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Justification for grouping of substances and read-across

In accordance with the specifications listed in Regulation (EC) No. 1907/2006 Annex XI, 1.5 Grouping of substances and read across, the similarity of category members has been shown to be justified based on the scope of variability and overlapping of composition, representative molecular structure, physico-chemical properties, tox-, ecotoxicological profiles and supporting Information by various validated QSAR methods. This information is given in further detail within the category justification for the grouping of chemicals and read-across (see IUCLID Section 13) for the dimerised fatty acids and its derivatives, and once more within the endpoint summary and discussion for Toxicokinetics.

For assessment of human health hazards of the category members, trends and similarities in toxicokinetic behaviour are most relevant. In particular, the molecular weight-dependent decrease in oral and dermal absorption and common metabolic pathways, which are explained by trends in molecular structure and common functional groups (monomers, dimers and trimers of similar long-chain fatty acids). This justifies the assumption that the toxicological profile of all category members is similar and effects or the lack of effects observed in toxicological studies of one ore more substances can also be expected and explained for the other substances in the category.

Therefore, in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, in order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of human health hazards. Thus where applicable, human health effects are predicted from adequate and reliable data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach).

All the available information from the substances within the category is taken into account for each endpoint to be assessed. Key studies are selected for assessment of the test substance and for read-across as to fulfil the requirements laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, i.e. in all cases the results are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3); cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3) if exposure duration is a relevant parameter; and adequate and reliable documentation of the applied method is provided.

 

Discussion

There are no data available on the genotoxic potential of fatty acids, C16-C18 and C18 unsaturated, branched and linear. However, there are reliable data for other members of the chemical category they belong to. Therefore, read-across was conducted based on a category approach.

The in vitro genetic toxicity of isooctadecanoic acid (CAS No. 30399-84-9) was investigated in a bacterial reverse mutation assay (Ames test) according to OECD Guideline 471 (adopted May 26, 1983) and complying with GLP. The test material was dissolved in 95% ethanol and the direct plate incorporation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations up to 3300 µg/plate. Cytotoxic effects were not observed in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9). The test material did not induce a dose-related increase in the number of revertant colonies both in the absence and in the presence of S-9 mix, in two independently repeated experiments.

It was concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay (van de Waart, 1996).

In support of these findings and following the category approach mentioned above, read-across from another member of the chemical category was also performed.

The in vitro genetic toxicity of fatty acids, C18-unsaturated, dimers (CAS No. 61788-89-4) was investigated in a bacterial reverse mutation assay (Ames test) according to EU Method B.13/14 and complying with GLP. The test material was dissolved in 95% ethanol and the direct plate incorporation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations up to 5000 µg/plate. Cytotoxic effects were not observed in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9) at test material concentrations up to 5000 µg/plate. The test material induced significant increases in revertant colonies in some strains, mostly at the higher concentration ranges and in the presence of S9. However, these results were not reproducible in further assays and the effects were not dose-dependent. The positive and negative controls included in the experiment showed the expected results. As the significant increases seen in revertant colonies were irreproducible, it was concluded that under the conditions of this study the test material did not induce mutations in Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98 with and without metabolic activation (Henderson, 1993).

Fatty acids, C18-unsaturated, dimers were also tested in another bacterial reverse mutation assay (Ames test) in accordance with OECD Guideline 471 and incompliance with GLP (Wollny, 2000, as cited in HPVIS – US EPA, 2005). Test material concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate were tested with and without metabolic activation (S9 mix) in S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level either in the presence or absence of metabolic activation. Thus, the test material was not considered to be mutagenic.

A study was performed to assess the ability of fatty acids, C18-unsaturated, dimers to induce chromosomal aberrations in human lymphocytes cultured in vitro in compliance with OECD Guideline 473 and GLP. Human lymphocytes from male blood donors, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test material both in the presence and absence of S-9 mix derived from Aroclor 1254-induced rat livers. Vehicle (DMSO) and positive control (ethylmethanesulphonate and cyclophosphamide) cultures were also prepared. After the appropriate treatment time cell division was arrested using colchicine, the cells harvested and slides prepared for metaphase analysis.

In order to assess the toxicity of fatty acids, C18-unsaturated, dimers to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test material and vehicle control. On the basis of these data concentrations were selected for metaphase analysis. The highest concentration was either at the limit of solubility (300 µg/mL) or at the limit of toxicity.

Two assays were performed: In the first assay, the mitotic index was reduced to 65% of the vehicle control value at 300 µg/mL (18 h harvest) in the absence of S-9 mix, and no decrease in its presence. Concentrations selected for metaphase analysis, with and without metabolic activation, were 75, 150 and 300 µg/mL. No statistically significant increases in the proportion of aberrant cells were observed at any concentration of test material analysed, in either the absence or presence of S-9 mix.

In the second assay, no significant reductions in mitotic index were observed at 300 µg/mL in the presence (18 and 32 h harvest) and in the absence of S-9 mix (18 h harvest). In the absence of S-9 mix (32 h harvest), 150 µg/mL caused a decrease in mitotic index to 63% of the vehicle control value. Concentrations selected for metaphase analysis, with and without metabolic activation (18 h harvest), were 75, 150 and 300 µg/mL. For the 32 h harvest, 150 µg/mL (without S-9 mix) and 300 µg/mL (with S-9 mix) were selected. No statistically significant increases in the proportion of aberrant cells were observed at any concentration of test material analysed, in either the absence or presence of S-9 mix. Both positive control compounds yielded the expected results in both assays.

It was concluded that the test material showed no evidence of clastogenic activity with and without metabolic activation in this in vitro cytogenetic test system (Akhurst, 1993).

Fatty acids, C18-unsaturated, dimers were also tested for mutagenic potential in another in vitro mammalian cell mutation assay following OECD Guideline 476 and in compliance with GLP. Mouse lymphoma L5178Y cells were treated with vehicle (DMSO) or the test material at end concentrations of up to 300 µg/mL in medium for 3 h, in two independent test with and without metabolic activation (S-9 mix from Aroclor 1254-induced rat livers). After an expression time of 48 h in growth medium, cells were incubated for 12 days with trifluorothymidine as selection agent for forward mutation at the thymidine kinase locus.

The test material was cytotoxic in both the absence and (more clearly) presence of S-9 mix (at 300 and 150 µg/mL, respectively).

A statistically significant increase in mutant frequency was observed at 250 µg/mL in one of the two tests without S-9 mix. This increase was small and not considered to be biologically significant. In the presence S-9 mix, no statistically significant increases in mutant frequency were observed in any of the two tests performed. The corresponding positive control substances (3-methylcholanthrene, with S-9 mix; ethylmethanesulphonate, without S-9 mix) induced highly significant increases in mutant frequency.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro gene mutation assay (Adams et al., 1993).

In addition, reviewed data on pure C18 fatty acids indicate that this substance class is not mutagenic (CIR, 1987). Thus, stearic acids yielded negative results in an Ames test performed with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538, with and without metabolic activation. In V79 Chinese hamster lung fibroblasts, oleic acid produced a mean number of sister chromatide exchanges per metaphase similar to that of the controls.


Short description of key information:
Based on read-across following a category approach, the available data indicate that fatty acids, C16-C18 and C18 unsaturated, branched and linear are not genotoxic.
Negative Ames test with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, with and without metabolic activation.
Negative results in mammalian chromosomal aberration test with cultured human lymphocytes.
Negative results in mammalian cell gene mutation tests using mouse lymphoma L5178Y cells, with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across following a category approach, the available information on the genotoxic potential of fatty acids, C16-C18 and C18 unsaturated, branched and linear is conclusive but not sufficient for classification.