Fatty acids, C18-22

Endpoint summary

Additional information

Human health effects in regard to genetic toxicity 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. No genotoxic potential for fatty acids is expected since fatty acids are found in all living organisms where they are fulfilling fundamental physiological functions. The negative genetic toxicity for fatty acids is demonstrated by gene mutation tests in bacteria (Ames test) with members of the fatty acid category including short-, mid- and long-chain fatty acids as well as mixtures of fatty acids, in cytogenicity tests with C9 fatty acid (nonanoic acid), C18:1 fatty acid (oleic acid), C22 fatty acid (docosanoic acid) and fatty acids, tall oil and in gene mutation assays in mammalian cells with C10 fatty acid (decanoic acid) and with C18 unsaturated fatty acids (linoleic and linolenic acid). Further, one exposure related observation study in humans is included which analyses the oxidative DNA damage after dietary supplementation with C18:2 fatty acid (linoleic acid) in peripheral blood lymphocytes. Due to the large number of available Ames tests of members of the fatty acids category only those which have been performed with the main constituents of fatty acids, C18-22, in particular C18 fatty acid (stearic acid) and C22 fatty acid (docosanoic acid) were summarised and presented.

Gene mutation in bacteria

Stearic acid (CAS# 57-11-4) was tested for genetic toxicity in S. typhimurium strains TA 97, TA 98, TA 100, TA 104 and TA 1538 with and without metabolic activation (NTP, 1989). For metabolic activation, cofactor supplemented post-mitochondrial fraction (S9 mix) were used, containing rat or hamster liver enzymes (S9) in a concentration of 10% or 30%. Concentrations of 10, 33, 100, 333 and 1000 µg stearic acid/plate were used in all strains without metabolic activation and with 10% S9 and in strain TA 97 with 30% rat liver S9 in the S9-mix. In addition concentrations of 100, 333, 1000, 3333 and 10000 µg stearic acid/plate were tested in all strains with 30% rat or hamster liver S9 in S9-mix and in strains TA 97, TA 98, TA 100 and TA 1535 without metabolic activation. Using the preincubation method, no genotoxicity was noted in any of the tested strains. Precipitation was observed at concentrations of 1000 µg/plate and above.

In another Ames test, Crebelli et al. (1985) tested the in vitro mutagenicity of stearic acid in the plate incorporation assay with S. typhimurium strains TA 97, TA 98, TA 100, TA 104 and TA 1538 in a concentration range of 40 – 1000 µg/plate with and without metabolic activation. Stearic acid did not show mutagenic activity.

Stearic acid (50 µg) was subjected to the spot test, a qualitative form of the Ames test, inS. typhimurium strainsTA 1535, TA 1537, TA 98 and TA 100 and TA 1538 with and without metabolic activation (Blevins and Taylor, 1982). There was no significant increase in the number of revertants compared to the controls and to other cosmetic ingredients which were tested in parallel.

Genetic toxicity of docosanoic acid (CAS# 112-85-6) in bacteria was evaluated in a study performed according to GLP and OECD Guideline 471 and 472 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (Nakajima, 2002). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were treated with docosanoic acid dissolved in DMSO at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation by S9 mix from livers of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone. No increase in revertants compared to the vehicle control was observed in any of the strains at all test concentrations. No toxicity was noted up to a concentration of 5000 µg/plate.

The same result was found in another study, where S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested with docosanoic acid (CAS# 112-85-6) concentrations of 4, 20, 100, 500, 1250 µg/plate with and without metabolic activation by S9 mix (Gloxhuber and Wallat, 1981).

Thus, based on these results stearic acid and docosanoic acid are not mutagenic in bacteria. Since stearic acid and docosanoic acid are main constituents of fatty acids, C18-22 and all substances belong to the same category based on structural and toxicological properties, the same result can be expected for fatty acids, C18-22. Moreover, all available Ames tests of fatty acids within the category were negative. Thus, fatty acids including fatty acids, C18-22 are considered to be not mutagenic in bacteria.

 

Cytogenicity in mammalian cells

Nonanoic cid (CAS# 112-05-1) was tested for its ability to induce chromosomal aberrations in cultured human peripheral lymphocytes in a study conducted according to OECD Guideline 473 and under GLP conditions (Meerts, 2001). Following a range finding pre-test, two independent experiments were conducted, both with and without metabolic activation (S9 mix; contained 9000 g supernatant from Aroclor 1254-induced male Wistar rat liver). The test concentrations in the definitive test ranged from 100 to 750 µg/mL. Precipitation was seen at 520 and 750 µg/mL. The pH at the highest non-precipitating dose level of 480 µg/mL was lowered to 7.34, compared to pH=7.48 of the solvent control. A mitotic index below 50% of the control indicated cytotoxicity at 750 µg/mL in the first experiment and at 240 µg/mL and above in the second.

A statistically significantly increased number of cells with chromosome aberrations was only seen at the toxic concentration of 750 µg/mL (mitotic index 38%, with and without metabolic activation), which was considered to be not biologically relevant. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

Therefore, it was concluded thatnonanoic acid did not induce chromosomal aberrations and was not clastogenic in human lymphocytes with and without metabolic activation.

 

A further in vitro mammalian chromosome aberration test was conducted with docosanoic acid (CAS# 112-85-6) in accordance with GLP and OECD Guideline 473 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (Nakajima, 2002). Properly maintained Chinese hamster lung (CHL) cells were treated with docosanoic acid dissolved in 1% carboxymethylcellulose sodium at concentrations of 875, 1750 and 3500 µg/mL for 6 hours with and without metabolic activation by S9-mix prepared from phenobarbital- and 5,6-benzoflavone-induced rat livers. The highest test concentration of 3500 µg/mL reflect 0.01 M of the test substance as required in the in OECD Guideline 473. In addition, the cells were incubated with 350, 700, 1400, 2800 µg/mL without metabolic activation for 24 hours and with 288, 575, 1150 and 2300 µg/mL without metabolic activation for 48 hours, respectively. The highest concentration of the test item used was set to the maximum one showing no apparent cytotoxic effects during continuous treatment. No increase in chromosomal aberrations nor polyploidy were observed up to the maximum concentration under short-term and continuous treatment with and without metabolic activation.The positive controls included during short-term and continuous exposure showed the expected results and thus verified the sensitivity of the assay.

 

Fatty acids, tall oil (CAS# 61790-12-3), which consists predominantly of C18 unsaturated and saturated fatty acids was tested for clastogenic activity in a chromosome aberration test according to OECD Guideline 473 (Murie, 2001).Chinese hamster ovary (CHO) cells were incubated with S9 mix at concentrations of 5, 10 and 20 µg fatty acids, tall oil/mL and without S9 mix at concentrations of 39, 78 and 156 µg fatty acids, tall oil/mL for 6 hours. Cells were harvested at 24 hours post-treatment. Chromosome aberrations were induced at cytoxic concentrations of 20 µg/mL with S9 mix and 156µg/mL without S9 mix.At these concentrations the cell count was reduced to ≤17% of the mean vehicle control values and there was consistent evidence of changes to cell morphology at 156 µg/mL without S9 mix.No chromosomal aberrations were observed at concentrations that were not cytotoxic.The increase in aberrant cells at the overtly toxic concentrations is considered as artefact and to be not biologically relevant. The positive controls substances cyclophosphamide and methyl methanesulphonate significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. Therefore, fatty acids, tall oil is considered not to be clastogenic.

 

The following study also indicates, that fatty acids do not have a genotoxic potential although not performed according to current guidelines.

 

Oleic acid (CAS# 112-80-1) was tested for the induction of sister chromatid exchanges in Indian muntjac fibroblasts (Higgins et al., 1999). The cells were incubated with 50 µM oleic acid in ethanol (equivalent to 14.1 µg/mL) for 24 h without metabolic activation. The cells were washed free of substance and cultured in the presence of BrdU for 48 h. Colcemid was added 3 h prior to harvesting. Chromosome preparations were made and stained with fluorescent plus Giemsa. The frequency of SCE was scored as the number of exchanges in 20 metaphases per slide and expressed as the number of SCEs/chromosome. Oleic acid did not induce increases in SCE frequencies above control levels and was therefore not considered genotoxic. No cytotoxicity was observed and the positive controls included showed the expected results.

 

In summary, no structural chromosomal aberrations were observed at non-cytotoxic concentrations for different members of the category and consequently fatty acids are considered not to be clastogenic.

 

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed with decanoic acid (CAS# 334-48-5) under GLP according to OECD Guideline 476 (Trenz, 2010). In two independent experiments, mouse lymphoma L5178Y cells were treated with decanoic acid at concentrations up to 1.18 mM without metabolic and up to 1.54 mM with metabolic activation by phenobarbital and beta-naphthoflavone-induced rat liver S9-mix, respectively. The exposure duration was 4 hours and 24 hours in experiments without S9 mix and 4 hours in the experiments with S9 mix. The treatment of cells in all experiments was followed by an expression period of 2 days and a selection period of 11-14 days in the presence of trifluorothymidine. Although cytotoxicity was observed at the highest concentrations tested, all mutant values were found to be within the range of the historical control data of the test facility, so that decanoic acid was regarded not to be mutagenic. In addition, colony sizing was performed for the highest concentrations used to detect potential clastogenic effects and/or chromosomal aberrations. As result, decanoic acid was found not to be clastogenic at all dose groups tested. The positive controls caused a pronounced increase in the mutation frequency demonstrating the sensitivity of the test system.

 

Another mouse lymphoma TK^+/-assay was performed with linoleic acid (CAS# 60-33-3) and linolenic acid (CAS# 463-40-1) similar to OECD Guideline 476 (Seifried et al., 2006). The applied test concentrations were very low and covered only a small dose range due to the insolubility of the test substances. Mouse lymphoma L5178Y cells were exposed for 4 hours to linoleic acid in a concentration range of 0.005 - 0.024 µL/mLwithout metabolic activation and to linoleic concentrations of 0.01 - 0.006 µL/mL with metabolic activation. Linolenic acid was tested in a concentration range of 0.021 - 0.025 µL/mL without S9 mix and in a range of 0.01 - 0.041 µL/mL with S9 mix. 2 days after treatment, treated cells were plated in soft agar medium containing TFT for 10 – 12 days. Cytotoxicity was observed for linoleic acid at the highest tested concentration with S9 mix (0.006 µL/mL). The relative total growth for linolenic aicd was decreased below 50% starting at 0.024 µL/mL without S9 mix and at 0.041 µL/mL with S9 mix. No increase in mutation frequency was observed for linoleic acid and linolenic acid compared to the control. Therefore, both C18 unsaturated fatty acids were considered not to be mutagenic in mammalian cells.

In summary, no gene mutation in mammalian cells was detected for different members of the category and consequently fatty acids are considered to be not genotoxic in vitro.

 

Investigations in humans

De Kok et al. (2003) analysed the oxidative DNA damage of linoleic acid after human dietary supplementation. Female volunteers (10/group), aged between 18 and 25 years, received either a high amount of linoleic acid (15 g linoleic acid/day), an intermediate amount of linoleic acid (7.5 g linoleic acid/day + 7.5 g palmitic acid/day) or a suspension containing only palmitic acid (15 g palmitic acid/day) for a period of 6 weeks. The average plasma linoleic concentration was significantly increased after two weeks and persisted until the end of the study in the two linoleic acid supplemented groups. No significant increase in oxidative DNA damage, measured as relative amount of 8-oxodG in DNA from peripheral lymphocytes, was noted in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05) comparing the average level of oxidative DNA damage before and after supplementation. Moreover, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group (23% decrease) were not significant. Additionally, no depletion of plasma antioxidants (alpha-tocopherol, retinol and beta-carotene) or total antioxidant status (TEAC) and no increase of plasma malondialdehyde, an important end product of lipid peroxidation were observed after the linoleic acid supplementation. Thus, based on this study, there is no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid.

 

Conclusion

Taking all the results together, the negative results of the available study data with different members of the fatty acids category do not provide any evidence that fatty acids are mutagenic or cytogenic as expected based on their physiological function within the body.

References

Blevins, R.D. and Taylor, D.E. (1982). MUTAGENICITY SCREENING OF TWENTY-FIVE COSMETIC INGREDIENTS WITH THE SALMONELLA/MICROSOME TEST. Journal of environmental science and health A17/502/51:217 – 239.

Crebelli, R. et al. (1985). Mutagenicity studies in a tyre plant: in vitro activity of workers' urinary concentrates and raw materials. British Journal of Industrial Medicine 42:481-487.

de Kok, T.M.C.M. et al.(2003). Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid. Food and Chemical Toxicology 41:351-358.

Gloxhuber and Wallat (1981). Edenor C 22 R: Testing for mutagenic activity in the Ames test. Testing laboratory: Henkel KGaA, Düsseldorf, Germany. Report no.: 263. Owner company: Henkel KGaA, Düsseldorf, Germany. Company study no.: TBD810106. Report date: 1981-12-04. Higgins, S. et al. (1999). Effects of oleic acid, docosahexanoic acid and eicosapentaenoic acid on background and genotoxin-induced frequencies of SCEs in Indian muntjac fibroblasts. Mutagenesis 14(3):335 – 338.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across based on a category approach. No study was selected, since all available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source substances and target substance and overall quality assessment (refer to endpoint discussion for further details).

Short description of key information:
in vitro:
- Gene mutation in bacteria (Bacterial reverse mutation assay/Ames test; OECD 471/OECD472): S. typhimurium TA 97, TA 98, TA 100, TA 104, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA: negative with and without metabolic activation; CAS# 57-11-4, C18 (NTP, 2005); CAS# 112-85-6, C22 (Nakajima, 2002)
- Chromosome aberration (OECD 473): negative with and without metabolic activation; CAS# 112-05-0, C9 (Meerts, 2001); CAS# 112-85-6, C22 (Nakajima, 2002); CAS# 61790-12-3, fatty acids, tall oil (Murie, 2001)
- Gene mutation in mammalian cells (TK locus; OECD 476): negative with and without metabolic activation; CAS# 334-48-5, C10 (Trenz, 2010)

No properties for genetic toxicity were observed for members of the fatty acids category.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All available data on genetic toxicity of the members of the fatty acids category do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.