Fatty acids, soya, conjugated

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 in peripheral blood lymphocytes after dietary supplementation with C18:2 fatty acid (linoleic acid). Due to the large number of available Ames tests of members of the fatty acids category only those which have been performed according or similarly to OECD Guideline 471 with constituents of fatty acids, soybean oil, conjugated, in particular the main constituents (>10%) C18:1 fatty acid (oleic acid) and C18:2 fatty acid (linoleic acid) were summarised and presented.

 

Genetic toxicity of oleic acid (CAS# 112-80-1) was examined inS. typhimuriumstrains TA 98, TA 100, TA 1535 and TA 1537, which were exposed to 0.1, 0.3, 1, 3.3 and 10 µg/plate without metabolic activation and to 3.3, 10, 33, 100 and 333 µg/plate with metabolic activation, respectively (NTP, 1981). Two different metabolic activation systems were used, one with 10% rat liver S9 and another one with 10% hamster liver S9. No genotoxicity was found for any strain at any concentration using the pre-incubation method. Cytotoxicity was detected starting at 3.3 µg oleic acid/plate for TA 98 and TA 100 without S9-mix, at 10 µg oleic acid/plate for TA 1537 without S9-mix and at 333 µg oleic acid/plate for all strains with S9-mix.

 

The mutagenicity of oleic acid was also analysed inS. typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and inE. coli WP2uvrAby Shimizu et al. (1985). The Ames test was carried out under the absence and presence of rat microsomal activation. Test concentrations of 1, 5, 10, 50, 100, 500, 1000 and 5000 µg oleic acid/plate for TA100, TA1535,E.coli WP2 uvrAand 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 µg oleic acid/plate for TA 1537 and TA 1538 were used in the preincubation method. No mutagenicity was noted in any of the strains. A decrease in revertants below 60% compared to the control plates was observed in strain TA 1537 starting at 5 µg/plate without S9-mix and at 100 µg/plate with S9-mix and at 100 µg/plate in TA 1535 without S9 mix. In strain TA 1538 and inE. coli WP2 uvrAcytotoxicity was determined at the highest concentrations tested.

 

Cotruvo (1978) reported, that 99 ppm oleic acid were not mutagenic in the preincubation/suspension or in the plate incorporation assay using S. typhimurium strains TA 98, TA 100, TA 1535, TA 1536, TA 1537 and TA 1538 with and without metabolic activation system.

 

Linoleic acid (CAS# 60-33-3) was tested for genetic toxicity inS. typhimuriumstrains TA 98, TA 100, TA 1535 and TA 1538 with and without metabolic activation (NTP, 1981). For metabolic activation cofactor supplemented post-mitochondrial fraction (S9 mix) were used, containing rat or hamster liver enzymes (S9) in a concentration of 10%. Concentrations of 0.1, 0.3, 1, 3.3 and 10 µg linoleic/plate without metabolic activation and concentrations of 3.3, 10, 33, 100 and 333 µg linoleic acid/plate with metabolic activation were tested in the preincubation method. No genotoxicity was noted in any of the tested strains. Slight toxicity at 10 µg/plate for all strains without S9-mix and at 333 µg/plate for strains TA 100, TA 1535 and TA 1537 with S9-mix was observed.

 

The mutagenicity of linoleic acid in bacteria was also tested at concentrations of 100, 500, 2500, 5000 and 10000 µg/plate with and without metabolic activation in theS. typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Seifried et al., 2006). Liver S9 homogenate was prepared form male Sprague-Dawley rats and Syrian golden hamsters, which had been injected with Aroclor 1254. Using the plate incorporation assay, no mutagenicity was noted in any of the strains. A decrease in revertants (< 60% compared to control) was observed in TA 100 at a concentration of 10000 µg linoleic acid/plate with and without S9 and in TA 1535 starting at a concentration of 5000 µg linoleic acid/plate without S9-mix. In strain TA1538 cytotoxicity was noted at a concentration of 500 µg linoleic acid/plate and above in tests performed with rat S9-mix and at a concentration of 5000 µg linoleic acid/plate and 10000 µg linoleic acid/plate in tests performed with hamster S9-mix.

 

Thus, based on these results linoleic acid and oleic acid are not mutagenic in bacteria. Since linoleic acid and oleic acid are main constituents of fatty acids, soybean oil, conjugated and all substances belong to the same category based on structural and toxicological properties, the same result can be expected for fatty acids, soybean oil, conjugated. Moreover, all available Ames tests of fatty acids within the category were negative. Thus, fatty acids, soybean oil, conjugated 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

Cotruvo, J.A. et al. (1978). INVESTIGATION OF MUTAGENIC EFFECTS OF PRODUCTS OF OZONATION REACTIONS IN WATER. Annals of the New York Academy of Sciences 298:124 – 40.

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.

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:
- Gene mutation in bacteria (Bacterial reverse mutation assay/Ames test; OECD 471): S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA: negative with and without metabolic activation; CAS# 112-80-1, C18:1 (NTP, 1981; Shimizu et al., 1985 ); CAS# 60-33-3 (NTP, 1981; Seifried et al., 2006 )
- 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.