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Administrative data

Description of key information

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 acids, C12-18

Taken together in a weight of evidence approach members of the fatty acids category are not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
not specified
Vehicle:
not specified
Concentration:
10, 25 and 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: in a human 4 h patch test (Basketter et al., 1997) octanoic acid showed a high irritation potential (68% of the panel responded to octanoic acid and 58% of the panel reacted to 25% sodium lauryl sulphate (positive control))

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: Evaluation criteria used were taken from Kimber and Basketter (1992)

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test or vehicle alone was applied to the dorsum of both ears. The application was repeated on days 2 and 3. On day 6, 20 µCi 3H-methyl thymidine in 250 µL phosphate buffered saline (PBS) was injected into the tail vein. 5 h later, the draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation and washed twice in PBS. After the final wash, pellets were resuspended in 5% trichloracetic acid (TCA) and incubated at 4 °C for 18 h. Pellets were resuspended in TCA and thymidine incorporation was measured.
Positive control substance(s):
other: analogue tests with other substances are also reported
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% test substance
Parameter:
SI
Value:
1
Test group / Remarks:
25% test substance
Parameter:
SI
Value:
1.6
Test group / Remarks:
50% test substance
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For octanoic acid an in vivo skin sensitisation study (LLNA) and a human maximization test is available.

Moreover, 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.

Fatty 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.

 

Skin sensitisation studies in animals

Octanoic 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 properties.

 

Azelaic 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 the challenge.

 

A 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.

 

Kreiling 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). 

In the 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.

In the 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.

In 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.

 

Furthermore, 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).

 

However, 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.

The 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.

The 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.

In 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 classification.

 

An 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.

 

Skin 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.

The 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.

 

Skin sensitisation predictions using Toxtree and OECD Toolbox

The 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.

Structural 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.

In 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.

By 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.

 

Skin sensitisation studies in humans

Octanoic 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.

 

Conclusion

Taken 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.

 

References:

Anderson, SE. et al. (2009). Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components.Journal of Immuntoxicology 6(1):19 - 29.

Basketter, D.A. et al. (1998).Strategies for Identifying False Positive Responses in Predictive Skin Sensitization Tests. Food and Chemical Toxicology 36(4):327 – 33.

Basketter, 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.

Kreiling, 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 Toxicology 46:1896 - 1904.

 

Opdyke, D.L.J. (1981). Monographs on Fragrance Raw Materials. Fd Cosmet. Toxicol. Vol. 19:237-254.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

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.