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In vitro Genotoxicity

Bacteria Gene Mutation

The mutagenic potential was determined in a GLP study by the method of Ames (1975) as described in OECD Guideline 471. The test item was tested up to the limit concentration 5000 µg/plate in S. typhimurium strains TA 98, TA 1535, TA 1537 and TA 100 as well as E. coli WP2uvrA with and without metabolic activation. Measurement of the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were performed to check for cytotoxicity. The test substance was negative for causing cytotoxic effects. There was no evidence of induced mutant colonies for the test item over background. The positive controls induced the appropriate responses in the corresponding strains. (BASF SE 2010; reliability = val 1).


Mammalian Cytogenicity

In the mammalian in vitro cytogenetic assay to test for the chromosome aberration potential of the test substance, peripheral human lymphocytes were exposed in culture medium at the concentrations of 333, 500, 1000, 1500, 2000 and 2363 µg/ml with and without metabolic activation (S9 mix) for a duration of 3, 24, 48 hours. The substance was tested up to the limit concentration 2363 µg/ml (0.001M). No precipitation was noted. Cytotoxicity was not evident at the tested concentrations. Positive controls induced the appropriate response. Both in the absence and presence of metabolic activation, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. (NOTOX B.V 2005; reliability = 1).

Mammalian Gene Mutation

In the GLP OECD 476 guideline study (BASF SE, 2010) the test substance did not induce gene mutations at the HPRT locus in V79 cells. Cells were incubated with the test substance with and without metabolic activation (S9 mix) for 4 h and 24 h with concentrations up to 2420 µg/mL. Minor increases of the mutation frequency occasionally occurred. However, these increases were generally not dose dependent as indicated by the lacking statistical significance. The only significant increase was judged as irrelevant fluctuation since the mutation frequency did not exceed the range of historical solvent controls. Furthermore, no cytotoxicity occured.

In vivo Genotoxicity

Mammalian chromosome aberration

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice according to the OECD TG 474 (BASF SE, 2011; Val 1, GLP; test substance purity 98.65%). For this purpose, the test substance, dissolved in deionized water, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. Animals treated with the vehicle only served as negative control.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

Treatment with vehicle gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. A slight change in the ratio of PCE/NCE was observed at 2000 mg/kg body weight at 24-hour preparation interval as indication for target organ toxicity. Therefore, it is shown that the test substance reached the blood respectively the bone marrow.

According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

- The highest dose of 2000 mg/kg bw led to 0.7‰ polychromatic erythrocytes containing micronuclei after 24 hours and 1.0‰ after 48 hours.

- In the two lower dose groups, rates of micronuclei of 1.1‰ (1000 mg/kg group) and 0.7‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.

- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.

- The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (18.9‰) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected.

- Vincristine sulfate, a spindle poison, also produced a statistically significant increase (31.7‰) in the number of polychromatic erythrocytes containing micronuclei, with a significant portion increase (11.7‰) attributable to large micronuclei.

Therefore, because the rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Taken together with the results of the available in vitro studies, it is concluded that the test substance does likely not have the potential to induce genetic toxicity in vitro and in vivo.

Short description of key information:
There is no indicaton for genotoxicity from the Ames test, an in vitro chromosomal aberration assay, a HPRT assay and an in vivo Micronucleus test in bone marrow cells of the mouse.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance does not meet the classification and labelling criteria as laid down in 67/548/EEC and 1272/2008/EEC (EU-GHS) for mutagenicity.