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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the overall weight of evidence, the test substance is considered to be non-genotoxic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

An in vitro study was conducted to determine the genetic toxicity of the test substance according to OECD Guideline 471 and EU Method B.13/14 (mutagenicity - reverse mutation test using bacteria), in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, to negative or positive control substances for 48 ± 4h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 512 and 1600 µg/plate (absence and presence of S9-mix, respectively) in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Since the test substance was severely cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without  S9-mix according to the pre-incubation method. In this dose range finding study, the test substance was initially tested up to concentrations of 512 and 1600 µg/plate in the tester strain TA100 (absence and presence of S9-mix, respectively) and up to 5000 µg/plate in tester strain WP2uvrA. The test substance precipitated on the plates at dose levels of 512 μg/plate and upwards. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix and in WP2uvrA in the absence of S9-mix. In tester strain WP2uvrA in the presence of S9-mix, no toxicity was observed at any of the dose levels tested. In the second mutation experiment, the test substance was tested up to concentrations of 52 and 512 µg/plate (absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The test substance precipitated on the plates at dose levels of 512 μg/plate. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in the tester strain TA98. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was considered not mutagenic in in a bacterial reverse mutation test with and without metabolic activation (Verspeek-Rip, 2006).

Chromosomal aberration (CA) test:

An in vitro study was conducted to determine the genetic toxicity of the test substance, according to OECD Guideline 473 (chromosome aberration study in mammalian cells), in compliance with GLP. Two experiments were performed on cultured peripheral human lymphocytes. In the first cytogenetic assay, the test substance was tested up to 125 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The test substance precipitated in the culture medium at this dose level. The cytotoxicity of the test substance in the lymphocyte cultures was determined using the mitotic index. No cytotoxicity was observed in the 3 h exposed duplicate cultures, which were subsequently scored for chromosome aberrations. In the second cytogenetic assay, the test substance was again tested up to 125 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. The test substance precipitated in the culture medium at this dose level. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was not clastogenic in cultured peripheral human lymphocytes (Verbaan, 2016).

Mouse Lymphoma (MLA) test:

An in vitro study was conducted to determine the genetic toxicity of the test substance according to OECD Guideline 490 (mouse lymphoma assay), in compliance with GLP. Two experiments were performed on mouse lymphoma L5178Y cells at the thymidine-kinase locus (TK-locus). In the first experiment, the test substance was tested up to concentrations of 100 µg/mL in the absence and presence of S9-mix. The incubation time was 3 h. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level. In the second experiment, the test substance was again tested up to concentrations of 100 µg/mL in the absence of S9-mix. The incubation time was 24 h. No toxicity was observed at this dose level. The test substance precipitated in the culture medium at this dose level. Per culture 8.0E06 cells (10E06 cells/mL for 3 hour treatment) or 6.0E06 cells (1.25E05 cells/mL for 24 hour treatment) were used. For expression of the mutant phenotype, cells were cultured for 2 d after the treatment period. During this culture period at least 4.0E06 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). The determination of the mutant colonies and the calculation of the survival or viability were recorded as well. No severe toxicity was observed up to and including the highest tested dose level of 100 µg/mL. In the first mutation experiment (3-h treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. This result was confirmed in an independent experiment with modification in the duration of treatment. In the second mutation experiment (24 -h treatment), no significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. The mutation frequency found in the solvent control (ethanol) cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was not mutagenic in mouse lymphoma L5178Y cells (Verspeek-Rip, 2017).

Justification for classification or non-classification

Based on the overall weight of evidence, the test substance does not meet the criteria for classification for this endpoint according to CLP (Regulation 1272/2008/EC) criteria.