Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro: Reverse mutation assay:

In a GLP compliant reverse mutation assay, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA,were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation (RCC 2006). The plates incubated with the test item showed normal background growth up to5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects occurred in the testgroups with and without metabolic activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 98 (without metabolic activation) and TA 100 with and without metabolic activation in both experiments. In experiment I, the required threshold of two times the number of the corresponding solvent control could not be reached. However, the number of colonies was above the laboratory's historical control range in strains TA 98 and TA 100 from 1000 up to 5000 µg/plate without metabolic activation and in strain TA 100 at 2500 and 5000 µg/plate with metabolic activation. Based on these equivocal results, the more sensitive pre-incubation design was chosen for experiment II. Again, a moderate but dose dependent increase in revertant colony numbers was observed in strain TA 98 without S9 mix and in strain TA 100 with S9 mix. The threshold was exceeded in strain TA 98 at 5000 µg/plate without S9 mix (factor 2.3). In strain TA 100, the required threshold was not quite reached but a dose dependent response was observed in the presence of metabolic activation. Therefore, a possible mutagenic potential of the test item cannot be excluded. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains TA 98 and TA 100.

In vitro: Chromosome aberration test:

In a GLP compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix (RCC 2006). The exposure period was 4 hours with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. Dose selection of the main experiments was based on a pretest. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated

concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, dose-related significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

In vitro: Gene mutation assay:

In a GLP-compliant mammalian cell gene mutation test, performed according to OECD guideline 476, V79 cells of the Chinese hamster were exposed to the test substance with and without metabolic activation to investigate the potential of the test substance to induce gene mutations at the HPRT locus (RCC 2006). The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

In vivo: Chromosome aberration:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period (RCC 2007). Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. 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 urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. 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. In conclusion, it can be stated that under the experimental conditions reported, the test item 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.


Short description of key information:
The test substance is considered to be mutagenic in the reverse mutation assay and in the in vitro chromosome aberration. However, the test substance was not mutagenic in the in vitro gene mutation assay and the in vivo micronucleus test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008