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There are reliable tests available investigating genetic toxicity in bacteria and mammalian cells in-vitro as well as an in-vivo genotoxicity study.

Mutagenicity in bacteria was assessed by two GLP-studies performed according to OECD 471 (Herbold, 2004; Herbold, 2006). In both studies the S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102; 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 and the bacteria were exposed to concentrations of 16, 50, 158, 500, 1581 and 5000 µg/plate in the plate incorporation assay in the absence and presence of metabolic activation by rat liver S9-mix in both studies. In the older study from 2004, a bacteriotoxic effect in TA102 was observed at 5000 µg/plate only in the first trial (Herbold, 2004). Therefore, this study was chosen as key study. In both studies, no precipitation was observed. The results of both studies corresponded well to each other and to the historical control data; in both studies the maximum numbers of revertants observed in the test substance-treated plates were comparable to those in the negative controls with and without metabolic activation in all strains tested. Appropriate positive controls were included into the study design, which gave the expected results.

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 (Herbold, 2004). The selection of the concentrations used for the main study was based on the results of a pre-tests. Based on the findings of cytotoxicity and precipitation, the following concentrations were chosen for the main test: The V79 cells were exposed for 4 h to 400, 800 and 1600 µg/mL without metabolic activation and to 500, 1000 and 2000 µg/mL with metabolic activation and harvested after 18 h. Additionally, cells exposed for 4 h to 1600 µg/mL without and to 2000 µg/mL with S9-mix were harvested after 30 h. At last, cells exposed for 18 h to concentrations of 400, 800 and 1600 µg/mL without S9-mix were harvested after 18 h. Precipitation was observed at 1600 µg/mL without S9 activation. Cytotoxicity was observed at 1600 µg/mL (4 h exposure and harvested after 30 h; 18 h exposure and harvest after 18 h) without metabolic activation. 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 according to OECD 476 under GLP-conditions (Herbold, 2006). In the pre-test Chinese hamster V79 cells were exposed for 5 h to test substance concentrations of 5, 10, 50, 100, 500, 1000, 1500 and 2000 µg/mL in the absence and presence of metabolic activation. Precipitation of the test substance in the culture medium was observed at 2000 µg/mL. No cytotoxicity was observed. Based on these results, the test substance was tested at the same concentrations in the main test, two trials were performed. A decrease in relative population growth was observed at precipitating concentrations of 2000 µg/mL. However, the test substance did not induce any statistically significant increases in mutant frequencies at any concentration tested. Therefore, the test substance was not mutagenic in mammalian cells in-vitro. The positive controls gave the expected results.

 

The genetic toxicity of the test substance in-vivo was tested in the micronucleus assay in mice, performed according to OECD 474 (Herbold, 2004). Five males per dose received 2 intraperitoneal injections 24 hours apart of 500, 1000 and 2000 mg/kg bw of the test substance. 24 h after the last application, the animals were sacrificed, and bone marrow smears were prepared. The slides were analysed microscopically, and the number of micronucleated polychromatic erythrocytes was determined. Clinical signs of toxicity were observed up to sacrifice after the last injection, demonstrating relevant systemic exposure of the animals to the test substance. One animal died due to acute intraperitoneal toxicity of 2000 mg/kg bw test substance. There was no biologically important or statistically significant variation in the number of micronucleated polychromatic erythrocytes noted for the test substance-treated groups. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with the test substance, being 2000: 1734 (1s=748) in the negative control, 2000: 2141 (1s=701) in the 500 mg/kg group, 2000: 3065 (1s=781) in the 1000 mg/kg group and 2000: 3018 (1s=1389) in the 2000 mg/kg group, respectively. This finding was assumed as a demonstration of relevant systemic exposure of the males to the test substance. However, the incidence of micronucleated cells was 2.4, 2.2, and 3.8 per 2000 polychromatic erythrocytes (PCE) in all treatment groups (500, 1000, and 2000 mg/kg bw) compared to 2.4 per 2000 PCE in the negative control group. These results gave no indication of a clastogenic effect of the test substance in male mice in-vivo after two injections of up to and including 2000 mg/kg bw. Cyclophosphamide (20 mg/kg bw), the positive control, had a clear clastogenic effect.

In conclusion, the test substance did not induce gene mutations in bacteria and mammalian cells in-vitro or clastogenicity both in-vitro and in-vivo.


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 TA102: negative with and without metabolic activation (according to OECD 471)
Chromosome aberration (in-vitro mammalian chromosome aberration test): negative with cultured V79 cells with and without metabolic activation (according to OECD 473).
Gene mutation (in-vitro mammalian cell gene mutation test): negative with cultured V79 cells with and without metabolic activation (according to OECD 476).

Genetic toxicity, in-vivo:
Chromosome aberration (micronucleus assay): negative (according to OECD 474; 3 concentrations (2 x 500 mg/kg bw, 2 x 1000 mg/kg bw, 2 x 2000 mg/kg bw) intraperitoneally injected to mice)

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.