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Administrative data

Key value for chemical safety assessment

Additional information

The following information was taken into account for any hazard / risk assessment:

The ability to induce mutations in bacterial strains (AMES, OECD TG 471)

The test material was tested in the Salmonella typhimuriurn reverse mutation assay with four histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) (Loveday, 1988). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Test substance was soluble in dimethyl sulfoxide, and it was tested up to concentrations of 5000 µg/plate.

Experiment 1 (without S9):

1) No biologically relevant increase in the number of revertants was observed at all dose levels tested up to the dose level of 5000 ug/plate in all strains in the absence of S9. However, severe cytotoxicity was observed in TA1537. Also, the positive control group in this strain failed to produce positive response.

2) The test was repeated at the same dose range (15, 50, 150, 500, 1500 and 5000 ug/plate), no mutagenic effects were found in all tested strains; cytotoxicity was observed in TA1537 strain.

3) TA1537 alone was tested at lower concentration range (0.5, 1.5 and 5 ug/plate) to avoid cytotoxicity. No mutagenic effect was observed in this test.

Experiment 2 (with S9):

1) No biologically relevant increase in the number of revertants was observed at all dose levels tested up to the dose level of 5000 ug/plate in all strains in the presence of S9, no cytotoxicity was observed in any test strains, but positive control group in this strain failed to produce positive response in TA1537.

2) The test was repeated at the same dose range (15, 50, 150, 500, 1500 and 5000 ug/plate) in the presence of S9, no mutagenic effects were found in all tested strains;

3) TA1537 alone was tested at concentrations of 15, 50, 150, 500, 1500 and 5000 ug/plate. No mutagenic effect was observed in this test.

Based on the results of this study it is concluded that the test substance is non-mutagenic in tester strains TA1535, TA1537, TA98, TA100 and E.Coli WP2, with/without metabolic activation.

Chromosome Aberration Assay in vitro (OECD TG 473)

The study was conducted according to a method that was designed to assess the potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chinese Hamster Ovary (CHO) cell line (Loveday, 1989). Duplicate cultures of CHO cells that had been treated with the test material in either the presence or absence of metabolic activation, were evaluated for induction of chromosome aberrations. Duplicate vehicle and positive controls were included in parallel in both experiments. The metabolic activation system was a liver S9 prepared from male rats induced with a IP injection of Arocolor 1254.

Four exposure conditions were used:

Experiment 1: the exposures were 10 h or 20 h without metabolic activation;

Experiment 2: cells were exposed to the test article and controls for 2 hour in serum free medium containing the 10x isocitrade cofactors and S9 microsome fractions. The test substance or control materials were removed after 10 or 20 hour continuous exposure, and the tissue cultures were allow to grow for 8 h or 18 h in medium supplemented with serum.

The vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for the CHO cell line. All of the positive control materials induced significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system.The test material did not induce any statistically significant increases in the frequency of cells with aberrations in any of the exposure groups. The dose levels of the test material were shown to be toxic to CHO cellsin vitroand toxicity was achieved in all exposure groups.

The test material was considered to be non-clastogenic to CHO cellsin vitro.

The ability to induce gene mutations in mammalian cells (tk+/-mouse lymphoma assay, OECD TG 476)

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK+/-, locus of the L5178Y mouse lymphoma cell line (Brown, 2011). The method used meets the requirements of the OECD (476) of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK+/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 30 µg/mL in the absence of metabolic activation and 15 to 50 µg/mL in the presence of metabolic activation. The dose range for Experiment 2 was 5 to 45 µg/mL in the absence of metabolic activation, and 1.25 to 40 µg/mL in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK+/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test

Conclusion

Negative results were from all the assays, and suggest that the test material is non-clastogenic and non-mutagenic.


Short description of key information:
The test material is non-clastogenic and non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No. 1272/2008) classification is not required for genotoxicity.