5-amino-6-methyl-1,3-dihydrobenzoimidazol-2-one

Endpoint summary

Additional information

Bacterial Reverse Mutation Assay

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2UvrA in two independent experiments. The study was conducted according to OECD guidelines 471 and 472. The test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. In tester strain TA98, in the presence of S9-mix, the test substance induced 2.9 and 2.6-fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment, respectively. In the absence of S9-mix, the test substance did not induce the number of revertant colonies compared to the solvent control in neither experiment. The other bacterial strains, TA1535, TA1537, TA100 and WP2UvrA, showed negative responses over the entire dose range, i.e. no dose-related, twofold, increase in the number of revertants in two independently repeated experiments.

Based on the results of this study, it is concluded that the test substance is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. (NOTOX B.V., 1998)

in vitro Mammalian Cell Gene Mutation Test

This report describes the effects of the test substance on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells in the presence and absence of S9-mix according to OECD guideline 476.

The test substance was tested up to concentrations of 2000 and 2500 µg/mL in the absence of S9-mix in the first and second experiment, respectively. The test substance was tested up to concentrations of 2500 µg/mL in the presence of S9-mix in both experiments. At these dose levels no cytotoxicity was observed.

In the absence of S9 metabolic activation, the test substance induced 2.3-fold increases in the mutant frequency at the TK-locus in both experiments. However, these increases were less than three-fold and showed no clear dose response relationship. Therefore, this increase was considered not biologically relevant and the test substance is considered to be not mutagenic in the absence of S9-mix.

In the presence of S9 metabolic activation, no significant increase in the mutant frequency at the TK-locus was observed in neither experiment. Mutant frequencies induced by positive control chemicals were increased by 8-fold for EMS in both experiments, and by 16- and 12-fold for DMN, in the first and second experiment, respectively.

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. It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report. (NOTOX B.V., 1998)

in vivo Micronucleus Assay

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was conducted according to OECD guideline 474. The test item was formulated in PEG400, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000. and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. However, the bedding of the treated animals showed orange colouration, which indicated that the urine of these animals contained the test item, confirming the bioavailability of the test item.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay. (RCC-CCR, 2007)

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study in vivo.

Short description of key information:
A positive mutagenic response was seen in the Bacterial Reverse mutation assay. However, this result could not be confirmed by an in vitro study with mammalian cell culture and an in vivo micronucleus assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available mutagenic study data, the test substance does not need to be classified and labelled according to Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC (DSD).