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EC number: 204-677-5
CAS number: 124-07-2
Reliable studies on skin sensitisation (animal sensitisation
tests) are available for the following fatty acids category members:
LLNA (OECD 429): CAS# 124-07-2, C8; CAS# 112-80-1, C18:1; CAS#
60-33-3, C18:2; CAS# 463-40-1, C18:3
GPMT (OECD 406): CAS# 123-99-9, C9d; CAS# 112-80-1, C18:1; CAS#
60-33-3, C18:2; CAS# 463-40-1, C18:3
Buehler test (OECD 406): CAS# 112-05-0, C9; CAS# 67701-01-3, fatty
Taken together in a weight of evidence approach members of the
fatty acids category are not skin sensitising.
octanoic acid an in vivo skin sensitisation study (LLNA) and a human
maximization test is available.
human health effects in regard to skin sensitisation are predicted from
adequate and reliable data for source substances by read-across to the
target substance within the group applying the group concept in
accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006.
acids are found in all living organisms fulfilling fundamental
physiological functions within the body (for details refer to
Toxicokinetics, metabolism and distribution). Based on this role within
the body no sensitisation potential of fatty acids is expected as it
could be demonstrated by animal studies with C8 fatty acid (octanoic
acid), C9 fatty acids (azelaic acid, nonanoic acid), C10 fatty acid
(decanoic acid), C12 fatty acid (lauric acid), C18:1 fatty acid (oleic
acid), C18:3 fatty acid (linolenic acid), C12-18 fatty acids (fatty
acids, C12-18), by QSAR calculations for C6 fatty acid (hexanoic acid)
and C22 fatty acid (docosanoic acid) and by human data with C8 fatty
acid (octanoic acid), respectively.
sensitisation studies in animals
acid (CAS# 124-07-2) was tested in the local lymph node assay (LLNA),
which measures the induction of sensitisation as a function of primary
proliferation of lymphocytes in the lymph node draining the site of
topical chemical application (Basketter et al., 1998). Three
experimental groups each of four CBA mice were treated once daily with
octanoic acid concentrations of 10%, 25% and 50% on three consecutive
days, respectively. The lymphocyte proliferation in the test animals was
compared with that of sham treated controls. The stimulation indices (SI
values) calculated for the substance concentrations 10%, 25% and 50%
were 0.7, 1.0 and 1.6, respectively. Since octanoic acid did not elicit
an SI ≥ 3 in the LLNA, it is not considered to have skin sensitising
acid (CAS# 123-99-9) was examined for its skin sensitisation potential
in a guinea pig maximisation test (GPMT) which was conducted under GLP
according to OECD guideline 406 (1995). Ten Dunkin-Hartley guinea pigs
received an intradermal injection of 0.25% azelaic acid for induction
followed by the second induction 8 days later as a topical application
of 50% azelaic acid on the same site, which had been previously treated
with 10% SDS in petrolatum. The used concentrations were based on a
preliminary range finding test, where the suitable concentrations for
the intradermal injection and patch testing were evaluated. Eleven days
after the last induction application, the test animals and the 5 control
animals were challenged with 50% test material by an occluded patch. As
result, no sensitising effects could be observed 24 and 48 hours after
dermal sensitisation study was performed with nonanoic acid (CAS#
112-05-1) according to OECD guideline 406 (Buehler test) (1981). Hartley
guinea pigs were induced nine-times within three weeks with an
epicutaneous occlusive application of the test substance (100% for
inductions 1-5; 75% for inductions 6-9 due to severe dermal irritation)
onto the right dorsal skin for 6 hours. 14 days after the last induction
exposure, epicutaneous challenge was conducted with 10% nonanoic acid in
acetone on the left flank under occlusive conditions for 6 hours. No
dermal sensitisation reaction was seen at 24 and 48 hours after
challenge. Following re-challenge (7 days after the first challenge)
with a concentration of 25% nonanoic acid in acetone, 3 and 1 out of 20
animals exhibited barely perceptible erythema after 24 and 48 hours,
respectively. Since barely perceptibly erythema (score +/-) is not
considered as positive sensitisation response (score ≥ 1) and since this
response also reversed in two out of three animals, nonanoic acid did
not elicit skin sensitisation.
et al. (2008) assessed the skin sensitisation potential of unsaturated
C18 fatty acids (oleic acid (CAS# 112-80-1), linoleic acid (CAS#
60-33-3), linolenic acid (CAS# 463-40-1) by the GPMT and LLNA.
Additional information for oleic acid is obtained from skin
sensitisation studies including LLNA and GPMT conducted by Anderson et
al. (2009) and Basketter et al. (2009).
studies performed by Kreiling et al. (2008) unsaturated C18 fatty acids
(oleic acid, linoleic acid and linolenic acid were assessed for their
skin sensitisation potential in the GPMT according to OECD guideline 406
and in the LLNA according to OECD guideline 429 both under GLP.
GPMT, first a range-finding test was performed to assess the irritation
effects of the test substances after intradermal and topical
application. 10 SPF-Hsd Poc:DH guinea pigs received each an intradermal
induction of 5% oleic acid, linoleic acid and linolenic acid (day 0). 7
days later the second induction treatment was performed by applying a
gauze patch with 50% oleic acid and 100% linoleic and linolenic acid,
respectively to the clipped skin under occlusive conditions for 48
hours. No sodium lauryl sulphate was applied 24 hours before the topical
induction, since the three unsaturated fatty acids itself caused a clear
skin irritation at the topical induction concentration used. On day 20
and day 28 the test animals and 5 control animals were challenged and
re-challenged by occluded patch with 25% oleic acid and 50% linoleic
acid and linolenic acid, respectively. 24, 48 and 72 hours after removal
of the challenge patches, skin reactions were recorded. For oleic acid
skin sensitisation was considered to be present in two animals. For
linolenic acid two test animals showed a skin reaction at the 24 hours
reading, but not after 48 and 72 hours, indicating a non-specific rather
than an allergic reaction, since also one control animal showed grade 1
skin reaction at the 48 hours reading. In another test animal a skin
reaction was observed at 48 and 72 hours. Since the response of at least
30% of the animals is not achieved in the GPMT for oleic acid (2/10
animals with skin sensitisation reaction) and linolenic acid (no animal
with skin sensitisation reaction), they are considered not to be skin
sensitiser. Linoleic acid caused skin reactions in the control group as
frequently or even at a higher incidence than in the test group after
challenge and re-challenge. Thus it was assumed, that the test
concentrations for challenge were too high and induced non-specific,
irritation skin reactions. As a consequence, no conclusion with regard
to skin sensitisation could be drawn for linoleic acid in the GPMT.
parallel to the GPMT the LLNA was performed in SPF-CBA/Ca01aHsd mice.
The highest tolerable exposure concentration was determined in
preliminary experiments. 10, 25 and 50% of the unsaturated C18 fatty
acids in acetone/olive oil (3+1 (v/v)) and the vehicle alone were
applied daily to the ears of 5 animals each for three consecutive days.
Local irritant effects expressed as ear swelling were assessed by
measuring the ear thickness. Oleic acid, linoleic acid and linolenic
acid gave clear positive results in the LLNA with SI values ≥ 3. For 10,
25 and 50% oleic acid the SI values were 2.6, 14.9 and 6.9, for 10, 25
and 50% linoleic acid 1.5, 7.0 and 9.1 and for linolenic acid the SI
values were 3.1, 9.3 and 10.3, respectively. There was a good agreement
between SI values > 3 and statistically significant lymph node weight
increase. No clinical signs of local irritation were noted and no
significant ear swelling (data not shown) was measured according to the
authors. However, these positive results are in discrepancy to the
negative results of the GPMT tests.
Basketter et al. (2009) evaluated the sensitising potential of oleic
acid including a GPMT and LLNA. In the GPMT assay according to OECD
guideline 406, 10 guinea pigs were induced via intradermal injection of
5% oleic acid followed by epicutaneous induction with undiluted oleic
acid. For challenge and re-challenge, 1% oleic acid was applied
topically. No positive skin reaction was observed in the test animals.
The validity of the test was confirmed by a positive control group
treated with 10% hexyl cinnamic aldehyde, in which 40% of the animals
showed a positive sensitisation reaction. The LLNA was conducted in
compliance with OECD guideline 429 with the deviation that no
pre-screening test was performed to determine the concentration level,
that does not induce systemic toxicity and/or excessive local skin
irritation. Topical application of oleic acid induced a dose-dependent
lymphoproliferative response as shown by stimulation indices of 3.4, 5.7
and 6.5 for 25, 50 and 100% oleic acid, respectively. Thus, the results
from the two test systems are also in conflict as the GPMT revealed a
negative and the LLNA a positive test result, as already shown by
Kreiling et al. (2008).
the positive LLNA result for oleic acid is considered a false positive
based on a LLNA performed by Anderson et al. (2009), in which an
excessive skin irritation was noted after application of 25 and 50%
oleic acid as indicated by ear swelling measurements. Beside increases
in the stimulation indices (SI: 1.6, 2.4 and 5.6 for 10, 25 and 50%
oleic acid, respectively, resulting in an EC3 value of 29.7), oleic acid
induced a dose-dependent increase in ear thickness: control animals
revealed no ear swelling (-1.8 ± 2%) whereas oleic acid exposed animals
showed ear swellings of 11.2 ± 2.6%, 29.2 ± 3.9% and 51.6 ± 6.2% for 10,
25 and 50%, respectively, with statistical significance for the
high-dose group. In compliance with OECD 429, an increase in ear
thickness of ≥ 25% has to be interpreted as excessive skin irritation.
Thus, positive results after treatment with more than 10% oleic acid
have to be considered as ambiguous due to the induction of excessive
skin irritation. Furthermore, 10% oleic acid, which seems not to induce
excessive skin irritation, did not reveal a stimulation index above 3.
Taken together, oleic acid seems to induce false positive results in the
LLNA when concentrations of ≥ 30% (EC3 value) are applied to the skin
due to skin irritation properties.
known limitation of the test system to produce false positive findings
with certain skin irritants as indicated in OECD guideline 429 supports
the hypothesis that the effects observed in the LLNA performed by
Anderson et al. (2009) and thus in the LLNAs performed by Kreiling et
al. (2008) and Basketter et al. (2009) are secondary effects due to
irritation and cannot clearly be attributed to a sensitisation potential
of the test substance. Kreiling et al. (2008) measured the ear swelling
in the LLNA, but the data was not presented or described in the result
part. It was only stated, that no significant increases in ear thickness
in the LLNA studies were measured for any of the test substance
concentrations. However, statistically non-significant increases can be
obtained even when ear thickness is greater than 25% (e.g. Andersons et
al. measured at a test concentration of 25% oleic acid, a statistical
non-significant ear swelling of 29.2 ± 3.9%). Thus, a biological
significant increase in ear thickness cannot be excluded. Further,
regarding the study performed by Basketter et al. (2009) no information
about the induction of skin irritation in the applied oleic acid
concentrations is available, which might be the reason for the
discrepancy between the two test models GPMT and LLNA. Thus it is not
comprehensible, if local irritant effects have been considered
adequately in the studies by Kreiling et al. and Basketter et al. For
linoleic acid and linolenic acid also skin irritation potential was
noted during the GPMT assay by Kreiling et al. (2008). Thus, the design
of the LLNA studies should be regarded as not suitable to evaluate the
sensitisation potential for the tested unsaturated C18 fatty acids,
since local irritation cannot be definitely excluded.
basic principle underlying the LLNA is that sensitisers induce
proliferation of lymphocytes in the lymph nodes draining the sited of
test substance application (OECD 429). Kreiling et al. (2008)
speculated, that unsaturated fatty acids may cause cell activation by a
number of several specific mechanisms (e.g. by induction of
intracellular Ca signals, activation of protein kinase C, inhibition
GTPase activity, induction of interleukin release etc.) leading to the
activation of epidermal cells. “This stimulation could have activated
Langerhans cells to migrate out of the skin to the auricular lymph node
where they induced a hapten-unrelated cell proliferation of lymph node
cells. The gathered information on possible activation of cells in the
skin by mechanisms different from a hapten-specific activation suggests
that, perhaps, at least part of the lymph node reactions observed after
application of oleic acid, linoleic acid and linolenic acid has been
caused in an unspecific way” (Kreiling et al., 2008). Furthermore,
regarding the fact that the LLNA assesses the induction/sensitization
phase of allergic responses only via 3H-thymidine incorporation in the
auricular draining lymphe node whereas the GPMT assess the
induction/sensitisation period in combination with the elicitation phase
of an allergic response via scoring of skin reactions after intradermal
induction including a powerful adjuvant which enhances the induction of
the immune response (Basketter et al., 2009), the result of the GPMT is
considered to be of stronger biological significance than the LLNA.
conclusion, taken into account (1) the discussions about possible false
positive findings in the LLNA due to irritating effects and non-specific
cell activation, (2) the fact, that unsaturated C18 fatty acids occur in
fatty acid constituents of foods and as natural building block in
membrane phospholipids and triglycerides, (3) the absence of reports on
human cases of allergic reactions, (4) negative QSAR predictions and (5)
the negative result in the GPMT for oleic acid and linolenic acid, the
unsaturated C18 fatty acids do not fulfill the criteria for skin
UVCB substance with C12-18 fatty acids (0.1% C8, 2.6% C10, 55.9% C12,
19.1% C14, 9.8% C16, 0.1% C17, 11.1% C18 and 0.3% C20) was examined in a
dermal sensitization study (Buehler test) (1979). Twenty Dunkin-Hartley
guinea pigs were induced with 40% of the test substance (CAS#
67701-01-3) in distilled water by applying an occlusive patch on the
left shoulder once a week for three consecutive weeks. This induction
concentration represented the highest concentration to form a homogenous
suspension and causing some irritation reactions at the dermal sites of
induction. 13 days after the last induction, the test animals and 10
control animals were challenged with 20% test substance under occlusive
conditions for 6 hours. Only one animal showed slight, but confluent to
moderate erythema at the 26 h reading after patch removal. No positive
sensitising reactions were observed at the 48 h reading after challenge.
8 days after challenge, a re-challenge was carried out including a new
naïve control group with ten 10 animals. In 6 animals confluent to
moderate erythema was noted at the 26 hours reading after re-challenge.
However, this response reversed within 22 hours in all animals and is
therefore not considered as sensitising effect.
sensitisation tests have also been performed with decanoic acid (CAS#
334-48-5) and lauric acid (CAS# 143-07-7). Since only short abstracts
are available, these studies have been judged with reliability 4 "not
assignable". However, the results of these studies confirm the
non-sensitising properties of fatty acids. The skin sensitisation
potential of decanoic acid was tested in a Buehler test, where 20 guinea
pigs were induced by an epicutaneous application of 5% decanoic acid in
40% ethanol under occlusion for 6 hours once a week for three
consecutive weeks (1975). Two weeks after the last induction, the
animals were challenged epicutaneously under occlusion with a
concentration of 5% decanoic acid in acetone for 6 hours. The readings
24 and 48 hours after removal of the patches revealed occasional very
slight degree of irritation in the test and control groups,
respectively. However, no signs of a sensitisation reaction were noted.
same negative result was obtained for lauric acid tested in a study
according to the method described by Magnusson and Kligman (1979). 20
female Pirbright-white guinea pigs received an induction by intradermal
injection and were challenged with a concentration of 2.5%
epicutaneously under occlusion for 24 hours. The readings of the skin
sites 24 and 48 hours later did not reveal any reaction so that lauric
acid is regarded as not sensitising to skin.
sensitisation predictions using Toxtree and OECD Toolbox
skin sensitisation potential of hexanoic acid (CAS# 142-62-1) and
docosanoic acid (CAS# 112-85-6) was estimated using QSAR calculations
(2012). These two substances are representing the fatty acids with the
shortest (C6 fatty acid) and the longest carbon chain length (C22 fatty
acid) within the fatty acids category.
alerts of hexanoic acid and docosanoic acid using Toxtree (Estimation of
Toxic Hazard – A Decision Tree Approach v.2.1.0) were examined. No
structural alerts were detected for the test substances.
addition a read-across approach based on organic functional groups was
conducted using the OECD Toolbox databases. For 5 read-across substances
which contain the same functional groups, skin sensitisation data could
be found in OECD Toolbox databases. The experimental results of the
surrogate substance were negative.
means of the OECD Toolbox (v 2.2) the sensitisation potential for
hexanoic acid and docosanoic acid was also predicted based on the (Q)SAR
Multicase commercial model A33 also referred to as Danish EPA QSAR
Database. The prediction for both substances C6 and C22 are negative.
sensitisation studies in humans
was not found to be a skin sensitizer in a published study with 25 human
subjects, who received an application of 0.3 g octanoic acid (CAS#
124-07-2) at 5% concentration under occlusion for induction onto the
forearm 5 times for 24 hours (Opdyke, 1981). The challenge with a
concentration of 1% did not result in any positive reaction when scored
72 and 96 hours later.
into account all available data on skin sensitisation testing in animals
and humans and by QSAR applications, members of the fatty acids category
do not fulfil the criteria for classification as skin sensitiser.
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D.A. et al. (1998).Strategies for Identifying False Positive Responses
in Predictive Skin Sensitization Tests. Food and Chemical Toxicology
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D. et al. (2009). Application of a weight of evidence approach to
assessing discordant sensitisation datasets: Implications for REACH.
Regulatory Toxicology and Pharmacology 55:90 - 96.
R. et al. (2008). Comparison of the skin sensitizing potential of
unsaturated compounds as assessed by the murine local lymphnode assay
(LLNA) and the guinea pig maximization test (GPMT).Food and Chemical
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Study not required according to Annex VII-X of Regulation (EC) No
All available data on skin sensitisation of
the members of the fatty acids category do not meet the criteria for
classification according to Regulation (EC) No 1272/2008, and are
therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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