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Bacterial mutagenicity

In an Ames assay equivalent to OECD TG 471, the Salmonella tester strains TA1535, TA 1537, TA 1538, TA 98, TA 100 and the E. coli WP2 uvr A were treated with 5, 10, 50, 100, 500, 1000 and 5000 µg test substance per plate (Ciba-Geigy, 1981). The experiments were performed in the presence and absence of a metabolic activation system (S9 fraction of liver from rats induced with Aroclor 1254). Solvent and positive controls were included in this study and have shown to be valid. In contrast to actual regulations (OECD TG 471, adopted 1997) 2-Aminoanthracene was the sole positive control in the presence of metabolic activation. In the experiments performed without and with microsomal activation, comparison of the number of histidine- or tryptophanprototrophic mutants in the controls and after treatment with test substance revealed no marked differences. Thus, the test substance showed no potential to induce gene mutations in bacteria.

Mutagenicity in mammalian cells (HPRT)

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (BASF, 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 1600 μg/mL was limited by the solubility properties of the test item in organic solvents and aqueous medium. The test item was dissolved in DMSO. The tested concentrations ranged from 100 to 1600 µg/ml. Precipitation of the test item visible to the naked eye at the end of treatment was noted at 1200 μg/mL and above in experiment I with metabolic activation and at 800 μg/mL and above in experiment II with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the first experiment at 1200 μg/mL and above without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 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 article is considered to be non-mutagenic in this HPRT assay.

Chromosome damage (in vivo)

In a Nucleus Anomaly Test, 3 Chinese hamsters per sex were treated with 1250, 2500, and 5000 mg/kg bw test substance by gavage (Ciba-Geigy, 1983). Treatments were performed once daily on 2 consecutive days. Cyclophosphamide was used as a positive control substance, and a vehicle control was used as negative control. 24 h after the second treatment the animals were sacrificed and the bone marrow was prepared for scoring. 1000 bone marrow cells were scored per animal, and the following parameters were investigated: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells. One male animal each of the low-dose, the intermediate-dose and the high-dose group and one female animal of the intermediate dose group died in the course of the experiment. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg bw) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 11.4, whereas the negative control yielded a percentage of 0.08.


Justification for selection of genetic toxicity endpoint
The in vivo study was selected, although all available studies are required to cover mutagenicity.

Short description of key information:
Ames test (Ciba-Geigy, 1981): negative
HPRT (BASG, 2012): negative
In vivo micronucleus test (Ciba-Geigy, 1983): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

-No classification required for genotoxicity.

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

- No classification required for genotoxicity.