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Additional information

In vitro, Ames test:

In a GLP compliant Ames test, 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 (RCC 2003). The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. A minor toxic effect, evident as a reduction in the number of revendants was only observed in strain TA 1535 at 2500 µg/plate in the presence of metabolic activation in experiment II. No substantial increase in revertant colony numbers of any of the Salmonella typhimurium strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The Escherichia coli strain showed an increase in revertant colony numbers in the preincubation test. In the first pre-incubation test the threshold of two was exceeded at 5000 µg/plate with and without metabolic activation. In the confirmatory experiment (pre-incubation test) the threshold was exceeded at 5000 µg/plate only without metabolic activation but there was also tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and others in the genome of the Escherichia coli strain WP2 uvrA used.

In vitro, chromosome abberation test:

In a GLP compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells (in vitro), were exposed to the test substance, with and without metabolic activation by S9 mix (RCC 2003). Two independent experiments were performed. The exposure period was 4 hrs 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, except for the test concentration 500 µg/mL with metabolic activation where 200 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data. In both experiments, clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Clear toxic effects indicated by reduced cell numbers of below 50% of control were observed in both experimental parts at the highest evaluated concentrations. In contrary no clearly reduced mitotic indices were observed at the test item concentrations evaluated. But after treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix extremely high mitotic indices were observed. In the absence and presence of S9 mix, biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item at concentrations showing clear reduced cell numbers. No increase in the frequencies of polypoid metaphases were found after treatment with the test item as compared to the frequencies of the controls. In conclusion, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and in the 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 2003). 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 4 hours. 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 item 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 vitro, UDS:

In a GLP-compliant unscheduled DNA synthesis assay, performed according to OECD guideline 482, the potential of the test substance to induce DNA repair synthesis in primary hepatocytes of rats in vitro was investigated (RCC 2004). The test was performed in two independent experiments, using identical procedures. The freshly isolated hepatocytes were exposed to the test item for 18 h in the presence of 3HTdR (methyl-3H-thymidine). The uptake of radioactivity was determined by autoradiography. For each concentration, including the controls, 100 cells were evaluated. The following concentrations were evaluated in both main experiments: 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49 µg/mL. The concentration ranges for the main experiments were estimated by pre-experiments for toxicity. In the two independent experiments, after treatment with the test substance, no reproducible concentration dependent increase in the number of nuclear and net grain counts was observed up to the highest concentration evaluated. In conclusion, it can be stated that during the described study and under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes used.

In vivo: micronucleus test:

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 2003). 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 substance, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control indicating that the test substance has no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. Therefore, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei in the bone marrow cells of the mouse.


Short description of key information:
The test substance is considered to be mutagenic in the in vitro chromosomal aberration test and in the Ames test. However, the in vitro gene mutation assay, the in vitro unscheduled DNA synthesis assay, and the in vivo erythrocyte micronucleus test did not show any genetic toxicity.

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