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Mutagenicity in bacteria was assessed in a GLP-study performed according to OECD 471 (Thompson, 2001). S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvr A were tested. A plate incorporation assay served as range-finder for the determination of the test concentrations and was considered as a pre-test for toxicity. The bacteria were exposed to concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the main test (plate incorporation assay) in the absence and presence of metabolic activation by rat liver S9-mix. Neither cytotoxicity nor precipitation was observed. Appropriate positive controls were included into the study design, which gave the expected results. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

The clastogenic potential of the test substance in-vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD 473 under GLP-conditions (Wright, 2002). The selection of the concentrations used for the main study was based on the results of a pre-test. Based on the findings of cytotoxicity and precipitation, the following concentrations were chosen for the main test: human lymphocytes were exposed for 4 h to 209, 418, 836 and 1672 µg/mL with and without metabolic activation and harvested after 20 hours. In this experiment, a slight dose-related inhibition of mitotic index was observed in the with-metabolic activation group treated only and that approximately 40% mitotic inhibition was achieved at 836 and 1672 µg/ml. During continuous treatment, cells were exposed for 24 h to 104.5, 209, 418, 627, 836 and 1672 µg/mL without and to 104.5, 209, 418, 836 and 1672 µg/mL with S9-mix for 4 h. A dose-related inhibition of mitotic index was observed in the 24-hour continuous exposure case, in the absence of S9. The mitotic index at 836 and 1672 µg/ml was greater than 50% at these dose levels and was in fact at the maximum acceptable level of approximately 80% mitotic inhibition. Therefore, the intermediate dose level of 627 µg/mL was included in the doses selected for evaluation. Precipitation was not observed. There were no biologically and statistically significant increases in numbers of metaphases with aberrations at any exposure duration and at any total culture time, irrespective of metabolic activation. The positive controls resulted in clear and statistically significant increases in metaphases with aberrations.

The mutagenic potential of the test substance in mammalian cells in-vitro was assessed by a HPRT-assay equivalent to OECD 476 under GLP-conditions (Robert and Young, 1990). In the pre-test, Chinese hamster ovary cells (CHO) were exposed for 4 h to test substance concentrations of 7.8 to 4000 µg/mL in the absence and presence of metabolic activation. No cytotoxicity was observed. Based on these results, the test substance was tested at 50, 100, 1000, 2000, 3000, and 4000 µg/mL in the main test with and without metabolic activation for an exposure period of 4 hours. No dose-related toxicity to CHO cells in culture as measured by either relative clonal survival or relative population growth was observed. In the assay without metabolic activation, three of the six cultures had mutant frequencies that were significantly elevated over the mutant frequencies of the concurrent vehicle control cultures. The mutant frequencies were within the range of acceptable background mutant frequencies and were consistent with normal assay variation. Therefore, the test substance was considered as negative for inducing forward mutations at the HPRT locus in CHO cells in the absence of S9 metabolic activation under the conditions of testing. In the trial with metabolic activation, no treated culture had a mutant frequency that was significantly elevated over the average background mutant frequency of the control. Therefore, the test substance was considered not mutagenic in mammalian cells in-vitro. The positive controls gave the expected results.


Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies were negative.

Short description of key information:
Genetic toxicity, in-vitro:
Gene mutation (Bacterial reverse mutation assay/Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A: negative with and without metabolic activation (according to OECD 471)
Chromosome aberration (in-vitro mammalian chromosome aberration test): negative with cultured human lymphocytes with and without metabolic activation (according to OECD 473).
Gene mutation (in-vitro mammalian cell gene mutation test): negative with cultured CHO cells with and without metabolic activation (equivalent to OECD 476).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance 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.