains were significantly reduced at that dose. (Additional details were not
provided.)
Groups of 12 gravid female Sprague-Dawley rats were dosed orally, by gavage, with 50-300 mg/kg bw/day DEA
(>98% purity) distilled water on days 6-19 of gestation, while controls were dosed with vehicle only.38 Dosing volume was 5
ml/kg. Surviving dams and pups were killed on PND 21. All females dosed with 300 mg/kg DEA were killed prior to study
termination due to excessive toxicity. Toxicity was also observed for one dam dosed with 200 mg/kg, and only 5 dams of the
250 mg/kg group delivered live litters and survived until study termination. The calculated LD50 was 218 mg/kg bw/day. No
significant maternal or developmental toxicity was seen with 50 mg/kg bw/day DEA. Signs of maternal and developmental
toxicity were seen at doses of ≥125 mg/kg bw/day, including reduced maternal weight gains, increased kidney weights in
dams, increased post-implantation and postnatal mortality, and reduced live pup weights. The NOAEL for maternal toxicity
and teratogenicity was 0.05 mg/l, and the LOAEL for these parameters were 125 mg/kg/day.
Inhalation
In a range-finding study, groups of 10 gravid Wistar rats were exposed, nose-only, to target concentrations of 0.10.4 mg /l DEA, 6 h/day, on days 6-15 of gestation.39 (DEA purity was >98.7%.) All animals survived until study termination. Relative liver weights were increased in animals of the 0.2 mg/l group, and absolute and relative liver weights were increased in animals of the 0.4 mg/l group. No treatment-related effects were observed with 0.1 mg/l DEA.
Groups of 25 gravid Wistar rats were exposed, nose-only, to target concentrations of 0.01-0.2 mg DEA aerosol/l air,
6 h/day, on days 6-15 of gestation.40,41 (DEA purity >98.7%; vehicle not specified.) Maternal toxicity, as indicated by
vaginal hemorrhage, was seen at the highest dose level. No treatment-related malformations were observed at 0.2 mg/m3.
11
The NOAEC for both maternal and developmental toxicity was 0.05 mg/l, and the NOAEC for teratogenicity was >0.2 mg/l,
which was the highest dose tested in this study.
GENOTOXICITY
In Vitro
DEA, with and without metabolic activation using liver preparations from rats induced with a
polychlorinated biphenyl mixture, was not mutagenic to Salmonella typhimurium TA100 or TA1535.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
With or without metabolic activation, DEA was not mutagenic in Ames test using Salmonella typhimurium TA98,
TA100, TA1535, or TA1537, was negative in a mouse lymphoma assay, and did not induce sister chromatid exchanges or
chromosomal aberrations in a Chinese hamster ovary cell cytogenetic assay.18 DEA was not clastogenic in a mouse micronucleus test. Positive results were reported in an in vitro assay for induction of DNA single-strand breaks in isolated hepatocytes for rats, hamsters (both at ≥25 µmol/tube), and pigs (≥12.5 µmol/tube).
CARCINOGENICITY
Dermal
The National Toxicology Program (NTP) evaluated the carcinogenic potential of DEA (>99% purity) in ethanol using B6C3F1 mice and F344/N rats.18 Groups of 50 male and 50 female mice were dosed dermally with 0-160 mg/kg/day, 5
days/wk, for 103 wks. Survival of dosed females, but not males, was significantly decreased in a dose-dependent manner.
Mean body weights of test animals were decreased at various intervals throughout the study. In male mice, the incidences of
hepatocellular adenoma and of hepatocellular adenoma and carcinoma (combined) in all dose groups, and the incidence of
hepatocellular carcinoma and hepatoblastoma in the 80 and 160 mg/kg group, were statistically significantly increased compared to controls. In female mice, the incidence of hepatocellular neoplasms was significantly increased. Male mice also had
a dose-related increase in the incidences of renal tubule hyperplasia and renal tubule adenoma or carcinoma (combined), and
an increase in the incidence of renal tubule adenoma. In male and female mice, incidences of thyroid gland follicular cell
hyperplasia were increased. Hyperkeratosis, acanthosis, and exudate were treatment-related changes observed at the application site. It was concluded that there was clear evidence of carcinogenic activity of DEA in male and female B6C3F1 mice
based on increased incidences of liver neoplasms in males and females and increased incidences of renal tubule neoplasms in
males.
Groups of 50 male rats were dosed dermally with 0-64 mg/kg bw DEA, and groups of 50 female rats with 0-32
mg/kg bw, 5 days/wk for 103 wks. The only treatment-related clinical finding was irritation at the application site. Minimal
to mild non-neoplastic lesions were found at the site of application of dosed males and females. The incidence and severity
of nephropathy in dosed females, but not males, was significantly greater in the treated groups compared to controls. There
was no evidence of carcinogenic activity of DEA in male or female F344/rats.
The carcinogenic potential of DEA was evaluated using a Tg·AC transgenic mouse model.42,43 Groups of 10-15
female homozygous mice were dosed dermally with 0-20 mg DEA/mouse in 95% ethanol, 5x/wk for 20 wks. DEA was
inactive in Tg·AC mice.
12
IRRITATION AND SENSITIZATION
Irritation
Skin
Non-Human
The primary skin irritation potential of DEA was determined using rabbits. Undiluted DEA, applied to an
unspecified number of rabbits using 10 open applications of 0.1 ml to the ears and 10 semi-occluded
applications to the abdomen, was moderately irritating. No irritation was observed with 10% aq. DEA
following the same protocol. Using groups of 6 rabbits, application of 30 and 50% DEA using semiocclusive patches to intact and abraded skin produced essentially no irritation.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Human
In a study in which an undiluted formulation containing 1.6% DEA was applied to the back of 12 female
subjects for 23 h/day for 21 days, the formulation was considered an experimental cumulative irritant. No
irritation was reported during the induction phase of sensitization studies for a formulation containing 2%
DEA, tested as a 10% solution in distilled water (165 subjects), for a formulation containing 2.7% DEA,
tested undiluted (100 subjects), or for a formulation containing 1.6% DEA, tested undiluted (25 subjects).
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Mucosal
Non-Human
The ocular irritation potential of 30-100% DEA was evaluated using rabbits. As a 30% aq. solution, DEA
was essentially non-irritating, while a 50% aq. solution was a severe irritant. Instillation of 0.02 ml
undiluted DEA produced severe injury to rabbit eyes. A hair preparation containing 1.6% DEA had a
maximum avg. irritation score of 0.7/110 for rinsed and unrinsed eyes.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Sensitization
In Vitro
DEA had an EC3 value of 40% in a mouse local lymph node assay, resulting in a categorization of weak potency of
for skin sensitization.44
Non-Human
In a maximization study using 15 Dunkin-Hartley guinea pigs, intradermal and epicutaneous induction used 1% and
17.6% aq. DEA, respectively, after 10% sodium lauryl sulfate pre-treatment.45 At challenge with 0.7, 3.5, or 7% DEA, 1/15
animals reacted to the lowest and highest challenge concentrations after 2, but not 3 days. In a second maximization test using 20 Himalayan spotted guinea pigs, the intradermal induction, epicutaneous induction, and epicutaneous challenge concentrations were 5%, 75%, and 25% DEA in physiological saline, respectively.45,46 Freund’s complete adjuvant (FCA) was
used at intradermal induction. Two animals had mild erythema at day 1, and one animals had mild erythema at day 2. DEA
was not a sensitizer.
A maximization study was performed on a test substance (known as FORAFAC 1203) containing 2% DEA using a
treatment group of 10 male and 10 female guinea pigs.47 (The type of product was not specified; it also contained 40% water,
30% butoxydiglycol, 11% fluorinated copolymer, and a few other ingredients with concentrations of ≤8%). The intradermal
induction, which included the use of 50% FCA, used a concentration of 5% in sterile saline, and the topical induction and
challenge used undiluted product. A control group of 5 males and 5 females was treated with vehicle. The test substance
containing 2% DEA induced delayed hypersensitivity in 100% of the test animals. No reactions were observed in the
controls.
13
Human
Formulations containing 1.6 and 2.7% DEA, tested undiluted, and a formulations containing 2% DEA,
tested at a 10% solution in distilled water, were not sensitizing in clinical studies.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
Provocative Testing
Over a 15-yr period, provocative patch testing using DEA was performed on 8791 patients.45 There were 157
(1.8%) positive reactions to DEA, and most of the reactions (129; 1.4%) were weak positives. There were 17 (0.2%) irritant
reactions reported. Cosensitization was reported; 77% of the patients that reacted to DEA also tested positive to MEA.
Occupational sensitization was reported; of 7112 male patients, 1.0% that did not work in the metal industry had positive
reactions to DEA, as opposed to 3.1 and 7.5% of those working in the metal industry and those exposed to water-based
metalworking fluids, respectively.
MISCELLANEOUS STUDIES
In male albino rats, following repeated oral administration of 320 mg/kg/day in drinking water of
radiolabeled DEA, a decrease was seen in the amount of injected MEA and choline incorporated in the
liver and the kidneys after 1, 2, and 3 wks as compared to 0 wks. MEA and choline phospholipid
derivatives were synthesized faster and in greater amounts, and were catabolized faster than DEA
phospholipid derivatives. This was not seen with a single 250 mg/kg injection of DEA. The effect of DEA
on the mitochondrial function was also investigated using male albino rats. Administration of neutralized
DEA in the drinking water at doses of 490 mg/kg/day for 3 days or of 160 mg/kg/day for 1 wk produced
alterations of hepatic mitochondrial function. Oral and i.p. administration of DEA my affect, directly or
indirectly, the serum enzyme levels, isozyme patterns, and concentrations of some amino acids and urea in
the male rat liver and kidney. Repeated DEA administration in the drinking water increased male rat
hepatic mitochondrial ATPase and altered mitochondrial structure and function.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and
Monoethanolamine.1
N-Nitrosodiethanolamine Formation
The formation of NDELA upon dermal dosing with DEA (99.7% purity), with and without supplemental oral
sodium nitrite, was determined in male B6C3F1 mice.48 Groups of 5-6 mice were dosed orally with aq. DEA or dermally
with DEA in acetone. DEA, 160 mg/kg/day, was applied for 7 days/wk for 2 wks. One group of mice dosed dermally was
allowed access to the application site, while the other was not. Studies were performed both with and without ~40 mg/kg/day
supplemental sodium nitrite in drinking water. NDELA was not found in the urine or blood of mice dosed with DEA without
nitrite or in the blood or gastric contents of those given supplemental nitrite with DEA.
NDELA formation from DEA (>99% purity) and nitrite was also examined in another study.49 Female B6C3F1
mice were dosed dermally or orally with 4 mg/kg DEA, in conjunction with oral exposure to sodium nitrite. Following 7
days of dermal dosing, no NDELA was detected in the blood, ingesta, or urine of test, vehicle control, or sodium nitrite
control mice. (The limits of detection for the blood, ingesta, and urine were 0.001, 0.006, and 0.47 µg/ml, respectively.)
With a single oral dose, NDELA was formed in all of the animals; the amount of NDELA detected in the blood and ingesta
of mice 2 h post-dosing were 0.008 ± 0.003 µg/g and 0.424 ± 0.374 µg/g, respectively.
Immunotoxicity
Groups of 48 female F344 female rats were dosed orally, by gavage, with 0-200 mg/kg bw DEA in distilled water
for 14 days.50 Rats exposed to 10 and 200 mg/kg DEA had significant decreases in body weight and/or body weight gains.
Liver and kidney weights were increased in a dose-dependent manner. Exposure to DEA did not alter the number of B-cells,
14
T-cells, or T-cell subsets. The proliferative response to allogenic cells was increased in a dose-dependent manner. Natural
killer cell response and cytotoxicity of resident macrophages were decreased in DEA-treated animals.
Inhibition of Choline Uptake
The ability of DEA to alter cellular choline levels was examined in a number of studies. In the study described
previously in which B6C3F1 mice were dosed orally and dermally with 160 mg/kg/day DEA in conjunction with oral sodium
nitrite, a pronounced decrease in choline and its metabolites was observed in the livers.48 The smallest decreases were seen
in the mice that were dosed dermally and not allowed access to the test site, and the greatest increase was seen in the mice
dosed orally.
In Syrian hamster embryo (SHE) cells, DEA inhibited choline uptake at concentrations ≥50 µg/ml, reaching a
maximum 80% inhibition at 250-500 µg/ml.4 DEA also reduced phosphatidylcholine in the phospholipds, was incorporated
into SHE lipids, and transformed SHE cells in a concentration-dependent manner. Excess choline blocked these biochemical
effects and inhibited cell transformation, and the researchers hypothesized there is a relationship between the effect of DEA
on intracellular choline availability and utilization and its ability to transform cells. In a study performed to test the hypothesis that DEA treatment could produce biochemical changes consistent with choline deficiency in mice, it was found that DEA
treatment caused a number of biochemical changes consistent with choline deficiency in mice.51 Hepatic concentrations of Sadenosymethionine (SAM) decreased. Biochemical changes were seen without fatty livers, an observation often associated
with choline deficiency.
To study the dose-response, reversibility and strain-dependence of DEA effects, B6C3F1 mice were dosed dermally
with DEA in ethanol for 4 wks.51 Control animals were either not dosed or dosed with ethanol only. The pattern of changes
observed in choline metabolites after DEA treatment was very similar to that observed in choline-deficient mice, and the
NOEL for DEA-induced changes in choline homeostasis was 10 mg/kg/day. Fatty livers were not observed. (LehmanMcKeeman hypothesized that the lack of fatty livers is the result of an age-dependence mechanism.52) The reactions were
dose-dependent, strain-dependent, and reversible. Dermal application of 95% ethanol decreased hepatic betaine levels,
suggesting that used of ethanol as a vehicle for dermal application of DEA could exacerbate the biochemical effects of DEA.
Species Selective Effect on DNA Synthesis
In studies examining species selectivity of effects caused by DEA, increases in DNA synthesis were observed in
mouse and rat, but not human, hepatocytes following treatment with DEA.53 Additionally, when the hepatocytes were
incubated in medium containing reduced choline, DNA synthesis was increased in mouse and rat hepatocytes, but not human
hepatocytes. Conversely, choline supplementation reduced DEA-induced DNA synthesis in mouse and rat hepatocytes.
Effect on Hippocampal Neurogenesis and Apoptosis
The effect of DEA on neurogenesis was investigated using C57BL/6 mice.54 DEA, 0-640 mg/kg bw in ethanol, was
applied to a 2 cm2 area on the backs of gravid female mice, 6 per group, on days 7-17 of gestation. A dose-related decrease
in litter size was observed at doses >80 mg/kg, and the decrease was statistically significant at doses of 160-640 mg/kg
bw/day. The livers of the maternal mice were analyzed on day 17 of gestation, and hepatic concentrations of choline and its
metabolites were statistically significantly decreased. In the fetal brain, treatment with 80 mg/kg bw/day DEA diminished
the proportion of cells that were in the mitotic phase to 50% of controls. The number of apoptotic cells of the hippocampal
area was >70% higher in fetuses of DEA-treated mice compared to controls. The researchers stated that the effect observed
in the mouse fetal brain after administration of DEA was likely to be secondary to diminished choline levels. The researchers
also hypothesized that a potential mechanism for the effect of choline deficiency, and maybe DEA, on progenitor cell
proliferation and apoptosis involves abnormal methylation of promoter regions of genes.
15
The doses used in the above study were based on expected concentrations of 1-25% DEA in cosmetic formulations.
However, based on comments from the Cosmetic, Toiletry, and Fragrance Association (now known as the Personal Care
Products Council) indicating the over-estimation of the amount of DEA contained in consumer products,8 the researchers
tested lower doses to establish a dose-response relationship. Groups of 7 mice were dosed dermally with 0-80 mg/kg bw
DEA (purity >99.5%) as described previously, with the exception that acetone was used as the vehicle. While the results
reported in the earlier study with 80 mg/kg bw DEA were confirmed, no differences were seen between treated and control
groups with <80 mg/kg bw.
In a study to identify the potential mechanism for the alterations described above, mouse neural precursor cells were
treated in vitro with DEA.55 Cells exposed to 3 mM DEA had less cell proliferation at 48 h and had increased apoptosis at 72
h. DEA treatment decreased choline uptake into the cells, resulting in diminished choline and phosphocholine. A three-fold
increase in choline concentration prevented the effects of DEA exposure on cell proliferation and apoptosis; intracellular
phosphocholine levels remained low. The researcher hypothesized that DEA interferes with choline transport and choline
phosphorylation in neural precursor cells. Additionally, it was suggested that DEA acts by altering intracellular choline
availability.
Effect on Cell Proliferation and Apoptosis in the Liver
To determine whether repeated dermal administration of DEA induced cell proliferation and/or apoptosis in the
livers of mice, male and female B6C3F1/Crl mice were dosed dermally with 0 and 160 mg/kg bw DEA (99.6% purity) in
96% ethanol for 1 wk followed by a 3-wk recovery period, and male mice were exposed dermally to 0 or 160 mg/kg for 1, 4,
or 13 wks.28 A dose response relationship was examined with application of 0-160 mg/kg DEA to male mice for 1 or 13
wks. After 1 wk of dosing, increased cell proliferation in the liver was observed in males and females; this effect was
reversible. Repeated application of ≥10 mg/kg DEA to male B6C3F1 mice caused increased liver cell proliferation. DEA
had no effect on the number of apoptotic cells in the liver.
Possible Mode of Action for Carcinogenic Effects
A non-genotoxic mode of tumorigenic action is indicated, because DEA is not mutagenic or clastogenic. Choline
deficiency has been shown to increase spontaneous carcinogenesis in rodents, and choline deficiency may promote liver
tumor formation.4 Since disposition data indicate that DEA is less readily absorbed across rat skin than mouse skin, resulting
in lower blood and tissue levels of DEA in rats than in mice, it is suggested that, in rats, the levels of DEA that occur are not
high enough to markedly alter choline homeostasis. If true, species differences observed in tumor susceptibility could be a
function of the internal dose of DEA. Alternatively, species differences in tumor susceptibility may explain the increased
incidence of hepatocarcinogenesis in B6C3F1 mice compared to rats exposed to DEA.. Additionally, rats and mice are
reported to be much more susceptible to choline deficiency than humans.56
As further support for this mode of action, a species-selective inhibition of gap junctional intercellular communication by DEA in mouse and rat, but not human, hepatocytes with medium containing reduced choline concentrations provided
additional support that the mechanism for DEA-induced carcinogenicity involves cellular choline deficiency.57 Also, Bachman et al. have hypothesized the DEA-induced choline deficiency leads to altered DNA methylation patterns, which
facilitates tumorigenesis.58
Two other hypotheses as to the mode of action of DEA carcinogenicity as possible alternatives to the intracellular
choline deficiency hypothesis have been proposed.52 One involves the nitrosation of DEA to NDELA and the other the formation of DEA-containing phospholipids. The researcher did not find these likely for the following reasons. Regarding the
first alternate hypothesis, nitrosation to NDELA; NDELA was not detected in mouse plasma or urine after cotreatment of
16
mice with DEA and nitrite. In regards to the second, altered phospholipids; while DEA has been shown to be incorporated
into phospholipids, without qualitative or quantitative differences between rats and mice, carcinogenic effects are seen only
in mice, making it unlikely that incorporation of DEA into the phospholipids is a major determinant of carcinogenic response.
Leung et al reviewed the information available and also felt that choline deficiency is the mechanism responsible for liver
tumor promotion in mice.59
The localization of β-cartenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F1 mice exposed
dermally to 0-160 mg/kg bw DEA for 2 yrs were characterized, and genetic alterations in the Catnb and H-ras genes were
evaluated.60 A lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas led the researchers to suggest that
the signal transduction pathway is not involved in the development of liver tumors following DEA administration.
RISK ASSESSMENT
The Occupational Safety and Health Administration time-weight average (TWA) for DEA is 3 ppm, and the American Conference of Industrial Hygienists threshold limit value-TWA is 0.46 ppm or 2 mg/m3.38
According to a review of DEA by the International Agency for Research on Cancer (IARC) Working Group, there is
inadequate evidence in humans, for the carcinogenicity of DEA.61 There is limited evidence in experimental animals for the
carcinogenicity of DEA. The overall evaluation of the IARC is that DEA is not classifiable as to its carcinogenicity to
humans (Group 3).
17
TABLES
Table 1. Definition and Structure
Ingredient
Definition
CAS No.
Diethanolamine The product of the ammono111-42-2
lysis of 2 stoichiometric
equivalents of ethylene oxide
Synonyms
N,N-diethanolamine
2,2’-dihydroxydiethylamine
2,2’-iminobisethanol
Function in
Cosmetics
pH adjuster
Formula/Structure
H
N
HO
OH
Table 2. Physical and Chemical Properties
Property
Physical Form
Color
Odor
Molecular Weight
/Specific Gravity
Vapor Pressure
Melting Point
Boiling Point
Water Solubility
Other Solubility
log Kow
Disassociation Constant
pKa
Value
clear viscous liquid
white crystalline solid at room temperature
viscous liquid above 28°C
colorless
ammoniacal
105.14
1.0966 @ 20°C
0.01 mm Hg (°C not specified)
28.0°C
268.8°C
soluble
soluble in alcohol, ethanol, and benzene
-2.18 @ 25°C
Reference
1
34
1
1
1
62
1
62
62
62
62
62
62
8.88 @ 25°C
Table 3. Current and historical frequency and concentration of use according to duration and type of exposure
# of Uses
Conc. of Use (%)
data year
19811
20106
19811
20107
Totals
18
30
≤5
0.008-0.3
Duration of Use
Leave-On
1
15
1-5
0.008-0.06
Rinse Off
17
15
≤5
0.009-0.3
Exposure Type
Eye Area
NR
NR
NR
NR
Possible Ingestion
NR
NR
NR
NR
Inhalation
NR
3
NR
NR
Dermal Contact
4
15
≤0.1
0.009-0.06
Deodorant (underarm)
NR
NR
NR
NR
Hair - Non-Coloring
1
13
1-5
0.03-0.3
Hair-Coloring
12
2
1-5
NR
Nail
1
NR
NR
NR
Mucous Membrane
NR
5
≤0.1
0.009
Bath Products
4
NR
NR
NR
Baby Products
NR
NR
NR
NR
NR - no reported use
18
Table 4. Percutaneous penetration study details (Study 1)
[14C]DEA (µCi)
0.020free DEA (%)
Cocamide DEA (%)
Lauramide DEA (%)
Oleamide DEA (%)
TEA (%)
product dose (mg/cm2)
DEA dose (µg)/cm2
Application Time
Monitoring Time (h)
amount absorbed (in
receptor fluid; % of
applied dose)
total penetration
(% of applied dose)
total found in skin
(% of applied dose)
found in epidermis and
dermis (% of dose)
number of samples
Shampoo A
0.49
0.092
x
Shampoo B
0.49
0.28
Hair Dye C
0.49
0.61
x
x
Hair Dye D
0.43
0.61
x
Body Lotion E
0.13
0.020
Body Lotion F
0.12
0.020
x
1.9
4.2
5 min
24 h
1.9
7.7
5 min
24 h
17.5
109.3
30 min
24 h
17.5
108.92
30 min
24 h
0.0115
3
1.02
24 h
24 h; 72 h
0.0115
3
1.2
24 h
24 h
0.1
0.1
0.1
0.1
0.6
1.2
2.5
3.3
2.5
3.6
15.4
7.8
2.4
3.2
2.4
3.5
14.8
6.6
0.9
1.0
1.3
2.3
10.0
4.3
9
8-12
9-12
9-12
21-28
9-12
Oxidative
Hairdye H
0.249
x – indicates that ingredient was part of the formulation
Table 5. Percutaneous penetration study details (Study 2)
DEA (%)
Cocamide DEA (%)
Lauramide DEA
(%)
TEA (%)
Dose Applied
Dilution
Application Time
Monitoring Time
(h)
penetration
(% of dose)
found in skin
(% of dose)
found in dermis
(% of dose)
number of samples
Shampoos A/B
Shampoo C/D
Bubble Bath E
Moisturizer F
0.98
4.02
0.25
-
0.25
-
0.008
-
Semi-Permanent
Hairdye G
0.075
-
-
-
4.75
4.75
-
1.420
4.737
100 µl/cm2
1:10 aq.
10 min
24 h (B) or 48 h
(A)
100 µl/cm2
1:10 aq.
10 min
100 µl/cm2
1:300
30 min
1.992
5 mg/cm2
48 h
100 mg/cm2
30 min
100 mg/cm2
1:1 w/H2O2
30 min
24 h
24 h
48 h
24 h
24 h
A: 0.019%
B: 0.025%
C: 0.034%
D: 0.011%
0.508%
0.063
0.024
A: 0.063%
B: 0.078%
C 0.037%
D: 0.04%
1.923%
0.0301
0.0609
-
-
-
-
-
12
12
12
10
12
cocamide DEA contained 11.0% free DEA
lauramide DEA contained 2.79% free DEA
TEA contained 0.28% free DEA
19
fresh skin: 0.605%
frozen skin:
0.456%
fresh skin: 10.773%
frozen skin:
12.723%
fresh skin: 0.877%
frozen skin:
0.534%
fresh = 13
frozen = 12
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35. Lee ID, Lee GS, Moon HJ, Seok JH, Yang JY, Chae SY, Lee YW, Kim SM, Kim SH, and Jeong SY. A study of the reproductive
and developmental toxicity of diethanolamine. (Unofficial translation.). The Annual Report of KFDA. 2007. 11:18021824. 8EHQ-0409-17501A; Submission by Dow Chemical Co.
36. Marty MS, Neeper-Bradely TL, Neptun DA, and Carney EW. Developmental toxicity of diethanolamine applied cutaneously to
CD rats and New Zealand White rabbits. REg Toxicol Pharmacol. 1999;30:169-181.
37.
Gulati DK, Grimes LK, Heindel J, and Schwetz BA. Range finding studies: Developmental toxicity of diethanolamine when
administered via gavage in CD Sprague-Dawley rats, NTP 89-RF/DT-002. 1990. Secondary reference in Marty et al.
1999.
38.
Center for Life Science and Technology. Final study report. Developmetnal toxicity screen for diethanolamine (CAS No. 11142-2) administered by gavage to Sprgue-Dawley (CD) rats on gestational days 6 through 19: Evaluaton of dams and
pups through postnatal day 21. 12-22-1999. Prepared for the National Toxicology Program; NTP Study No. TER-96001.
39.
BASF AG. Range-finding study on the prenatial inhalation toxicity of diethanolamin in pregnant rats. BASF Project No
11R0233/90011. 8-20-1991. Secondary reference in OECD 2008.
21
40.
Gamer AO, Hellwig J, and Hildebrand B. Study of the prenatal toxicity of diethanolamin n rats after inhalation, Project No.
31R0233/90010; BASF. 1993. Secondary reference in Marty et al. 1999.
41.
BASF AG. Study of the prentatl inhalation toxicit of diethanolamine in rats after inhalation. BASF Project No. 31R0233/90010.
7-8-1993. Secondary reference in OECD 2008.
42.
Tennant RW, French JE, and Spalding JW. Identifying chemical carcinogens and assessing potential risk in short-term bioassays
using transgenic mouse models. Environ Health Perspect. 1995;103:942-950.
43.
Spalding JW, French J, Stasiewicz S, Furedi-Machacek M, Conner F, Tice RR, and Tennant RW. Responses of transgenic mouse
lines p53+/- and Tg·AC to agents tested in conventional carcinogenicity bioassays. Toxicol Sci. 2000;53:213-223.
44.
Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Gerberick GR, Newsome C, Steiling W, and Vohr HW. Classification
of contact allergens to potency. Proposals, Food Chemical Toxciol. 2003. 41:1799-1809. Secondary reference in
OECD 2008.
45.
Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine.
Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60:243-255.
46.
RCC. Contact hypersensitivity to Diethanolamin, rein in albino Guinea pigs, Maximization test, RCC Project 264903.
Unpublished report. 1990. Secondary reference in OECD 1990.
47.
CIT. Skin sensitization test in guinea pigs on FORAFAC 1203. (Maximization method of Magnusson and Kligman.) Laboratory
study umber 20023 TSG. 12-14-2000. 8EHQ-01-14860.
48.
Stott WT, Bartels MJ, Brzak KA, Mar M-H, Markham DA, Thorton CM, and Zeisel SH. Potential mechanisms of tumorigenic
action of diethanolamine in mice. Toxicol Lett. 2000;114:67-75.
49.
Saghir SA, Brzak A, Markham DA, Bartels MJ, and Stott WT. Investigation of the formation of N-nitrosodiethanolamine in
B6C3F1 mice following topical administration of triethanolamine. REg Toxicol Pharmacol. 2005;43:10-18.
50.
Munson AE, White KL, and McCay JA. Immunotoxicity of diethanolamine in female Fischer 344 rats. Report to the National
Toxicology Program. 1992. Secondary reference in OECD 2008.
51. Lehman-McKeeman LD, Gamsky EA, Hicks SM, Vassalo JD, Mar M-H, and Zeisel SH. Diethanolamine induces hepatic choline
deficiency in mice. Toxicol Sci. 2002;67:38-45.
52.
Lehman-McKeeman LD. Incorporating mechanistic data into risk assessment. Toxicology. 2002;181-182:271-274.
53.
Kamendulis LM and Klaunig JE. Species differences in the induction of hepatocellular DNA synthesis by diethanolamine.
Toxicol Sci. 2005;87:(2):328-336.
54.
Craciunescu CN, Wu R, and Zeisel SH. Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus.
FASEB J. 2006;20:1635-1640.
55.
Niculescu MD, Wu R, Guo Z, da Costa KA, and Zeisel SH. Diethanolamine alters proliferation and choline metabolism in mouse
neural precursor cells. Toxicol Sci. 2007;96:(2):321-326.
56.
Zeisel SH and Blusztajn JK. Choline and human nutrition. Annu Rev Nutr. 1994;14:269-296.
57.
Kamendulis LM, Smith DJ, and Klaunig JE. Species differences in the inhibition of gap junctional intercellular communication
(GJIC) by diethanolamine. Toxicologist. 2004. 78:(S-1):
58. Bachman AN, Kamednulis LM, and Goodman JI. Diethanolamine and phenobarbital produce an altered pattern of methylation in
GC-rich regions of DNA in B6C3F1 mouse hepatocytes similar to that resulting from choline deficiency. Toxicol Sci.
2006;90:(2):317-325.
59.
Leung H-W, Kamendulis LM, and Stott WT. Review of carcinogenic activity of diethanolamine and evidence of choline
deficiency as a plausible mode of action. REg Toxicol Pharmacol. 2005;43:260-271.
60. Hayashi S-M, Ton VT, Hong H-HL, Irwin RD, Haseman JK, Devereux TR, and Sills RC. Genetic alterations in the Catnb gene
but not the H-ras gene in hepatocellular neoplasms and hepatoblastomas of B6C3Fa mice following exposure to
diethanolamine for 2 years. Chemico-Biological Interactions. 2003. 146:251-261. Secondary reference in OECD 2008.
22
61.
International Agency for Research on Cancer (IARC). Diethanolamine. IARC Monographs on the evaluation of the carcinogenic
risk of chemicals to humans . 2000;77:349-379.
62.
Knaak JB, Leung H-W, Stott WT, Busch J, and Bilsky J. Toxicology of mono-, di-, and triethanolamine. Review of
Environmental Contamination and Toxicology. 1997;149:1-86.
23
Re-Review of Ethanolamine
Introduction ................................................................................................................................................................................. 1 Chemistry..................................................................................................................................................................................... 1 Use ............................................................................................................................................................................................... 1 Cosmetic ................................................................................................................................................................................. 1 Non-Cosmetic ......................................................................................................................................................................... 1 Toxicokinetics.............................................................................................................................................................................. 2 Absorption, Distribution, Metabolism and Excretion ............................................................................................................. 2 In-Vitro ............................................................................................................................................................................... 2 Dermal ................................................................................................................................................................................ 2 Non-Human ................................................................................................................................................................... 2 Other................................................................................................................................................................................... 3 Non-Human ................................................................................................................................................................... 3 Toxicological Studies .................................................................................................................................................................. 3 Acute (Single Dose) Toxicity ................................................................................................................................................. 3 Dermal ................................................................................................................................................................................ 3 Oral..................................................................................................................................................................................... 3 Inhalation............................................................................................................................................................................ 3 Other................................................................................................................................................................................... 3 Repeated Dose Toxicity .......................................................................................................................................................... 3 Dermal ................................................................................................................................................................................ 3 Oral..................................................................................................................................................................................... 4 Inhalation............................................................................................................................................................................ 4 Reproductive and developmental studies..................................................................................................................................... 4 Dermal ................................................................................................................................................................................ 4 Oral..................................................................................................................................................................................... 4 Genotoxicity ................................................................................................................................................................................ 5 In Vitro.................................................................................................................................................................................... 5 Irritation and Sensitization ........................................................................................................................................................... 5 Irritation .................................................................................................................................................................................. 5 Skin .................................................................................................................................................................................... 5 Mucosal .............................................................................................................................................................................. 5 Sensitization ............................................................................................................................................................................ 6 Provocative Testing ....................................................................................................................................................... 6 Clinical Use ................................................................................................................................................................................. 6 Tables........................................................................................................................................................................................... 7 Table 1. Definition and Structure .......................................................................................................................................... 7 Table 2. Physical and Chemical Properties of MEA ............................................................................................................. 7 Table 3. Current and historical frequency and concentration of use of MEA according to duration and type of exposure .. 7 References ................................................................................................................................................................................... 8 INTRODUCTION
Ethanolamine (MEA), an ingredient that functions in cosmetics as a pH adjuster, has previously been reviewed by
the CIR Expert Panel. At that time, the INCI name for the ingredient was monoethanolamine. In 1983, the Expert Panel
concluded that MEA is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin, and MEA should only be used in rinse-off products. In that review, it was shown that MEA
is a skin and eye irritant in animals, and clinical studies with formulations containing MEA indicated that it is a human skin
irritant. Also, there was indication that the longer MEA was in contact with the skin, the greater the likelihood of irritation.
This current document contains summaries of data obtained from a search of published literature, as well as summary information from the original safety assessment
CHEMISTRY
MEA is an amino alcohol. The structure and synonyms of MEA are given in Table 1, and chemical and physical properties are given in Table 2. MEA is produced commercially by aminating ethylene oxide with ammonia. The replacement of one hydrogen of ammonia with an ethanol group produces MEA. MEA typically contains a small amount of
diethanolamine (DEA).
MEA is reactive and bifunctional, combining the properties of alcohols and amines. At temperatures of 140°-160°C,
MEA will react with fatty acids to form ethanolamides. Additionally, the reaction of ethanolamines and sulfuric acid
produces sulfates, and, under anhydrous conditions, MEA may react with carbon dioxide to form carbamates.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine1
USE
Cosmetic
2
MEA functions in cosmetics as a pH adjuster. In 1981, data provided through to the Food and Drug Administration
(FDA) Voluntary Cosmetic Registration Program (VCRP) indicated that MEA was used in 51 formulations.1 All but 2 of
those products were rinse-off formulations, and 28 uses were in hair coloring products. Products contained MEA at
concentrations ≤10%. VCRP data obtained in 2010 indicate that the use of MEA has increased significantly, as it is now
reported to be used in 795 formulations. All of the uses, except 1, are rinse-off products, and 782 uses are in hair coloring
formulations.3 According to data submitted by industry in response to a recent survey conducted by the Personal Care
Products Council (Council), MEA is used at concentrations of 0.0002-18%.4 All uses reported are in rinse-off products, with
the exception of 3% in an aerosol hair color spray. The complete current and historical use data for MEA are shown in Table
3. According to data obtained from Health Canada, MEA is used at up to 10%.5 Most of the uses are rinse-off, but some
leave-on type products are reported. For example MEA is reported to be included at 3-10% in an eye make-up.
MEA is listed by the European Commission in Annex III Part 1: the list of substances which cosmetic products must
not contain, except subject to the restrictions and conditions laid down.6 Monoalkylamines, monoalkanolamines, and their
salts, including MEA, are allowed a maximum secondary amine content of 0.5% in finished product. Other limitations
include: do not use with nitrosating agents; minimum purity of 99%; maximum secondary amine content of 0.5% (for raw
materials); maximum nitrosamine content of 50 µg/kg; and must be kept in nitrite-free containers.
Non-Cosmetic
MEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes,
mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate
for resins, plasticizers, and rubber chemicals. It is also used as a lubricant in the textile industry, as a humectant and softening
agent for hides, as an alkalizing agent and surfactant in pharmaceuticals, as an absorbent for acid gases, and in organic
syntheses.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
1
MEA has uses as an indirect food additive; according to 21 CFR 175.105 MEA is allowed for use as a component of
adhexives.7 MEA is also used as a rust inhibitor in water-based metalworking fluids.8
TOXICOKINETICS
MEA is the only naturally occurring ethanolamine in mammals, and it is excreted in the urine. MEA was converted to
phosphatidylethanolamine (PE) in the liver, blood, and brain of female rats that were dosed intraperitoneally (i.p.) with
radiolabeled MEA. The step-wise methylation of PE that converts it to phosphatidylcholine (PC) did not occur in the
brain. Labeled respiratory carbon dioxide has been found in rats after i.p. administration of labeled MEA.
A coenzyme-B12-dependent ethanolamine deaminase mediated the conversion of MEA to acetaldehyle and ammonia
was demonstrated. Intraperitoneal administration of MEA to rats increased blood urea and blood glutamine, and the
researchers suggested that ethanolamine was an ammonia source. Additional researchers, upon detection of labeled
acetate in the urine of rats fed labeled MEA, suggested that MEA is phosphorylated by ATP in vivo, converted to
acetaldehyde, ammonia, and inorganic phosphate, and the acetaldehyde is oxidized to acetate. The researchers further
hypothesized that the removal of phosphorylated MEA by its conversion to acetate may exert a regulatory effect on PE
biosynthesis.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Ethanolamine.1
Absorption, Distribution, Metabolism and Excretion
In-Vitro
The dermal penetration of [1,2-14C]ethanolamine HCl was evaluated in vitro using split-thickness skin from weanling Yorkshire pigs.9 An ethanolic solution was prepared so that a 5 µl application to 0.8 cm2 of skin gave a chemical dose of
4 µg/cm2 and a radioactive dose of approximately 0.05 µCi. After 50 h, 11% of the dose was lost to evaporation, 5% penetrated percutaneously, and 62% was recovered in the skin residue. Seven to nine times more radioactivity was recovered in
the upper 100 µm layer, compared to recovery from the remaining dermis.
Three full thickness skin preparations from CD rats, CD-1 mice, and New Zealand White rabbits, and 6 from female
mammoplasty patients were used to compare the dermal penetration of MEA through skin of different species.10 [14C]MEA
(98.8% purity; specific activity [sp. act.] 15.0 mCi/mmol) was applied to the skin sample undiluted or as an aqueous (aq.)
solution at a dose of 4 mg/cm2. Dose volumes of 7 µl undiluted [14C]MEA or 32 µl of the aq. solution (22% w/w) were
applied to the exposed surface of the skin (1.77 cm2). These volumes maintained an infinite dose during the 6 h exposure
period. With undiluted MEA, the cumulative dose absorbed was 5.98, 16.92, 8.66, and 0.61% through rat, mouse, rabbit, and
human skin, respectively. With aq. MEA, 1.32, 24.79, 1.87, and 1.11%, was the cumulative dose absorbed through rat,
mouse, rabbit, and human skin, respectively. In vitro absorption of undiluted and aq. MEA was much greater through mouse
skin than human skin, and human skin was the least permeable of all the skin samples. With human skin samples, the cumulative dose absorbed was greater with aq. MEA as compared to undiluted MEA. The researchers hypothesized that enhanced
penetration of aq. MEA may be attributable to elevated skin hydration.
Dermal
Non-Human
The distribution and metabolism of MEA was determined using groups of 5 male athymic nude mice with human
skin grafts and ungrafted athymic nude mice.9 [1,2-14C]Ethanolamine HCl in ethanol was applied at a dose of 4.0 µg (3.6
µCi) to a 1.45 cm2 area of grafted or non-grafted skin. Penetration appeared similar for both groups. Radioactivity in expired
carbon dioxide (CO2) appeared 30 min after dosing, and the amount recovered at 30 min was 0.76-0.83% of the dose for
grafted and ungrafted skin, respectively. After 24 h, 18.5-19% of the dose was in expired CO2. Distribution was also similar
for both groups. At 24 h after dosing, 24.3-25.8% of the dose was recovered in the liver and 2.24-2.53% in the kidneys. At
the application site, 18.4% of the radioactivity was recovered in the grafted skin, and 12.1% was recovered in the ungrafted
skin. The amount of radioactivity recovered in the urine was 4.6 and 5.2% for grafted and ungrafted nude mice, respectively.
2
Unchanged MEA comprised 10.2% of the urinary radioactivity. The major urinary metabolites were urea and glycine,
comprising 39.9 and 20.4% of the urinary radioactivity. Minor urinary metabolites were serine, uric acid, choline, and
unidentified ninhydrin-positive compounds.
The radioactivity in proteins and amino acids isolated from the liver, human skin grafts, and mouse skin was determined as an evaluation of the metabolism of MEA. The researchers stated that the appearance of 14C in skin and hepatic
amino acids and proteins, and the incorporation of MEA into phospholipids, was evidence of extensive metabolism of the
absorbed MEA. The liver was the most active site of MEA metabolism.
Other
Non-Human
Five male athymic nude mice were dosed intraperitoneally (i.p.) with 4.0 µg (3.6 µCi) of [1,2-14C]ethanolamine
HCl in ethanol, and the distribution and metabolism were examined following dosing.7 Radioactivity was detected in expired
CO2, increasing gradually over time. After 24 h, 18% of the radioactivity was detected in expired air.
TOXICOLOGICAL STUDIES
Acute (Single Dose) Toxicity
Dermal
The acute dermal toxicity of a mixture containing MEA (as well as hydroxylamine, diglycolamine, propylene
glycol, catechol, and water; percentages not specified) was determined by applying 2g/kg of the mixture to the shaved back
of 5 male and 5 female rabbits.11 The dermal LD50 of the mixture was >2 g/kg. In a similar study with a formulation containing MEA (and hydroxylamine, diglycolamine, gallic acid, and water; percentages not specified), the LD50 of the mixture in
rabbits was 1.24 g/kg bw.12
Oral
The acute oral toxicity of MEA, concentration not specified, ranged from 1.72-2.75 g/kg for rats. Using 90-120 rats,
20% aq. MEA had an LD50 of 2.14-2.74 g/kg, based on results of studies performed over a 10 yr time period. With
groups of 10 rats, a hair preparation containing 5.9% MEA had an LD50 of 14.1 g/kg when diluted and 12.9 ml/kg when
undiluted.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
The acute oral toxicity of a mixture containing MEA (as well as hydroxylamine, diglycolamine, propylene glycol,
catechol, glycolic acid and water; percentages not specified) was determined using groups of 5 male and 5 female rats.13 The
oral LD50 of the mixture was 0.95 g/kg. In a similar study with a formulation containing MEA (and hydroxylamine, diglycolamine, gallic acid, and water; percent in formulation not specified), the oral LD50 of the mixture was 0.815 g/kg bw.
Inhalation
The acute inhalation toxicity of a mixture containing MEA (as well as hydroxylamine, 1-amino-propano-2-ol, catechol, and water; percent of each not specified) was determined using groups of 5 male and 5 female rats. The inhalation LC50
of the mixture was estimated to be 2.48 mg/l.13
Other
The intraperitoneal (i.p.) LD50 of MEA was 1.05 g/kg for mice.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Repeated Dose Toxicity
Dermal
Percutaneous application of 4 mg/kg/day MEA to rats resulted in nonspecific histological changes in the heart and lung,
fatty degeneration of the liver parenchyma, and subsequent focal liver necrosis. The duration of dosing was not
specified.
3
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Oral
Groups of 10 rats were fed 0-2.67 g/kg/day MEA for 90 days. Heavy livers and kidneys were observed at ≥0.64
g/kg/day, and deaths and “major pathology” occurred with ≥1.25 g/kg/day.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Inhalation
The dominant effects of “continuous exposure” of dogs, guinea pigs, and rats to 5-6 ppm MEA vapor were skin
irritation and lethargy. No dogs or rodents exposed to 12-26 ppm MEA vapor for 90 days died, but some dogs exposed
to 102 MEA vapor for 2 days and some rodents exposed to 66-75 ppm MEA vapor for 24-28 days did die. Dogs and
rodents exposed to 66-102 ppm MEA vapor had behavioral changes, pulmonary and hepatic inflammation, hepatic and
renal damage, and hematological changes.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
REPRODUCTIVE AND DEVELOPMENTAL STUDIES
Dermal
The developmental toxicity of dermally-applied aq. MEA was evaluated using groups of 30-45 gravid SpragueDawley rats and 15 gravid New Zealand White rabbits.14 MEA was applied at doses of 0, 10, 25, 75, and 225 mg/kg bw/day
to the backs of rats on days 6-15 of gestation, with a dose volume of 1 ml/kg. Significant skin irritation, consisting of erythema followed by necrosis, scabs, and scar formation, occurred with 225 mg/kg MEA, but not at the other dose levels. No
effects on kidney or liver weights were reported. Despite maternal effects in the 225 mg/kg dose group, no effects on reproductive parameters were observed at any dose, and there were no treatment-related increases in the visceral or skeletal malformations. The no-observed effect levels (NOELs) for maternal and embryonal/fetal toxicity in rats were 75 and 225
mg/kg/day, respectively.
In rabbits, 0, 10, 25, and 75 mg/kg bw/day MEA was applied to the back (2 ml/kg). Severe skin irritation at the
application site, consisting of necrosis, exfoliation, and crusting, was observed in the 75 mg/kg group, and crusting, transient
erythema, and edema were observed in a few rabbits of the 25 mg/kg dose group. No effects on kidney or liver weights were
reported. As with the rats, no effects on reproductive parameters were observed at any dose, and there were no treatmentrelated increases in the visceral or skeletal malformations. The NOEL for both maternal and embryonal/fetal toxicity in rabbits was 75 mg/kg/day.
Oral
No reproductive or developmental effects were observed in groups of gravid rats given a hair dye and base containing
22% MEA in the diet at concentrations ≤7800 ppm on days 6-15 of gestation. No differences in reproductive or
developmental parameters were observed when male rats were fed treated diet for 8 wks prior to and during mating
with undosed females or when female rats were fed treated feed for 8 wks prior to dosing until day 21 of lactation.
These females were mated with undosed males. Additionally, no teratologic effects were induced by dosing gravid
rabbits with ≤19.5 mg/kg/day of the hair dye and base by gavage on days 6-18 of gestation.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Ethanolamine.1
Groups of 10 gravid Long-Evans rats were dosed by gavage with 50-500 mg/kg aq. MEA on days 6-15 of gestation,
and a negative control group of 34 gravid rats were dosed with water only.15 The animals were killed and examined on day
20 of gestation. Significant maternal toxicity was not noted. Embryolethality was significantly increased in the 500 mg/kg
group, with male pups affected more than female pups. Male pups were more severely affected than female pups at all dose
levels in regards to intrauterine growth retardation and increased gross structural anomalies that were considered indicative of
depressed fetal growth. However, pups of either sex who were contiguous to male siblings were more adversely affected that
those contiguous to one or more female siblings. Male pups contiguous to male siblings (mMm) were, apparently, resorbed
4
(mRm). The number of malformed pups per dam was significantly increased in animals dosed with 300 mg/kg MEA. The
number of malformed pups, when evaluated as a percent of the litter affects, was significantly increased for all 3 dose groups.
The teratogenic potential of MEA was evaluated in a Chernoff-Kavlock postnatal mouse screening assay.16 Gravid
CD-1 mice were dosed orally with 850 mg/kg/day MEA on days 6-15 of gestation, resulting in 16% mortality of maternal
mice and reduced numbers of viable litters. Litter size, percentage survival of pups, birth weight, and pup weight gains were
not affected.
A preliminary study was performed in which gravid Wistar rats were dosed orally with 0-500 mg/kg by/day aq.
MEA; details not provided.17 Maternal effects were observed in the high dose animals, but no embryonal/fetal effects were
observed at any dose. Based on these results, groups of 40 Wistar rats were dosed by gavage, with 0, 40, 120, or 450 mg/kg
bw/day aq. MEA on days 6-15 of gestation. On day 20 of gestation, 25 dams/group were killed and necropsied, while the
remaining 15 were allowed to litter, and then killed on day 21 of lactation. Despite evidence of maternal toxicity in the 450
mg/kg group, as indicated by statistically significant decreases in maternal body weights and feed consumption, no effects on
reproductive parameters, incidences of visceral or skeletal malformations, or postnatal growth were observed at any dose
The NOELs for maternal and developmental toxicity were 120 and 450 mg/kg/day, respectively.
GENOTOXICITY
In Vitro
MEA, with and without metabolic activation using liver preparations from rats induced with a polychlorinated biphenyl
mixture, was not mutagenic to Salmonella typhimurium TA100 or TA1535.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
MEA was negative in most Ames tests;18 however “weak mutagenic effects” were reported in one study.19,20 MEA,
25-500 µg/ml, was negative in a cell transformation assay using hamster embryo cells.21 MEA was clearly cytotoxic at 500
µg/ml. MEA did not produce chromosomal aberrations in rat liver cells,18 but a “weak positive” response was reported in
human lymphocytes.19
IRRITATION AND SENSITIZATION
Irritation
Skin
Non-Human
MEA was corrosive to rabbit skin with single semi-occlusive patches of 30, 85 and 100% on intact and abraded skin.
When applied to rabbit ears using non-occlusive applications and to shaved skin of the abdomen under semi-occlusive
patches, ≥10% was corrosive, >1% was extremely irritating, and 1% was irritating..
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Human
In a study in which a formulation containing 5.9% MEA was applied to the backs of 12 female subjects for 23 h/day for
21 days, the formulation was considered an experimental cumulative irritant. Results observed during the induction
phase of a patch test of 165 subjects using a formulation containing 11.47% MEA were interpreted by the Expert Panel
as irritation.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Mucosal
Non-Human
Using 6 rabbits, 30% aq. MEA was moderately irritating to rabbit eyes. Undiluted MEA, instilled as a volume of 0.005
ml, produced severe injury to rabbit eyes. A hair preparation containing 5.96% MEA had a maximum avg. irritation
score of 0.7/110 for rinsed and unrinsed eyes.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
5
Sensitization
In Vitro
A local lymph node assay was performed to evaluate the sensitization potential of MEA (as the hydrochloride
salt).
8,22
Groups of 6 female CBA/Ca mice were treated on the ear with 10, 40, or 70% w/w aq. MEA HCl. MEA HCl did
not have a skin sensitizing effect.
Non-Human
In a maximization study using 15 Dunkin-Hartley guinea pigs, intradermal and epicutaneous inductions with 0.6%
and 10.3% aq. MEA, respectively, were performed after 10% sodium lauryl sulfate pre-treatment.8 (MEA contained less than
0.1% DEA and TEA.) At challenge with 0.41, 2.05, or 4.1% MEA, 3, 2, and 3 animals, respectively, reacted positively after
3 days. Two of the 15 test animals reacted to the vehicle, but none of the control animals reacted to MEA or the vehicle.
Possible cross-reactions to 5% TEA occurred in 3 animals, and to 7% DEA in 2 animals. In a second test using the same
protocol, no animals reacted to 0.41% MEA, 2 reacted to 2.05% MEA, and 1 reacted to 4.1% MEA. Additionally, 1 animal
reacted to 10% TEA and 2 reacted to 7% DEA. The control animals did not react to any of the ethanolamines.
Human
A formulation containing 5.9% MEA, tested undiluted, and one containing 11.47% MEA, tested at 5% in 25% alcohol,
were not sensitizing in clinical studies.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
Provocative Testing
A patch test using 2% MEA in petrolatum (pet.) was performed on 370 patients with suspected dermatitis to MEAcontaining metal-working fluids and on 452 control subjects.23 A 10-fold higher reaction was seen in the patient group, with
12.2% of the patients having positive reactions, as compared to 1.3% in the control group.
Over a 3-yr period, 11/595 (1.9%) hairdresser clients and 7/401 (1.8%) female hairdressers reacted positively to 2%
MEA pet.8 In 22 patients with suspected intolerance to oxidative hair dye components, 4 had a positive reaction to 2% MEA
pet. Over a 15-yr period, provocative patch testing using MEA was performed on 9602 subjects. There were 363 (3.8%)
positive reactions to MEA, and most of the reactions (277; 2.9%) were weak positives. There were 55 (0.6%) irritant
reactions reported. Cosensitization was reported; 38% of the patients that reacted to MEA also tested positive to DEA.
Occupational sensitization was reported; of 5884 male patients, 2.9% that did not work in the metal industry had positive
reactions as opposed to 7.0 and 15.2% of those working in the metal industry and those exposed to water-based metalworking
fluids, respectively.
CLINICAL USE
MEA inhalation by humans has been reported to cause immediate allergic responses of dyspnea and asthma and
clinical symptoms of acute liver damage and chronic hepatitis.
From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine.1
A case of occupational asthma in an industrial worker exposed to a detergent containing 8% ethanolamine was
reported.
24
Since the detergent was used in hot water, released\ of MEA vapor was greater than if the detergent temperature
was lower.. Exposure to vapors from MEA in cleaning solutions can irritate the nose, throat, and lungs.25 The Occupational
Safety and Health Administration Permissible Exposure Level for MEA is 3 ppm, and the American Conference of
Governmental Industrial Hygienists 15 min short term exposure limit is 6 ppm.
6
TABLES
Table 1. Definition and Structure
Ingredient
CAS No.
Ethanolamine
141-43-5
Definition
Synonyms
Function(s) in Cosmetics
The product of the ammonolysis of one stoichiometric equivalent of
ethylene oxide
2-aminoethanol
2-hydroxyethylamine
monoethanolamine
pH adjuster
HO
NH2
Table 2. Physical and Chemical Properties of MEA
Property
Physical Form
Color
Odor
Molecular Weight
Specific Gravity
Viscosity
Vapor Pressure
Melting Point
Boiling Point
Water Solubility
Other Solubility
Formula/Structure
Value
clear viscous liquid
colorless
ammoniacal
61.08
1.0117 (25°C)
18.95 (25°C)
0.48 mm Hg (20°C)
10.3-10.5 (20°C)
171°C
miscible with water
miscible with methanol and acetone
Reference
1
1
1
1
1
1
1
1
1
14
14
Table 3. Current and historical frequency and concentration of use of MEA according to duration and type of exposure
# of Uses
data year
Totals
Duration of Use
Conc. of Use (%)
19811
20103
19811
20104
51
795
≤10
0.0002-18
Leave-On
2
1
0.1-1
3 (coloring hair spray)
Rinse Off
Exposure Type
Eye Area
Possible Ingestion
Inhalation
49
794
≤10
0.0002-18
1
NR
NR
NR
1
NR
0.1-1
NR
NR
NR
NR
3
Dermal Contact
Deodorant (underarm)
Hair - Non-Coloring
Hair-Coloring
Nail
5
NR
17
28
NR
2
NR
11
782
NR
≤5
NR
0.1-10
0.1-10
NR
0.0002-11
NR
0.05-6
3-18
NR
Mucous Membrane
Bath Products
Baby Products
3
NR
NR
NR
NR
NR
≤5
NR
NR
0.02-0.05
NR
NR
NR - no reported use
7
REFERENCES
1.
Elder RE (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll
Toxicol. 1983;2:(7).
2. Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal
Care Products Council, 2010.
3. Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database. 2010. Washington, DC: FDA.
4.
Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey. 10-13-2010.
5.
Heatlh Canada.Cosmetic Ingredient Hotlist. 2010. http://www.hc-sc.gc.ca/cps-spc/person/cosmet/info-ind-prof/_hot-listcritique/hotlist-liste_1-eng.php. Accessed 10-29-2010.
6.
European Commission.European Commission Enterprise and Industry. Cosmetics - Cosing. Annex III/Part 1, 61 Monoalkylamines, nomoalkanolamines and their salts. 2010.
http://ec.europa.eu/consumers/cosmetics/cosing/index.cfm?fuseaction=search.details&id=33796. Accessed 10-5-2010.
7.
Food and Drug Administration.Everything Added to Food in the United States (EAFUS). 5-27-0010.
http://www.fda.gov/Food/FoodIngredientsPackaging/ucm115326.htm. Accessed 6-8-2010.
8.
Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine. Data
analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60:243-255.
9. Klain GC, Reifenrath WG, and Black KE. Distribution and metabolism of topically applied ethanolamine. Fund Appl Toxicol.
1985;5:S127-S133.
10. Sun JD, Beskitt JL, Tallant MJ, and Frantz SW. In vitro skin penetration of monoethanolamine and diethanolamine using excised
skin from rats, mice, rabbits, and humans. J Toxicol -- Cut & Ocular Toxicol. 1996;15:(2):131-146.
11.
Anonymous. Letter to the Environmental Protection Agency reporting an actue dermal toxcity study of a formulation containing
DEA. 6-7-2010.
12.
Anonymous. Letter to the Environmental Protection Agency reporting actue dermal and oral toxcity studies of a formulation
containing DEA. 6-9-2010.
13.
Anonymous. Letter to the Environmental Protection Agency reporting an actue inhalaton toxcity studies of a formulation
containing DEA. 6-8-2010.
14.
Liberacki AB, Neeper-Bradley TL, Breslin WJ, and Zielke GJ. Evaluation of developmental toxicity of dermally applied
monoethanolamine in rats and rabbits. Fund Appl Toxicol. 1996;31:117-123.
15. Mankes RF. Studies on the embryopathic effects of ethanolamine in Long-Evans rats: Preferential embryopathy in pups contiguous
with male siblings in utero. Teratog.Carcinog Mutagen. 1986;6:403-417.
16.
Pereira M, Barnwell P, and Bailes W. Screening of priority chemicals for reproductive hazards, monoethanolamine,
diethanolamine, , triethanolamine. NIOSH Contract No. 200-84-2735. Environmental Health Research and Testing, Inc.
REport No. ETOX-85-1002. 1987.
17.
Hellwig J and Liberacki AB. Short communication. Evaluation of the pre-, peri-, and postnatal toxicity of monothanolamine in rats
following repeated oral adminstration during organogenesis. Fund Appl Toxicol. 1997;40:158-162.
18. Dean BJ, Brooks TM, Hodson-Walker G, and Hutson DH. Genetic toxicology testing of 41 industrial chemicals. Mutat Res.
1985;153:57-77.
19.
Arutyunyan RM, Zalinyan RM, Mugnetsyan EG, and Gukasyan LA. Mutagenic action of latex polumerization stabilizers in
different test systems. Tsitol Genet. 1987. 21:450-456.
20.
Mortelmans K, Haworth S, Lawlor T, Speck W, Tainer B, and Zeiger E. Salmonella mutagenicity tests: II. Results from the testing
of 270 chemicals. Environ Mutagen. 1986;8:(Suppl 7):1-119.
8
21.
Inoue K, Sunakawa T, Okamoto K, and Tanaka Y. Mutagenicity tests and in vitro transformation assays on triethanolamine. Mutat
Res. 1982;101:305-313.
22.
BASF. Ethanolamine hydrochloride Murine Local Lymph Node Assay (LLNA). 2007.
23.
Geier J, Lessmann H, Schnuch A, and Uter W. Diagnostic quality of patch test preparation monoethanolamine 2% pet. Contact
Derm. 2005;52:171-173.
24.
Savonius B, Keskinen H, Tupperainen M, and Kanerva L. Occupational asthma caused by ethanolamines. Alergy. 1994;49:877-881.
25.
Bello A, Quinn MM, Perry MJ, and Milton DK. Characterization of occupational exposures to cleaning products used for common
cleaning tasks - A pilot study of hospital cleaners. Envron Health. 2009;8:11.
9
Personal Care
Products Council
Committed to Safety,
Quality & Innovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDffiNT REVWW (CIR)
-
FROM:
John Bailey, Ph.D.
Industry Liaison to the CW Expert Panel
DATE:
October 13, 2010
SUBJECT:
Concentration of Use Ethanolamine, Diethanolamine and Triethanolamine
11011 7th Street, N.W., Suite 300
-
Washington, D.C. 20036-4702
202.331.1770
202.331.1969 (fax)
www.personalcarecouncil.org
Concentration of Use by FDA Product Category
Ethanolamine, Diethanolamine and Triethanolamine
Ingredient
Product Category
Concentration
of Use
Ethanolamine
Hair conditioners
4%
Ethanolamine
Hair straighteners
3%
Ethanolamine
Permanent waves
4-6%
Ethanolamine
Shampoos (noncolonng)
0.05%
Ethanolamine
Hair dyes and colors (all types requiring caution
statement and patch testing)
3-13%
Ethanolamine
Hair color sprays (aerosol)
3%
Ethanolamine
Hair bleaches
4-18%
Ethanolamine
Bath soaps and detergents
0.02-0.05%
Ethanolamine
Shaving cream (aerosol, brushless and lather)
0.0002%
Ethanolamine
Skin cleansing (cold creams, cleansing lotions, liquids
and_pads)
0.5-11%
Diethanolamine
Shampoos (noncoloring)
0.03-0.3%
Diethanolamine
Tonics, dressings and other hair grooming aids
0.008%
Diethanolamine
Bath soaps and detergents
0.009%
Diethanolamine
Shaving cream (aerosol, brushless and lather)
0.06%
Diethanolamine
Moisturizing creams, lotions and powders
0.06%
Triethanolamine
Baby lotions, oils, powders and creams
0.2-2%
Tnethanolamine
Bubble baths
0.4-0.7%
Triethanolamine
Other bath preparations
1-2%
Triethanolamine
Eyebrow pencil
1-3%
Triethanolamine
Eyeliner
0.3-3%
Tnethanolamine
Eye shadow
0.4-3%
Triethanolamine
Eye lotion
0.2-3%
Page 1 of 3
Triethanolamine
Eye makeup remover
0.4-0.6%
Triethanolamine
Mascara
2-4%
Triethanolamine
Other eye makeup preparations
0.8-3%
Triethanolamine
Colognes and toilet waters
0.001-1%
Tnethanolamine
Perfumes
0.05-0.4%
Triethanolamine
Hair conditioners
0.0003-0.3%
Triethanolamine
Hair sprays (aerosol fixatives)
0.05-2%
Triethanolamine
Hair straighteners
3%
Triethanolamine
Permanent waves
0.01%
Triethanolamine
Shampoos (noncoloring)
0.0003-6%
Triethanolamine
Tonics, dressings and other hair grooming aids
0.0003-3%
Triethanolamine
Other hair preparations (noncoloring)
0.5-3%
Triethanolamine
Hair dyes and colors (all types requiring caution
statement and patch testing)
2-13%
Triethanolamine
Hair tints
1%
Triethanolamine
Hair rinses (coloring)
5%
Triethanolamine
Other hair coloring preparations
3%
Triethanolamine
Blushers (all types)
0.0002-3%
Triethanolamine
Face powders
0.06%
Triethanolamine
Foundations
1-2%
Triethanolamine
Leg and body paints
6%
Tnethanolamine
Lipstick
0.2-1%
Triethanolamine
Makeup bases
1-3%
Triethanolamine
Other makeup preparations
0.7-2%
Triethanolamine
Basecoats and undercoats (manicuring preparations)
3%
Tnethanolamine
Cuticle softeners
0.5-2%
Tnethanolamine
Nail creams and lotions
0.2-1%
Page 2 of 3
Triethanolamine
Other manicuring preparations
0.5-2%
Triethanolamine
Bath soaps and detergents
0.5-19%
Triethanolamine
Deodorants (underarm)
0.1-0.4%
Triethanolamine
Feminine hygiene deodorants
0.1%
Triethanolamine
Other personal cleanliness products
0.2-3%
Triethanolamine
Aftershave lotions
0.5-2%
Triethanolamine
Beard softeners
0.7%
Triethanolamine
Preshave lotions (all types)
0.3%
Triethanolamine
Shaving cream (aerosol, brushless and lather)
3-10%
Triethanolamine
Other shaving preparations
0.3-6%
Triethanolamine
Skin cleansing (cold creams, cleansing lotions, liquids
and pads)
0.1-11%
Triethanolamine
Depilatories
0.8%
Triethanolamine
Face and neck creams, lotions and powders
0.4-6%
Triethanolamine
Face and neck sprays
0.07-1%
Triethanolamine
Body and hand creams, lotions and powders
0.3-3%
Triethanolamine
Body and hand sprays
0.2-0.4%
Tnethanolamine
Foot powders and sprays
0.4-1%
Triethanolamine
Moisturizing creams, lotions and powders
0.2-2%
Triethanolamine
Night creams, lotions and powders
0.3-5%
Triethanolamine
Paste masks (mud packs)
1-3%
Triethanolamine
Skin fresheners
2%
Triethanolamine
Other skin care preparations’
0.4-2%, 7%
Triethanolamine
Suntan gels, creams and liquids
2%
Triethanolamine
Indoor tanning preparations
‘7% in a rinse-off skin care preparation
0.3-2%
Information collected in 2010
Table prepared October 13, 2010
Page 3 of 3
Personal Care
Products Council
Committed to Safety,
Quality & Innovation
Memorandum
TO:
F. Alan Andersen, Ph.D.
Director COSMETIC INGREDIENT REVIEW (Cifi)
-
FROM:
John Bailey, Ph.D.
Industry Liaison to the CJR Expert Panel
DATE:
November 8, 2010
SUBJECT:
Updated Concentration of Use Triethanolamine
1101 17th Street, N.W., Suite 30O Washington, D.C. 20036-4702
202.331.1770
202.331.1969 (fax)
www.personalcarecouncil.org
Concentration of Use by FDA Product Category
Ethanolamine, Diethanolamine and Triethanolamine
Ingredient
Product Category
Concentration of
Use
Ethanolamine
Hair conditioners
4%
Ethanolamine
Hair straighteners
3%
Ethanolamine
Permanent waves
4-6%
Ethanolamine
Shampoos (noncoloring)
0.05%
Ethanolamine
Hair dyes and colors (all types requiring caution statement and
patch testing)
3-13%
Ethanolamine
Hair color sprays (aerosol)
3%
Ethanolamine
Hair bleaches
4-18%
Ethanolamine
Bath soaps and detergents
0.02-0.05%
Ethanolamine
Shaving cream (aerosol, brushless and lather)
0.0002%
Ethanolamine
Skin cleansine (cold creams, cleansine lotions, liquids and cads)
0.5-11%
Diethanolamine
Shampoos (noncoloring)
0.03-0.3%
Diethanolamine
Tonics, dressings and other hair grooming aids
0.008%
Diethanolamine
Bath soaps and detergents
0.009%
Diethanolamine
Shaving cream (aerosol, brushless and lather)
0.06%
Diethanolamine
Moisturizine creams, lotions and nowders
0.06%
Triethanolamine
Baby lotions, oils, powders and creams
0.2-2%
Triethanolamine
Bubble baths
0.4-0.7%
Triethanolamine
Other bath preparations
1-2%
Triethanolamine
Eyebrow pencil
1-3%
Triethanolamine
Eyeliner
0.3-3%
Triethanolamine
Eye shadow
0.4-3%
Triethanolamine
Eye lotion
0.2-3%
Triethanolamine
Eye makeup remover
0.4-0.6%
Triethanolamine
Mascara
0.7-4%
Triethanolamine
Other eye makeup preparations
0.8-3%
Triethanolamine
Colognes and toilet waters
0.00 1-1%
Page 1 of 3
Triethanolamine
Perfumes
0.05-0.4%
Triethanolamine
Hair conditioners
0.0003-0.3%
Triethanolamine
Hair sprays (aerosol fixatives)
0.05-2%
Triethanolamine
Hair straighteners
3%
Triethanolamine
Permanent waves
0.0 1%
Triethanolamine
Shampoos (noncoloring)
0.0003-6%
Triethanolamine
Tonics, dressings and other hair grooming aids
0.0003-3%
Triethanolamine
Other hair preparations (noncoloring)
0.5-3%
Triethanolamine
Hair dyes and colors (all types requiring caution statement and
patch testing)
2-13%
Triethanolamine
Hair tints
1%
Triethanolamine
Hair rinses (coloring)
5%
Triethanolamine
Other hair coloring preparations
3%
Triethanolamine
Blushers (all types)
0.0002-3%
Triethanolamine
Face powders
0.06%
Triethanolamine
Foundations
1-2%
Triethanolamine
Leg and body paints
6%
Triethanolamine
Lipstick
0.2-1%
Triethanolamine
Makeup bases
1-3%
Triethanolamine
Other makeup preparations
0.7-2%
Triethanolamine
Basecoats and undercoats (manicuring preparations)
3%
Triethanolamine
Cuticle softeners
0.5-2%
Triethanolamine
Nail creams and lotions
0.2-1%
Triethanolamine
Other manicuring preparations
0.5-2%
Triethanolamine
Bath soaps and detergents
0.5-19%
Triethanolamine
Deodorants (underarm)
0.1-0.4%
Triethanolamine
Feminine hygiene deodorants
0.1%
Triethanolamine
Other personal cleanliness products
0.2-3%
Triethanolamine
Aftershave lotions
0.5-2%
Triethanolamine
Beard softeners
0.7%
.
Page 2 of 3
Triethanolamine
Preshave lotions (all types)
0.3%
Triethanolamine
Shaving cream (aerosol, brushless and lather)
3-10%
Triethanolamine
Other shaving preparations
0.3-6%
Triethanolamine
Skin cleansing (cold creams, cleansing lotions, liquids and pads)
0.1-11%
Triethanolamine
Depilatories
0.8%
Triethanolamine
Face and neck creams, lotions and powders
0.4-6%
Triethanolamine
Face and neck sprays
0.07-1%
Triethanolamine
Body and hand creams, lotions and powders
0.3-3%
Triethanolamine
Body and hand sprays
0.2-0.4%
Triethanolamine
Foot powders and sprays
0.4-1%
Triethanolamine
Moisturizing creams, lotions and powders
0.2-3%
Triethanolamine
Night creams, lotions and powders
0.3-5%
Triethanolamine
Paste masks (mud packs)
1-3%
Triethanolamine
Skin fresheners
Triethanolamine
Other skin care preparations’
0.4-2%, 7%
Triethanolamine
Suntan gels, creams and liquids
2%
Triethanolamine
Indoor tanning preparations
0.3-2%
17%
-
2%
in a rinse-off skin care preparation
Information collected in 2010
Table prepared October 13, 2010
Updated November 8, 2010 (Triethanolamine: lowered minimum for Mascara to 0.7; increased maximum
Moisturizing to 3%)
Page 3 of 3
TITLE
PAGE
PAGETLE
PAGE
STUDY TITLE
Report
Ethanolamine hydrochloride
Murine Local Lymph Node Assay (LLNA)
DATA REQUIREMENT
OECD 429
2004/73/EC, B.42.
US EPA OPPTS 870.2600
AUTHOR(S)
Dr. Armin O. Gamer (Study Director)
Dr. Robert Landsiedel
STUDY COMPLETED ON
11 Jan 2007
PERFORMING LABORATORY
Experimental Toxicology and Ecology
BASF Aktiengesellschaft
67056 Ludwigshafen, Germany
LABORATORY PROJECT IDENTIFICATION
58H0372/052301
SPONSOR
Alkanolamines CHEMSTAR Panel
American Chemistry Council
Arlington, VA 22209
This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be used
only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction,
publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory agencies
acting within the limits of their administrative authority.
This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors
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Report; Project No.: 58H0372/052301
BLANK PAGE
THIS PAGE IS INTENTIONALLY LEFT BLANK
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Report; Project No.: 58H0372/052301
The following statement is for US EPA submissions only:
FLAGGING CRITERIA
The flagging criteria of 40 CFR 158.34 are not applicable for this study type
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CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL
Head of Experimental Toxicology and
Ecology:
Dr. rer. nat. B. v. Ravenzwaay
Management:
Dr. rer. nat. R. Landsiedel
Study director:
Dr. med. vet. A. O. Gamer
Statistics:
Dipl. Statistician M. Dammann
Analytical Chemistry:
Dr. rer. nat. E. Fabian
Quality Assurance Unit (QAU):
J. Hajok
Acute Toxicology:
M. Remmele
This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors
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GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY)
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Report; Project No.: 58H0372/052301
CONTENTS
TITLE PAGE
1
BLANK PAGE
2
GLP COMPLIANCE STATEMENT
3
FLAGGING CRITERIA
4
SIGNATURE PAGE
5
CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL
6
STATEMENT OF THE QUALITY ASSURANCE UNIT
7
GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY)
8
CONTENTS
9
LIST OF ABBREVIATIONS
11
1.
SUMMARY
13
2.
INTRODUCTION
14
2.1.
OBJECTIVES
14
2.2.
SELECTION OF DOSES/CONCENTRATIONS
14
2.3.
TEST GUIDELINES
15
2.4.
TIME SCHEDULE
16
2.5.
RETENTION OF RECORDS
16
3.
MATERIALS AND METHODS
17
3.1.
TEST SUBSTANCE
17
3.2.
TEST ANIMALS
17
3.3.
HOUSING CONDITIONS
18
3.4.
TEST GROUPS AND DOSES/CONCENTRATIONS
18
3.5.
TEST-SUBSTANCE PREPARATION
19
3.6.
ANALYSES
19
3.7.
EXPERIMENTAL PROCEDURE
20
3.7.1.
Conduct of the study
20
3.7.2.
³H-thymidine injection
21
3.7.3.
Terminal procedures
21
3.7.4.
Data evaluation
22
3.7.5.
Calculations and statistical analyses
22
3.7.6.
Historical control data
22
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3.7.7.
Positive control
22
3.8.
EVALUATION OF RESULTS
23
4.
RESULTS
24
4.1.
ANALYSES
24
4.2.
CLINICAL OBSERVATIONS AND ASSESSMENT OF FINDINGS
25
4.2.1.
Cell counts, 3H-thymidine incorporation and lymph node weights
25
4.2.2.
Ear weights
26
4.2.3.
Body weights
27
4.2.4.
Other findings
27
5.
DISCUSSION AND CONCLUSION
27
APPENDIX
28
3
CELL COUNT, H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT:
TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION
INDICES
29
EAR WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND
STIMULATION INDICES
29
CELL COUNT, 3H-THYMIDINE INCORPORATION, LYMPH NODE WEIGHT AND
EAR WEIGHT: INDIVIDUAL VALUES
30
BODY WEIGHT DATA
31
HISTORICAL CONTROL DATA
32
POSITIVE CONTROL
34
α-Hexylcinnamaldehyde, techn. 85%, vehicle: acetone
34
α-Hexylcinnamaldehyde, techn. 85%, vehicle 1% aqueous Pluronic® L92
36
FLOW CHART FOR EVALUATION OF THE LLNA
37
ANALYSES
38
Analytical characterization of the test substance (Certificate of Analysis)
39
Concentration Control Analysis of Ethanolamine hydrochloride in 1 % Pluronic® L 92
in doubly distilled water
40
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LIST OF ABBREVIATIONS
a.m.
approx.
= ante meridiem (before noon)
= approximately
ca.
cm
= circa
= centimeter
d
DPM
= day
= disintegrations per minute
EC
e.g.
= estimated concentration that leads to the respective stimulation index
= for example
g
= gram
h
= hour
L
LLNA
LSC
= liter
= Local Lymph Node Assay
= liquid scintillation counter
µCi
µL
µm
mg
mL
mm
=
=
=
=
=
=
No./Nos.
= number/s
p
PBS
p.m.
= significance level
= phosphate buffered saline
= post meridiem (after noon)
S.D.
SPF
SI
= standard deviation
= specific pathogen free
= stimulation index
TCA
= trichloro-acetic acid
v
v/v
= volume
= volume per volume
w
w/w
= weight
= weight per weight
micro Curie
microliter
micrometer
milligram
milliliter
millimeter
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Symbols:
°C
~
=
%
-
=
=
=
=
=
degree celsius
circa
equivalent
per cent
not observed, not determined
This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors
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Report; Project No.: 58H0372/052301
1. SUMMARY
The skin sensitizing potential of Ethanolamine hydrochloride was assessed using the
radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for
skin sensitization in mice. It determines the response of the auricular lymph nodes on
repeated application of the test substance to the dorsal skin of the ears.
Groups of 6 female CBA/Ca mice each were treated with 10%, 30% and 70% w/w
preparations of the test substance in 1% aqueous Pluronic® (1% Pluronic® L 92 Surfactant
in doubly distilled water) or with the vehicle alone. The study used 3 test groups and 1 control
group. Each test animal was applied with 2 x 12.5 µL per ear of the respective test-substance
preparation to the dorsum of both ears for three consecutive days. The control group was
treated with 2 x 12.5 µL per ear of the vehicle alone. The total volume of 25 µL per ear and
application was split into two portions in order to prevent run off of the test-substance
preparations due to high volume. The second daily application was performed immediately
after the first daily application was finished in all animals of the study collective.
Three days after the last application the mice were injected intravenously with 20 µCi of 3Hthymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine
injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node
response was evaluated by measuring the cellular content and 3H-thymidine incorporation
(indicator of cell proliferation) as well as the weight of each animal’s pooled lymph nodes.
Moreover a defined area with a diameter of 0.8 cm was punched out of the apical part of
each ear and for each animal the weight of the pooled punches was determined in order to
obtain an indication of possible skin irritation.
The mean stimulation indices (fold of change as compared to the vehicle control) for cell
count, 3H-thymidine incorporation, lymph node weight and ear weight are summarized for
each test group in the table below.
Test
group
Treatment
1
2
3
4
1% aqueous Pluronic®
10% in 1% aqueous Pluronic®
30% in 1% aqueous Pluronic®
70% in 1% aqueous Pluronic®
Cell Count
Stimulation Index
1.00
1.13
1.04
1.26
#
³H-thymidine
Lymph Node
Ear Weight
incorporation
Weight
Stimulation Index
Stimulation Index Stimulation Index
1.00
1.00
1.00
1.34
1.10
1.07
#
1.47
#
1.06
1.04
2.03
##
1.11
1.05
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )
No signs of systemic toxicity were noticed.
The statistically significant increases in cellularity, caused by the 70% concentration and in
H-thymidine incorporation into the lymph node cells, caused by the 70% and 30%
preparations, failed to reach the cut off stimulation index of 1.5 or 3, respectively, and thus lie
below the threshold of immunologic relevance. Lymph node weights were not statistically
significantly increased.
3
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Though not concentration related and statistically significant in mice treated with the 10% test
substance preparation, only, the slight increases in ear weight stimulation indices indicate
minimal ear skin irritation at all concentrations.
Thus it is concluded that Ethanolamine hydrochloride does not show a skin sensitizing
effect in the Murine Local Lymph Node Assay under the test conditions chosen.
2. INTRODUCTION
2.1. OBJECTIVES
The Murine Local Lymph Node Assay (LLNA) is recommended by international test
guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans.
The study was performed based on the method of Kimber I. et al. (1994)1. The study
procedure was extended according to the publications described in section “Test Guidelines”,
which evaluate the skin sensitizing potential of test substances by determination of lymph
node response using lymph node weight and lymph node cell count as parameters which are
set into perspective to ear weight or thickness as an indicator of skin irritation.
2.2. SELECTION OF DOSES/CONCENTRATIONS
The selection of concentrations took into account available information on the
chemical/physical properties and the composition of the test substance. In addition the
results of a pretest were considered, which showed slightly increased ear weights after
application of a 70% test substance preparation in 1% aqueous Pluronic® as indication of
some ear irritation. No increase in lymph node weights was observed (details are archived
with the raw data).
The following dose levels were selected:
10%, 30% and 70% in 1% aqueous Pluronic®
The control and test group animals were treated according to the following treatment
scheme:
Induction
Control group 1
Test group 2
Test group 3
Test group 4
________
1
Vehicle: 1% aqueous Pluronic®
Test substance 10% in 1% aqueous Pluronic®
Test substance 30% in 1% aqueous Pluronic®
Test substance 70% in 1% aqueous Pluronic®
Kimber I., Dearman R.J., Scholes E.W., Basketter D.A. (1994). The Local Lymph Node Assay:
Developments and Applications. Toxicology, 93, 13-31.
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2.3. TEST GUIDELINES
The study was conducted according to the following test guidelines and publications:
-
-
-
-
OECD Guideline for Testing of Chemicals No. 429, April 24, 2002 (“Skin Sensitization:
Local Lymph Node Assay”)
EC directive 2004/73, No. L 216, B.42., June 16, 2004 (“Skin Sensitization – Local Lymph
Node Assay”)
U. S. EPA Health Effects Test Guidelines OPPTS 870.2600, March 2003 (“Skin
Sensitization“)
Vohr H.-W., Blümel J., Blotz A., Homey B. and Ahr H.J., An intralaboratory validation of
the integrated Model for the differentiation of skin reaction (IMDS): discrimination between
(Photo)allergic and (photo)irritant skin reactions in mice. Arch. Toxicol. 73: 501-509, 2000.
Ulrich, P., Streich, J. and Suter, W., Intralaboratory validation of alternative endpoints in
the murine local lymph node assay for the identification of contact allergic potential:
primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical
chemicals. Arch. Toxicol. 74: 733-744, 2001.
Ehling G., Hecht M., Heusener A., Huesler J., Gamer A.O., van Loveren H., Maurer Th.,
Riecke K., Ullmann L., Ulrich P., Vandebriel R. and Vohr H.-W., An European interlaboratory validation of alternative endpoints of the murine local lymph node assay: First
round, Toxicology 212, 60-68, 2005.
Ehling G. Hecht M., Heusener A., Huesler J., Gamer A.O., van Loveren H., Maurer Th.,
Riecke K., Ullmann L., Ulrich P., Vandebriel R. and Vohr H.-W., An European interlaboratory validation of alternative endpoints of the murine local lymph node assay 2nd
round, Toxicology 212, 69-79, 2005.
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2.4. TIME SCHEDULE
Test dates:
Study initiation date
(date of signature of the study protocol)
05 Jul 2006
Arrival of the animals and start of acclimatization period
(at least 5 days before application)
20 Jun 2006
Experimental starting date
(selection of animals (randomization) for the first
treatment within a study)
11 Jul 2006
1st induction
12 Jul 2006
nd
2
induction
13 Jul 2006
rd
3 induction
14 Jul 2006
3
H-thymidine injection. Sacrifice of the animals.
Determination ear weight, lymph node weight and cell
count. Cell washing and precipitation.
17 Jul 2006
Measurement of 3H-thymidine incorporation
18 Jul 2006
Experimental completion date
(documentation of check of raw data)
09 Nov 2006
2.5. RETENTION OF RECORDS
GLP – relevant records and materials are stored at BASF Aktiengesellschaft
for at least the period of time specified in the GLP principles. Details concerning
responsibilities or locations of archiving can be seen from the respective SOPs and from the
raw data.
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3. MATERIALS AND METHODS
3.1. TEST SUBSTANCE
The analyses of the test substance were carried out at SIGMA-ALDRICH.
Name of test substance:
Ethanolamine hydrochloride
Test-substance No.:
05/0372-1
Batch identification:
092K5318
CAS No.:
2002-24-6
Purity:
100% (for details see Certificate of Analysis in the Appendix)
Homogeneity:
The test substance was homogeneous by visual inspection.
Storage stability:
The stability under storage conditions over the study period
was guaranteed (for details see Certificate of Analysis).
3.2. TEST ANIMALS
Test species / strain /
quality:
Mouse / CBA/CaOlaHsd
The female animals were nulliparous and non-pregnant.
Reasons for the selection of The test method was developed in mice.
the test species:
Age at day 0:
7 – 10 weeks
The somewhat younger age of the animals as compared to the
range given in the test guideline does not influence the test
according to our experience.
Sex:
Female
Supplier:
Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen,
Germany
Arrival in the testing facility:
Acclimatization period (22 days before the first test-substance
application)
Identification:
The single housed animals were identified by cage cards
Body weight at day 0:
18.3 g – 24.2 g
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3.3. HOUSING CONDITIONS
Room temperature /
relative humidity:
The animals were housed in fully air-conditioned rooms.
Central air-conditioning guaranteed a range of 20 – 24°C for
temperature and of 30 – 70% for relative humidity. There were
no deviations from these ranges, which influenced the results
of the study.
Day / night rhythm:
12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage:
Makrolon cage, type I
No. of animals per cage:
1
Feeding:
Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba
SA, Kaiseraugst, Basel, Switzerland, ad libitum
Drinking water:
Tap water ad libitum
Bedding:
Granulat Typ ¾ (staubfrei); SSNIFF
3.4. TEST GROUPS AND DOSES/CONCENTRATIONS
Groups
Control group 1
Test group 2
Test group 3
Test group 4
Number of animals
6
6
6
6
Animal No.
1–6
7 – 12
13 – 18
19 – 24
Control group 1 was used to rule out any influence of the vehicle, used for production of the
test-substance preparations, on the parameters measured in the test.
The concentrations used for induction are given in section 2.2 Selection of
doses/concentrations.
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3.5. TEST-SUBSTANCE PREPARATION
Test-substance preparation: The test-substance preparation was produced on a weight per
weight basis shortly before each application. After stirring with
a magnetic stirrer the test substance is soluble in the vehicle.
Vehicle:
1% Pluronic® L 92 Surfactant in doubly distilled water
Reason for the vehicle:
The test substance is highly soluble in the aqueous vehicle.
By means of the hydrophobic surface-active agent, Pluronic®
L 92, run-off of the test-substance preparations was
prevented. Pluronic® L 92 was recommended by Ryan C.A. et
al. as suitable to produce aqueous preparations for use in the
Murine Local Lymph Node Assay (LLNA).2
Form of application:
Solution
3.6. ANALYSES
Conduct of analyses:
The analyses were carried out at the Analytical Chemistry
Laboratory of the Experimental Toxicology and Ecology of
BASF Aktiengesellschaft.
Stability of the testsubstance preparation:
The stability of the test substance in the vehicle was
determined indirectly by the concentration analysis. For this
purpose, the samples taken were stored at room temperature
over the maximum duration of the administration period and
were subsequently deep-frozen. Afterwards, these samples
were analyzed.
Homogeneity of the testsubstance preparation:
The homogeneity of the test-substance preparation in the
vehicle was not determined by analysis.
Concentration analysis of
the test-substance
preparation:
The verification of the concentration of the test-substance
preparations (10%, 30%, 70%) was investigated once by
analysis during the study.
________
2
Ryan C.A. et al., Examination of a vehicle for use with water soluble materials in the murine local lymph node
assay, Food Chem. Toxicol 40, 1719-1725, 2002
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Analysis of feed:
The feed used in the study was assayed for chemical and
microbiological contaminants.
Analysis of drinking water:
The drinking water is regularly assayed for chemical
contaminants by the municipal authorities of Frankenthal and
the Technical Services of BASF Aktiengesellschaft as well as
for the presence of microbes by a contract laboratory.
Analysis of bedding:
The bedding is regularly assayed for contaminants
(chlorinated hydrocarbons, heavy metals).
Records on analyses of feed, drinking water and bedding are stored at Experimental
Toxicology and Ecology of BASF Aktiengesellschaft for at least the period of time specified
in the GLP principles.
3.7. EXPERIMENTAL PROCEDURE
3.7.1. Conduct of the study
The study comprised three treatment groups and a vehicle control group. Each group
consisted of 6 mice.
Randomization:
Prior to first application, the animals were distributed to the
individual groups, received their animal numbers and were
allocated to the respective cages according to the
randomization instructions of ”Nijenhuis, A. and Wilf, H.S.:
Combinatorial Algorithms, Academic Press, New York,
San Francisco, London, 1978, pp. 62 – 64“.
Body weight determination:
Individual body weights on day 0 prior to the first application
and on day 5 prior to the sacrifice of the animals.
Signs and symptoms:
No detailed clinical examination of the individual animals was
performed but any obvious signs of systemic toxicity and/or
local inflammation at the application sites were noted in the
raw data.
Form of application:
Epicutaneous application is simulating dermal contact with the
compound which is possible to occur under practical use
conditions.
Application volume:
2 x 12.5 μL per ear
The total volume of 25 µL per ear and application was split into
two portions in order to prevent run off of the test-substance
preparations due to high volume. The second daily application
was performed immediately after the first daily application was
finished in all animals of the study.
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Site of application:
Dorsal part of both ears
Frequency of application:
3 consecutive applications (day 0 – day 2) to the same
application site
Mortality:
Twice on Mondays through Fridays (beginning and end) and
once on Saturdays, Sundays and on public holidays.
The animals of test groups 1-4 were treated with vehicle or test-substance preparation as
given in section 2.2.
3.7.2. ³H-thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the
ears) the mice were injected intravenously with 20 µCi of 3H-thymidine in 250 µl of sterile
saline into a tail vein.
3.7.3. Terminal procedures
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by
cervical dislocation.
Determination of the ear
weight:
Immediately after the death of each animal a circular piece of
tissue (diameter 0.8 cm) was punched out of the apical part of
each ear of all animals. The weight of the pooled punches was
determined for each animal.
Removal and weight
determination of the lymph
nodes:
Immediately after removal of the ear punches the left and right
auricular lymph nodes were dissected. The weight of the
pooled lymph nodes from both sides was determined for each
animal.
Preparation of cell
suspension and
determination of cell count:
After weight determination, the pooled lymph nodes of each
animal were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension
was prepared as soon as possible after dissection by carefully
passing the lymph nodes through an iron mesh (mesh size
200 µm) into 6 mL of phosphate-buffered physiological saline.
For determination of cell counts, an aliquot of each suspension
was further diluted with Casy®ton in a ratio 1:500. The cell
count was determined using a Casy®-Counter.
Measurement of 3Hthymidine incorporation of
the lymph node cells:
The remaining cell suspensions were washed twice with
phosphate buffered saline (PBS) and precipitated with 5%
trichloro-acetic acid (TCA). Each precipitate was transferred to
scintillation fluid and incorporation of 3H-thymidine into the
cells was measured in a ß-scintillation counter.
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3.7.4. Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced
using PC based tabular calculation software. The mean and individual data were not always
rounded but the significant digits were produced by changing the display format. As a
consequence, calculation of mean values using the individual data presented in the report
will, in some instances, yield minor variations in value.
3.7.5. Calculations and statistical analyses
Mean values and standard deviations of the measured parameters were calculated for the
test and control groups from the individual values. The stimulation indices of cell count,
3
H-thymidine incorporation, lymph node weight and ear weight measurements were
calculated as the ratio of the test group mean values for these parameters divided by those of
the vehicle control group.
Further statistical analyses were performed according to the table below.
Parameter
Cell count, 3H-thymidine incorporation, lymph
node weight as well as ear weight
Statistical test
WILCOXON - Test3
3.7.6. Historical control data
Vehicle related historical control data, gathered over an appropriate time period, are
presented in the Appendix. These data may be used as described in section 3.8 for the
evaluation of the results in addition to the concurrent control values. The comparison of the
present data to the historical controls was not necessary for evaluation of this study.
3.7.7. Positive control
A concurrent positive control (reliability check) with a known sensitizer was not included into
this study.
Studies using the positive control substance α-Hexylcinnamaldehyde, techn. 85% are
performed twice a year in the laboratory in order to show that the test system is able to detect
sensitizing compounds under the test conditions chosen.
The results of the most recent studies are included in the Appendix.
________
3
Siegel, S. (1956): Non-parametric statistics for behavioural sciences. McGraw-Hill New York
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3.8. EVALUATION OF RESULTS
In order to reveal a possible induction of sensitization, the response in the draining lymph
node after epicutaneous application of several concentrations of the test substance to the
skin of the ear backs is determined. The parameters used to characterize the response are
lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain
extent lymph node weight. Because not only sensitization induction but also irritation of the
ear skin by the test substance may induce lymph node responses, the weight of ear punches
taken from the area of test-substance application is determined as a parameter for
inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
The increase stimulation index (SI) of cell count by a factor of >1.5 and/or of 3H-thymidine
incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is
generally considered as indicating a sensitizing potential of a test substance.
If applicable, the EC (estimated concentration) leading to the respective SI values were
calculated by linear regression between the data points directly below and above the SI if
possible or using the two nearest points below or above the SI.
In addition the evaluation uses the following considerations:
If a test substance does not show a statistically significant and/or biologically relevant
increase in cell count, 3H-thymidine incorporation and/or lymph node weight as compared to
the vehicle control in the presence of statistically significant and/or biologically relevant
increased ear weights as indication of skin irritation, it is considered not to be a sensitizer.
If at least one concentration tested causes a concentration dependent statistically significant
and/or biologically relevant increase in cell count, 3H-thymidine incorporation and/or lymph
node weight without being accompanied by a statistically significant and/or biologically
relevant increase in ear weight, the test substance is considered to be a sensitizer.
If statistically significant and/or biologically relevant increases in ear weights are running in
parallel to the increase in cell count, 3H-thymidine incorporation and/or lymph node weight, it
cannot be ruled out, that the lymph node response was caused by irritation and not by skin
sensitization. Then, for identification of the relevance of the statistical evaluation, a
comparison of the results of the present test to appropriate historical control values (cf.
section “Historical control data”) may be performed. If one or a combination of the measured
parameters change statistical significance, evaluation on basis of the criteria described above
may be possible. If the statistical comparison with the historical control does not yield results
useful for evaluation, further investigations may be necessary to differentiate between
irritation and sensitization response.
If a test substance does not elicit a biological relevant increase in cell count, 3H-thymidine
incorporation but shows a clear concentration related increase in response, further
investigation of the sensitization potential at higher concentrations should be considered.
A flow chart on the evaluation procedure is given in the Appendix.
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4. RESULTS
Tables on the results of the clinical examinations are presented in the Appendix.
4.1. ANALYSES
Stability of the testsubstance preparation:
The stability of the test substance in the vehicle for the
maximum application period was confirmed indirectly by the
concentration analysis.
Homogeneity of the testsubstance preparation:
The test-substance preparation was visually homogeneous
(solution).
Concentration analysis of
the test-substance
preparation:
The verification of the concentration of the test-substance
preparations (70%, 30% and 10%) for the first application was
confirmed by analysis (details are available in the Appendix ).
Analysis of feed:
In view of the aim and duration of the study the contaminants
occurring in commercial feed should not influence the results.
Analysis of drinking water:
In view of the aim and duration of the study there are no
special requirements exceeding the specification of drinking
water.
Analysis of bedding:
In view of the aim and duration of the study there are no
special requirements exceeding the specification of a
commercial grade monitored by the manufacturer.
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4.2. CLINICAL OBSERVATIONS AND ASSESSMENT OF FINDINGS
4.2.1. Cell counts, 3H-thymidine incorporation and lymph node weights
The statistically significant increases in cellularity, caused by the 70% concentration and in
3
H-thymidine incorporation into the lymph node cells, caused by the 70% and 30%
preparations, failed to reach the cut off stimulation index of 1.5 and 3, respectively, and thus
lie below the threshold of immunologic relevance. Lymph node weights were not statistically
significantly increased.
The group mean values are presented in the figures below. The corresponding data, the cell
count, 3H-thymidine incorporation and lymph node weight stimulation indices as well as the
individual values are given in the Appendix.
Cell Count
70% in 1% aqueous
Pluronic®
#
30% in 1% aqueous
Pluronic®
10% in 1% aqueous
Pluronic®
1% aqueous Pluronic®
0
5,000,000
10,000,000
15,000,000
Cell Counts [Counts / lymph node pair]
³H-thymidine incorporation
70% in 1% aqueous
Pluronic®
##
30% in 1% aqueous
Pluronic®
#
10% in 1% aqueous
Pluronic®
1% aqueous Pluronic®
0
500
1,000
1,500
³H-thymidine [DPM / lymph node pair]
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )
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Lymph Node Weight
70% in 1% aqueous
Pluronic®
30% in 1% aqueous
Pluronic®
10% in 1% aqueous
Pluronic®
1% aqueous Pluronic®
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Weight [mg / lymph node pair]
4.2.2. Ear weights
The ear weight stimulation indices indicate minimal ear skin irritation at all concentrations
though not concentration related. A slight but statistically significant increase in ear weights
was observed in mice treated with the 10% test substance preparation, only.
The group mean values are presented in the figures below. The corresponding data, the ear
weight stimulation index as well as the individual values are given in the Appendix.
Ear Weight
70% in 1% aqueous
Pluronic®
30% in 1% aqueous
Pluronic®
10% in 1% aqueous
Pluronic®
#
1% aqueous Pluronic®
0.0
10.0
20.0
30.0
40.0
50.0
Weight [mg / animal]
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )
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4.2.3. Body weights
The expected body weight gain was generally observed in the course of the study.
The individual body weights are shown in the Appendix.
4.2.4. Other findings
No abnormalities were observed during general observation.
5. DISCUSSION AND CONCLUSION
No signs of systemic toxicity were noticed.
The statistically significant increases in cellularity, caused by the 70% concentration and in
3
H-thymidine incorporation into the lymph node cells, caused by the 70% and 30%
preparations, failed to reach the stimulation index criterion of 1.5 or 3, respectively, and thus
lie below the threshold of immunologic relevance. Lymph node weights were not statistically
significantly increased.
Though not concentration related and statistically significant in mice treated with the 10% test
substance preparation, only, the slight increases in ear weight stimulation indices indicate
minimal ear skin irritation at all concentrations.
Thus it is concluded that Ethanolamine hydrochloride does not show a skin sensitizing
effect in the Murine Local Lymph Node Assay under the test conditions chosen.
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APPENDIX
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CELL COUNT, 3H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT: TEST
GROUP MEAN VALUES, STANDARD DEVIATIONS AND STIMULATION INDICES
Treatment
1% aqueous Pluronic®
10% in 1% aqueous Pluronic®
30% in 1% aqueous Pluronic®
70% in 1% aqueous Pluronic®
Cell Counts [Counts/Lymph Node Pair]
Mean
S.D.
Stimulation Index1
8,460,000.0
1,321,206.3
1.00
9,549,600.0
568,475.9
1.13
8,760,000.0
1,238,333.7
1.04
10,639,000.0
1,767,655.1
1.26 #
Test Group
1
2
3
4
Treatment
1% aqueous Pluronic®
10% in 1% aqueous Pluronic®
30% in 1% aqueous Pluronic®
70% in 1% aqueous Pluronic®
³H-thymidine incorporation [DPM/Lymph Node Pair]
Mean
S.D.
Stimulation Index1
332.2
124.8
1.00
443.8
142.4
1.34
486.9
128.4
1.47 #
673.7
233.1
2.03 ##
Test Group
1
2
3
4
Treatment
1% aqueous Pluronic®
10% in 1% aqueous Pluronic®
30% in 1% aqueous Pluronic®
70% in 1% aqueous Pluronic®
Lymph Node Weight [mg/Lymph Node Pair]
Mean
S.D.
Stimulation Index1
5.0
0.7
1.00
5.5
0.7
1.10
5.3
0.7
1.06
5.6
0.8
1.11
Test Group
1
2
3
4
EAR WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS AND
STIMULATION INDICES
Test Group
1
2
3
4
Treatment
1% aqueous Pluronic®
10% in 1% aqueous Pluronic®
30% in 1% aqueous Pluronic®
70% in 1% aqueous Pluronic®
Mean
32.8
35.2
34.2
34.5
Ear Weight [mg/animal]
S.D.
Stimulation Index1
2.2
1.00
1.2
1.07 #
2.1
1.04
2.7
1.05
1
test group x / test group 1 (vehicle control)
# = statistically significant for the value p ≤ 0.05
## = statistically significant for the value p ≤ 0.01
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Report; Project No.: 58H0372/052301
CELL COUNT, 3H-THYMIDINE INCORPORATION, LYMPH NODE WEIGHT AND EAR
WEIGHT: INDIVIDUAL VALUES
Group
Treatment
1
1% aqueous Pluronic®
2
10% in 1% aqueous Pluronic®
3
30% in 1% aqueous Pluronic®
4
70% in 1% aqueous Pluronic®
Animal
No.
Cell Counts/
Lymph Node Pair
9,456,000
7,806,000
8,148,000
6,792,000
5,646,000
10,098,000
8,910,000
³H-thymidine
incorporation
[DPM/Lymph Node Pair]
288.7
258.7
364.4
215.8
129.8
533.3
497.3
Lymph Node
Weight
[mg//Lymph Node Pair]
5.7
4.5
5.0
4.1
3.5
5.7
4.8
Ear
Weight
[mg/animal]
33.4
30.5
30.9
36.0
31.1
33.3
34.7
1
2
3
4
5*
6
7
8**
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8,952,000
9,870,000
10,050,000
9,966,000
9,846,000
7,992,000
9,240,000
10,212,000
6,918,000
8,352,000
11,460,000
9,960,000
7,848,000
11,742,000
12,876,000
9,948,000
250.3
380.0
456.3
635.1
580.3
394.0
527.6
602.9
271.1
545.4
552.7
796.7
308.4
728.6
998.2
657.4
5.8
4.8
6.2
6.0
5.5
4.8
4.5
6.0
6.3
4.8
5.5
4.7
5.2
6.1
6.8
5.0
34.8
34.4
34.9
37.4
32.5
32.6
36.1
34.0
37.5
32.5
33.7
30.9
34.8
35.0
39.1
33.7
* Values of this animal were not used for calculation of the mean values and evaluation. The body weight of the
animal decreased during the study period and unusually small lymph nodes were dissected. These effects
were not considered to be test-substance related.
** Animal died due to irregularities during i.v. application.
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Report; Project No.: 58H0372/052301
BODY WEIGHT DATA
Group
1
2
3
4
Animal No.
1
2
3
4
5*
6
7
8**
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Body Weight
d0 [g]
22.2
19.3
21.7
21.0
19.1
19.8
20.6
24.2
21.7
21.3
21.4
19.9
19.6
20.8
21.4
19.4
20.1
19.6
19.7
18.3
23.6
20.7
20.1
20.3
Mean
S.D.
20.8
1.23
21.0
0.73
20.2
0.79
20.5
1.75
Body Weight
d5 [g]
22.9
19.9
22.7
20.6
18.5
19.6
21.3
24.7
22.8
22.1
21.6
21.7
19.9
21.8
23.0
19.5
20.8
19.6
21.0
19.4
23.2
21.7
20.4
20.9
Mean
S.D.
21.1
1.56
21.9
0.58
20.8
1.40
21.1
1.28
Body Weight Change
d5-d0 [g]
0.7
0.6
1.0
-0.4
-0.6
-0.2
0.7
0.5
1.1
0.8
0.2
1.8
0.3
1.0
1.6
0.1
0.7
0.0
1.3
1.1
-0.4
1.0
0.3
0.6
Mean
S.D.
0.3
0.61
0.9
0.59
0.6
0.61
0.6
0.63
* Values of this animal were not used for calculation of the mean values and evaluation. The body weight of the
animal decreased during the study period and unusually small lymph nodes were dissected. These effects
were not considered to be test-substance related.
** Animal died due to irregularities during i.v. application.
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Report; Project No.: 58H0372/052301
HISTORICAL CONTROL DATA
The historical control data gathered in the laboratory with the vehicle used (1% aqueous
Pluronic®) are presented in the following table:
Project-No.
Date
45H0056/052011
45H0450/012303
Apr-05
45H0113/022264
45H0375/052048
Jun-05
45H0359/052068
45H0511/052083
Oct 05
45H0502/032222
Oct 05
45H0100/042289
Oct 05
45H0666/052189
Feb-06
45H0666/052189
Feb-06
58H0651/052124
Sep-05
Animal No.
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
25
26
27
28
29
30
25
26
27
28
29
30
37
38
39
40
41
42
1
2
3
4
5
6
Cell Counts/
Lymph Node Pair
8,942,000
14,216,000
6,268,000
5,061,000
10,518,000
15,440,000
5,219,000
4,955,000
8,094,000
6,996,000
4,246,000
5,677,000
11,406,000
8,296,000
8,884,000
10,788,000
9,524,000
9,252,000
12,330,000
10,278,000
12,176,000
6,684,000
8,760,000
12,968,000
13,656,000
11,250,000
8,930,000
11,442,000
6,497,000
9,494,000
11,370,000
8,316,000
12,430,000
7,174,000
11,404,000
10,054,000
11,960,000
12,500,000
10,358,000
10,924,000
6,360,000
15,600,000
12,762,000
12,492,000
12,222,000
8,082,000
8,892,000
8,130,000
³H-thymidine
incorporation
[DPM]
242.4
275.6
275.0
167.0
218.5
195.9
Lymph Node
Weight
[mg]
4.8
6.7
4.0
4.8
4.7
6.0
4.2
4.6
5.0
5.2
4.1
4.8
6.2
4.3
3.9
5.0
4.8
4.8
6.3
5.4
6.2
4.0
4.5
5.7
6.3
7.3
5.7
6.2
4.5
5.5
6.3
4.5
6.4
4.8
5.3
5.5
5.3
5.1
5.3
5.1
4.1
6.2
7.0
6.0
5.3
4.5
4.6
5.3
Ear Weight
[mg]
29.7
29.6
27.5
30.2
28.1
30.5
29.9
28.9
31.1
29.4
29.8
30.6
27.1
29.1
29.3
28.9
29.5
28.7
29.1
28.8
28.2
28.9
25.4
26.1
27.3
27.4
28.9
29.1
28.6
28.4
28.6
28.9
28.4
29.1
27.2
27.9
29.8
28.9
30.3
29.5
28.1
30.4
29.3
29.9
28.4
30.0
27.8
27.4
Ear Thickness
[µm]
240
230
230
230
220
220
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Report; Project No.: 58H0372/052301
HISTORICAL CONTROL DATA (continued)
58H0041/052249
Mar 06
45H0727/052257
45H0728/052258
Mar 06
45H0835/052233
Mar 06
Historical control data
Median
Mean
S.D.
Present study
70% in 1% aqueous Pluronic®
Median
Mean
S.D.
Positive control study
10% in acetone
58H0288/982075
Median
Mean
S.D.
Positive control study
10% in 1% aqueous Pluronic®
45H0288/982036
Median
Mean
S.D.
25
26
27
28
29
30
1
2
3
4
5
6
25
26
27
28
29
30
10,086,000
9,378,000
11,628,000
12,906,000
12,636,000
9,702,000
11,706,000
12,498,000
11,378,000
11,378,000
11,320,000
11,016,000
11,232,000
9,534,000
11,910,000
9,290,000
9,584,000
10,400,000
870.8
258.9
665.1
518.4
779.5
661.2
5.0
5.8
5.7
5.7
5.5
4.8
6.2
6.0
5.6
5.0
5.7
5.2
5.3
5.3
5.7
4.5
4.8
4.8
31.0
34.6
30.6
30.7
29.6
34.5
28.3
29.4
28.7
29.5
28.6
26.6
30.0
30.2
28.1
28.6
29.0
28.1
220
220
210
230
210
220
10,379,000
10,103,924
2,520,181
275.3
427.4
254.8
5.3
5.3
0.8
29.0
29.1
1.5
220.0
223.3
8.9
10,710,000
10,639,000
1,767,655
693.0
673.7
233.1
5.4
5.6
0.8
34.3
34.5
2.7
22,287,000
20,823,000
5,074,730
4571.2
4659.2
1409.4
8.9
8.8
1.2
30.0
29.9
0.9
10.2
9.4
2.1
38.5
39.2
4.5
26,536,000
25,013,667
6,849,692
230.0
226.7
5.2
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Report; Project No.: 58H0372/052301
POSITIVE CONTROL
α-Hexylcinnamaldehyde, techn. 85%, vehicle: acetone
As recommended by the respective testing guidelines, the following study was performed to
evaluate the sensitivity of experimental animals (mice, CBA/CaOlaHsd, Harlan Winkelmann
GmbH, Borchen, Germany) and the ability of procedures to detect a known mild to moderate
skin sensitizer.
Name of test substance:
CAS number:
Substance number:
Project No.:
Alpha-Hexylcinnamaldehyde, techn. 85%
101-86-0
98/288-1
58H0288/982075
Experimental starting date:
Experimental completion date:
14 Feb 2006
03 Mar 2006
The mean stimulation indices (fold of change as compared to the vehicle control) for cell
count,
3
H-thymidine incorporation, lymph node weight, ear weight and ear thickness are
summarized for each test group in the table below.
Test
group
Treatment
1
2
3
4
vehicle acetone
3% in acetone
10% in acetone
30% in acetone
Cell Count
Stimulation
Index
1.00
1.75
2.36
2.98
#
##
##
³H-thymidine
incorporation
Stimulation
Index
1.00
4.56
##
6.63
##
9.86
##
Lymph Node
Weight
Stimulation
Index
1.00
1.47
#
1.98
##
2.46
##
Ear Weight
Stimulation
Index
1.00
1.00
1.03
1.22
##
Ear Thickness
Stimulation
Index
1.00
1.02
1.05
1.15
#
##
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )
Discussion and Conclusion
No signs of systemic toxicity were noticed. One animal of test group 2 died after 3H-thymidine
injection.
The test substance induced a statistically significant and biologically relevant response
(increase to 1.5 fold or above of control value = stimulation index (SI) 1.5) in the auricular
lymph node cell counts when applied as 3%, 10% or 30% preparation in acetone.
Concomitantly, the increase of 3H-thymidine incorporation into the cells was statistically
significant and biologically relevant (increase above the cut off stimulation index of 3) at these
concentrations, which additionally caused statistically significantly increased lymph node
weights.
The statistically significant increase in ear weights at 30%, which was accompanied by an
increase in ear thickness, indicates some irritation of the ear skin.
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Report; Project No.: 58H0372/052301
A small but statistically significant increase in ear thickness was caused by the 10%
preparation. This mild irritation, however, does not explain the lymph node response
observed at this concentration.
Thus it is concluded that the Murine Local Lymph Node Assay is able to show the skin
sensitizing effect of α-Hexylcinnamaldehyde, techn. 85% under the test conditions
chosen.
The threshold concentration for sensitization induction was < 3% under the test conditions
chosen. The EC 1.5 for cell count and the EC 3 for 3H-thymidine incorporation was calculated
by semi-logarithmical regression from the results of all concentrations to be 1.9% and 1.7%,
respectively.
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Report; Project No.: 58H0372/052301
α-Hexylcinnamaldehyde, techn. 85%, vehicle 1% aqueous Pluronic® L92
Evaluation of the sensitivity of mice, CBA/CaOlaHsd, Harlan Winkelmann GmbH, Borchen,
Germany) to known sensitizers as recommended by the guideline.
Name of test substance:
CAS number:
Substance number:
Project No.:
α-Hexylcinnamaldehyde, techn. 85%
101-86-0
98/288-1
45H0288/982036
Experimental starting date:
Experimental completion date:
April 13, 2004
May 19, 2004
The indices for lymph node weight, cell count and ear weight in relation to the vehicle control
group are summarized in the table below.
Test
group
Treatment
Lymph node Weight
Index
Cell Count
Index
1
2
3
4
vehicle 1% Pluronic® L 92 in doubly distilled water
3% in 1% Pluronic® L 92 in doubly distilled water
10% in 1% Pluronic® L 92 in doubly distilled water
30% in 1% Pluronic® L 92 in doubly distilled water
1.00
1.28 #
1.81 ##
2.39 ##
1.00
1.43 #
2.28 ##
2.92 ##
Ear Weight
Index
1.00
1.10 ##
1.41 ##
1.94 ##
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )
Discussion and Conclusion:
No signs of systemic toxicity were noticed. The test substance induced a statistically
significant response in the auricular lymph nodes, when applied as 3%, 10% or 30%
preparations in 1% Pluronic® L 92 in doubly distilled water. The statistically significant
increases in ear weights indicate irritation of the ear skin at all concentrations. The high ear
weights in test groups 3 and 4 were accompanied by scaling and incrustations, which were
observed on the day of lymph node removal.
The increase in cell count index induced by the 3% test substance preparation was at the
border of biological significance. The marked increase at 10% and 30% cannot be explained
by skin irritation alone.
Based on the results of this study, it is concluded that the test conditions and animal strain
chosen are able to reveal the known skin sensitizing effect of α-Hexylcinnamaldehyde,
techn. 85% in the Murine Local Lymph Node Assay.
The threshold of skin sensitization induction of the compound lies just below 3% under the
test conditions used.
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Report; Project No.: 58H0372/052301
FLOW CHART FOR EVALUATION OF THE LLNA
The evaluation procedure described in section 3.8. of the report is presented here in
graphical form.
Statistically significant and/or
biologically relevant increase
of ³H-Thymidine incorporation,
cell count and/or lymph node
weight
Yes
No
Statistically significant
and/or biologically
relevant increase of ear
punch weight and/or ear
thickness
(Ear skin irritation)
Yes
No
Statistically significant and/or
biologically relevant increase
of ear punch weight and/or ear
thickness at concentrations
without lymph node response
Sufficiently high
concentration tested?
No
No
Conclusive evaluation of
lymph node responseear response-relation
possible by using
historical control data
Perform test with higher
concentrations
Yes
Yes
Yes
Further investigations
(e.g. challenge
experiment)
No
sensitizing
not
sensitizing
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ANALYSES
Analytical characterization of the test substance (Certificate of Analysis)
Concentration Control Analysis of Ethanolamine hydrochloride in 1 % Pluronic® L 92 in
doubly distilled water
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Report; Project No.: 58H0372/052301
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Report; Project No.: 58H0372/052301
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Report; Project No.: 58H0372/052301
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