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In vitro

Three key studies are available to address the in vitro genetic toxicity of the test material. All were awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 dissolved in dimethyl sulfoxide 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 metabolic activation (rat liver S9-mix induced by Aroclor 1254). Experiment 1 was performed using the plate incorporation method and Experiment 2 was performed using the pre-incubation method.

A dose range finding study was performed as part of Experiment 1; the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the rest of the first mutation experiment, the test material was tested up to concentrations of 1600 μg/plate in the strains TA1535, TA1537 and TA98. The test material precipitated on the plates at the top dose of 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test material was tested up to concentrations of 1600 μg/plate in all tester strains in the pre-incubation assay. The test material precipitated on the plates at the top dose of 1600 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix at the highest tested concentration.

The test material 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.

 Acceptable responses were obtained for the negative and positive control materials, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

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 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. An additional experiment was performed with a 3-hour treatment period in the absence and presence of S9-mix.

The test material was dissolved in dimethyl sulfoxide; in the first experiment, the test material was tested up to concentrations of 45 and 100 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 11 and 13 % in the absence and presence of S9-mix, respectively.

Since not all acceptability criteria were met in the first experiment, an additional experiment was performed. In the second experiment, the test material was tested up to concentrations of 25 and 60 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 25 and 13 % in the absence and presence of S9-mix, respectively.

In the third experiment, the test material was tested up to concentrations of 30 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 23 %.

The spontaneous mutation frequencies in the solvent-treated control cultures were not all within the laboratory historical control data ranges, specifically the responses in first experiment in the absence of S9-mix (159 and 126 per 10⁶ survivors in the solvent control treated groups against the range 50 to 119 per 10⁶ survivors of the historical control data range). Since the repeat experiment was within historical range, it was considered to be valid.

Mutation frequencies in cultures treated with positive control chemicals were increased by 4.7- to 13-fold for MMS in the absence of S9-mix, and by 15-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control materials were within 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 and second experiment. This result was confirmed in a follow-up 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.

It is concluded that the test material is not mutagenic in the mouse lymphoma L5178Y test system under the conditions of this study.

A was conducted to assess the clastogenic potential of the test material in Chinese Hamster Ovary cells. The study was conducted in accordance with the standardised Japanese MHLW guidelines, Notification No.24 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare (JMHW), 1989 under GLP conditions.

The test material was evaluated in the chromosome aberration assay in the absence and presence of an Aroclor-induced S9-activation system in CHO cells. In the absence of S-9 6, 24 and 48 hour exposure times were used and in the presence of S9 activation, a 6 hour exposure period was used.

In the absence of metabolic activation, dose levels were as follows: 6 hour treatment, 18 hour recovery period: 0.015, 0.022, 0.024, 0.026 mg/mL; 24 hour treatment: 0.0098, 0.013, 0.017, 0.022 mg/mL; and 48 hour treatment: 0.010, 0.020, 0.025, 0.030 mg/mL

In the presence of metabolic activation, dose levels were as follows: 6 hour treatment, 18 hour recovery period: 0.026, 0.035, 0.047, 0.062, 0.083, 0.11, 0.15 mg/mL.

All cells were collected at either 24 or 48 hours after treatment initiation. Visible precipitate was observed in treatment medium at concentrations of ≥0.035 mg/mL in the S9-activated system.

Concentrations ≤0.026 mg/mL in the S9-activated systems were soluble in treatment medium. All concentrations tested in the 6, 24 and the 48 hour treatment groups of the non-activated system were soluble in the treatment medium. Toxicity, as measured by cell growth inhibition relative to the solvent control, was 64, 56 and 73 % at the highest dose levels evaluated for chromosome aberrations, 0.026, 0.022 and 0.030 mg/mL in the non-activated 6, 24, and 48 hour exposure groups, respectively. Toxicity was 67 % at the highest dose level evaluated for chromosome aberrations, 0.062 mg/mL, in the S9-activated group.

No statistically significant increases in structural or numerical chromosome aberrations relative to the solvent control were observed at any dose level in either the non-activated or S9-activated test systems (p>0.05, Fisher's exact test).

Under the conditions of this study, the test material was negative for structural and numerical chromosome aberrations using Chinese hamster ovary (CHO) cells.


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 studies were conducted in accordance with standardised guidelines under GLP conditions.

Short description of key information:
IN VITRO
- Non mutagenic with and without metabolic activation (Ames test)
- Non mutagenic with and without metabolic activation (mouse lymphoma assay)
- Non clastogenic with and without metabolic activation (chromosome aberration test)

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.