Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro

Three key studies and one supporting study are available to address the in vitro genetic toxicity of the test material.

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). To obtain more information about the possible mutagenicity of the test material, additional experiments were performed with the tester strain TA98 in the absence of S9-mix.

In the first mutation assay, the test material was tested up to the concentration of 5000 μg/plate in DMSO in the absence and presence of 5 % (v/v) S9-mix. The test material precipitated heavily on the plates at the dose level of 5000 μg/plate and due to this heavy precipitate, the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 and TA100 in the presence of S9-mix. In tester strain TA98, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix. This increase was observed at two intermediate dose levels (52 and 164 μg/plate). In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material.

In the second mutation assay, the test material was tested up to the concentration of 2800 μg/plate in the absence of S9-mix and up to 5000 μg/plate in the presence of 10 % (v/v) S9-mix. The test material precipitated on the plates at dose levels of 2800 μg/plate and above. In the tester strain TA1537 in the absence of S9-mix, precipitate was already observed at the concentration of 1600 μg/plate. Due to the heavy precipitate on the plates at the test material concentration of 5000 μg/plate, the number of revertants at this dose level could not be determined. Cytotoxicity was observed in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in TA1537 in the presence of S9-mix. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

To verify the mutagenic response observed in the first mutation experiment in the tester strain TA98 in the absence of S9-mix, two additional experiments (experiments 3 and 4) were performed. The test material was tested up to the concentration of 5000 μg/plate in the absence of S9-mix. The test material precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in the third mutation experiment at dose levels of 164 μg/plate and above and in the fourth mutation experiment at dose levels of 512 μg/plate and above. No increase in the number of revertants was observed upon treatment with the test material under all conditions tested

In the tester strain TA98 in the absence of S9-mix in the first experiment, the test material induced an up to 4.9-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was observed only at two intermediate dose levels (52 and 164 μg/plate). Furthermore, this increase could not be repeated in three repeat experiments. Therefore, this increase is considered to be an incidental finding and not biologically relevant.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The ability of the test material to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted in accordance with the standardised guidelines OECD 473 and EU Method B.10 under GLP conditions.

The study investigates the effect of the test material on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of the test material was tested in two independent experiments.

Experiments were carried out in duplicate and concurrent solvent (DMSO) and positive controls took place.

In the first cytogenetic assay, the test material was tested up to 164 μg/mL for a 3 hour exposure time with a 24 hour fixation time in the absence and presence of 1.8 % (v/v) S9-fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to 50 μg/mL for a 24 hour continuous exposure time with a 24 hour fixation time and up to 30 μg/mL for a 48 hour continuous exposure time with a 48 hour fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test material 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 material on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that the test material does not disturb cell cycle progression under the experimental conditions described in this report.

It was noted that the test material increased the number of polyploid cells at the highest tested concentration of 164 μg/mL and in the presence of S9-mix only. This might indicate that it has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. However it is generally considered that in this sort of assay, polyploidy should only be considered to be significant if it occurs at levels relevant to human exposure.

Under the conditions of this study the test material is not clastogenic in human lymphocytes with and without metabolic activation. The test material may have the potential to disturb mitotic processes.

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The study was conducted in accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions.

The study investigated the effects of the test material on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period (rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone). Concurrent solvent (DMSO) and positive controls took place.

In the first experiment, the test material was tested up to concentrations of 37.5 and 80 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 13 and 24 % in the absence and presence of S9-mix, respectively.

In the second experiment, the test material was tested up to a concentration of 30 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11 %.

The spontaneous mutation frequencies in the solvent-treated control cultures were within the historical control data range and fulfilled the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 9.4- and 8.4-fold for methyl methanesulfonate in the absence of S9-mix, and by 19-fold for cyclophosphamide in the presence of S9-mix. In addition the observed mutation frequencies of the positive control materials fulfilled the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

In the GLP supporting study conducted according to the standardised guidelines OECD 471 (dated 1983) and EU Method B.13/14, the test material (approximately 65 % active substance, 35 % xylene) was assessed for mutagenicity in a bacterial reverse mutation assay (Ames test).

The Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were tested in two independent plate incorporation assays, with and without S9, at concentrations of 0, 5, 15.8, 50, 158 or 500 µg/plate of the test material and plated in triplicate for enumeration of the revertant colony forming units. This test system does not have the ability to detect certain oxidising or cross-linking mutagens. Plates incorporating the appropriate positive controls were used to validate the assay. Cytotoxicity was determined by thinning of the background lawn. The test substance showed no mutagenic activity at any of the concentrations tested; cytotoxicity was evident at 500 µg/plate. The positive control substances all showed mutagenic activity, demonstrating that the test was valid.

In conclusion, the test material showed no potential mutagenicity when tested at concentrations of up to 500 µg/plate in the Ames test with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 (with and without S9).


Justification for selection of genetic toxicity endpoint
No single key study was selected on the basis that the available studies all address different aspects of genetic toxicity and the data should be considered as a whole. All three key studies were conducted in accordance with standardised guidelines under GLP conditions and were awarded a reliability score of 1 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

Short description of key information:
IN VITRO
- Non mutagenic with and without metabolic activation (Ames test); OECD 471 and EU Method B.13/14
- Non clastogenic with and without metabolic activation (chromosome aberration test); OECD 473 and EU Method B.10
- Non mutagenic with and without metabolic activation (mouse lymphoma assay); OECD 476 and EU Method B.17

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.