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Key value for chemical safety assessment

Additional information

Genetic toxicity in vitro, Ames test:

In two independent GLP compliant Ames test studies, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) were used to test the mutagenic potential of the test substance (20, 80, 320, 1280 and 5120 µg/plate), both with and without metabolic activation system (BATTELLE 1987). The test substance did not show any mutagenic activity in any strain.

Genetic toxicity in vitro, Ames test:

In an Ames test study, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) were used to the test the mutagenic potential of the test substance (20, 80, 320, 1280 and 5120 µg/plate), both with and without metabolic activation system (BATTELLE 1986). The test substance did not show any mutagenic activity in any strain.

Genetic toxicity in vitro, Chromosome aberration test:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 743, V79 cells of the Chinese Hamster were exposed to the test substance with and without metabolic activation (CCR 1987). Different exposure times (up to 28 hours) and dosages (up to 3.00 mg/mL) were chosen in this study.The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells with 3.00 mg/mL reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced with the highest concentrations in the presence and absence of S9 mix. There were relevant enhancements of cells with structural aberrations after treatment with the test article at fixation intervals 7 h and 28 h with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese Hamster cell line. Therefore, the test substance is considered to be mutagenic in this chromosomal aberration assay.

Genetic toxicity in vitro, HPRT mutation assay:

In a GLP-compliant mammalian cell gene mutation assay, tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed (BSL Bioservice 2013). The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4h short-term exposure assay. Experiment II without metabolic activation was performed as 20h long time exposure assay. The following concentrations were used. Experiment I without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500µg/mL; Experiment I with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, and 2500µg/mL; Experiment II without metabolic activation: 10, 25, 50, 100, 200, 400, 600 and 800µg/mL; Experiment II with metabolic activation: 100, 316, 1000, 1250, 1500, 2000, 2400, 2800, and 3000µg/mL. No precipitation was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 16.5% for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4%. In experiment II without metabolic activation the relative growth was 12.2% for the highest concentration (800 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8%. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. The positive controls showed distinct biologically relevant effects in mutation frequency. In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chines hamster.

Genetic toxicity in vivo, micronucleus test:

In a GLP-compliant micronucleus test, tested according to OECD guideline 474, 5 non-consanguinous OF-1 albino mice originating from an SPF colony per sex per treatment group were treated once by oral gavage with the test substance 5000 mg/kg bw dissolved in distilled water followed by a 20, 44, 68 hour exposure period. In a preliminary range finding study the concentration of 5000 mg/kg bw was chosen for the main-test. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg bw showed pronounced evidence of mutagenicity 44 h after administration. No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance.


Short description of key information:
The test substance is considered mutagenic in the in vitro chromosomal aberration test, but not in the Ames test and the HPRT mutation assay. The in vivo erythrocyte micronucleus test did not show any genetic toxicity. Therefore it can be concluded that the substance is not genotoxic.

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