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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test (OECD TG 471)


The substance is negative in the In vitro micronucleus test (OECD TG 473)


The substance is negative in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay (OECD TG 476)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames
The ability of the test substance to induce mutations was investigated in the Bacterial Reverse Mutation Assay (Ames, OECD TG 471, GLP) by using 4 histidine-requiring strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537), and a tryptophan-requiring strain of Escherichia coli (WP2 uvrA) with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The study procedures described in this reverse mutation assay were equivalent to OECD guideline 471. Test concentrations used in the main test were as follows: - S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate; + S9 mix: 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate. The mutagenicity of the test compound was judged negative because the number of revertant colonies in the test compound treatment groups was less than twice that of each negative control in all test strains. The number of revertant colonies in the positive control groups were more than twice that of negative control groups. The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory, indicating that the test can be considered valid. It is concluded that the test substance has no ability to induce mutations in bacteria under the present test conditions. 


CA
The test substance was examined for its genotoxicity in the micronucleus test with human lymphocytes in vitro (OECD TG 473) in the presence and absence of an exogenous metabolizing system containing S9-fractions of Arochlor 1254 induced rats, and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses as indicated by the nuclear division index (NDI). The doses used were as follows: human peripheral lymphocytes ± S9: 0.06, 0.61, 6.1, 61, 122, and 242.5 µM, and human hepatoma cell line Hep G2: 0.1, 1.2, 12.2, 122, 242.5, and 485 µM. The nuclear division index (NDI) was calculated as NDI = MI + 2x MII +3 x (MIII+MIV)/500 cells, where MI–MIV represent the number of lymphocytes with 1 to 4 nuclei. Positive controls used in the human peripheral lymphocyte cultures were: -S9 mix: Mitomycin C and +S9 mix: Cyclophosphamide; in the human hepatoma cell line Hep G2: Cyclophosphamide. DMSO was used as solvent control. The test substance did not induce a significant increase in the frequency of micronuclei in both human lymphocytes and human hepatocytes. Positive control response indicated that the test was valid. It is concluded that the substance is not cytogenic in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.


MLA
The substance was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+/- mouse lymphoma cells in vitro (OECD TG 476, GLP). The concentrations of substance tested with and without S-9 activation in the Range Finding Test were 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 ug/mL. Relative Suspension Growth (RSG, i.e., growth in culture after treatment compared to the growth in culture of the corresponding solvent control cultures) was used to measure toxicity. The RSG for cultures treated with test item without activation indicated that test item was toxic at 50 ug/mL and above. Cultures treated with 50 ug/mL and above had 0% RSG. The cultures treated with 10 ug/mL had 89% RSG. The RSG for cultures treated with test substance with S-9 activation indicated that the substance was completely toxic, i.e., 0% RSG, at 5.0 µg/mL and above. The cultures treated with 0.1, 0.5, and 1.0 µg/mL had RSG values of 60%, 13% and 15%, resp.


The Main Mutation Assay was performed using a 4-hour treatment period at test article concentrations ranging from 5.0 to 100 ug/mL without activation and from 0.01 to 50 ug/mL with S-9 activation. Cultures were selected for cloning for mutant selection based on their RSG. All of the cloned cultures, both with and without activation, had Mutant Frequencies (MF = mutants per 10*6 viable cells cloned) that were similar to the average MF of their concurrent solvent control cultures. The Relative Total Growth (RTG = the combination of suspension growth and clonal growth compared to that of the corresponding solvent control cultures) for the cloned cultures ranged from 12% to 112% for cultures treated without activation and from 32% to 167% for cultures treated in conjunction with exogenous activation. Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a Repeat Assay with a 4-hour exposure period with activation was conducted. Cultures were treated with concentrations ranging from 10 to 155 ug/mL. The results of the Repeat for the S-9 activation portion of the Definitive Mutation Assay also showed that the treated cultures all had MFs that were similar to the average MF of the solvent control cultures. The RTG for these cultures ranged from 7% to 28%. Under the test conditions, the results of the Definitive Mutation Assay are considered negative. The Confirmatory Mutation Assay was conducted without activation with a 24-hour exposure period. Cultures were treated with concentrations ranging from 0.5 to 55 µg/mL. The cultures treated with 10 to 55 ug/mL were cloned for mutant selection. All of the cultures had MFs that were similar to the average MF of the solvent controls. The RTG for the cloned cultures ranged from 5% to 67%. Under the test conditions, the results of the Main and Confirmatory Mutation Assays are considered negative and it is therefore concluded that the test substance is negative in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay.

Justification for classification or non-classification

Based on the results of the genemutations in bacterial cells (Ames test), cytogenicity information (In vitro micronucleus test) and the genemutations in mammalian cells (L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay), the substance is not genotoxic and therefore does not have to be classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).