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Additional information

Genetic toxicity in vitro

- Gene mutation in bacteria

To characterize the mutagenic potential of the test substance in bacteria, an Ames test was conducted according to OECD 471 and under GLP conditions (Wallner, 2012). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were exposed to test substance concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, the tester strains were exposed for 48 h using the plate incorporation method, while in the second experiment the same conditions were applied, except for an additional preincubation period of 30 min. No signs of cytotoxicity and no precipitation were observed in all experiments up to the limit concentration of 5000 µg/plate. The mean number of revertants was not increased in the tester strains at any concentrations and in any experiment performed, except for TA 100 which showed a dose-dependent increase in the mean number of revertants at 2500 µg/plate and higher without metabolic activation in the first experiment. Based on the ambiguous results, a repetition experiment with the test substance at concentrations of 31.6, 100, 316, 1000, 1750, 2500, 3750 and 5000 µg/plate was performed in TA 100 only in the absence of S9 mix. Since no increase in the mean number of revertants was observed in TA 100 in the repetition experiment, the positive results obtained in the first experiment were not considered biologically relevant. In addition, clear negative results in TA 1535, which covers the same mutation spectrum as TA 100, further confirmed the non-mutagenic potential of the test substance. The positive and negative controls included showed the expected results in each experiment. Under the conditions of this assay, the test substance is not mutagenic in the selected strains of S. typhimurium.

- Chromosome aberration in mammalian cells

An in vitro mammalian chromosome aberration test was performed with the test substance in Chinese hamster V79 cells according to OECD guideline 473 and in compliance with GLP (Oppong-Nketiah, 2012). Based on a preliminary toxicity test, cells were treated with concentrations ranging from 125-5000 µg/mL in the presence and absence of metabolic activation (S9 mix) in the main experiment. Cytotoxicity was observed at concentrations of ≥ 3000 µg/mL without S9 mix, whereas no toxic effect was observed in the presence of S9 mix. Based on the data on cytotoxicity, concentrations of 1000, 2000, 3000 and 4000 µg/mL in the presence of metabolic activation (S9 mix) and concentrations of 2500, 3750 and 5000 µg/mL in the absence of metabolic activation were used to analyse chromosomal aberrations after exposure to the test substance. A clear and dose-dependent (5.5 and 7.5%, respectively) increase in the rate of chromosomal aberrations compared to negative control was observed at test concentrations of 2000 and 3000 µg/plate without S9 mix. The aberration rates within the other treatment concentrations were in the range of the historical control data (0-4%). No increase in the number of cells with chromosomal aberrations was observed at any test concentration in the presence of S9 mix. Precipitation of the test substance was observed at concentrations of ≥ 2000 µg/mL without S9 mix and ≥ 2500 µg/mL with S9 mix, respectively. The analysis of polyploid metaphases did not reveal any biologically relevant increase in the frequency of polyploidy cells after exposure to the test substance, neither in the presence nor in the absence of metabolic activation. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay. In conclusion, the test substance was clastogenic in Chinese hamster lung (CHL/IU) cells in the absence of metabolic activation, which, however, was in the precipitating concentration range of the test substance. No clastogenicity was observed after treatment of the cells with the test substance in the presence of metabolic activation.

Genetic toxicity in vivo

The genetic toxicity of the test substance was investigated in an in vivo mammalian erythrocyte micronucleus test according to OECD guideline 474 and in compliance with GLP (Donath, 2012). Based on a preliminary range-finding experiment, 5 NMRI mice per sex and group were intraperitoneally administered with the test substance at dose levels of 100, 250 and 500 mg/kg bw or 0.9% NaCl as vehicle. All dose groups were exposed to the test substance for 44 h and additionally groups for the negative control and the high dose were included for a prolonged exposure of 68 h. After sacrifice of the animals, the bone marrow was extracted and at least 10000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. For the assessment of cytotoxicity, the ratio between poly- and normochromatic cells was determined. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treatment groups compared to the vehicle control, both at the 44 and the 66 h sampling interval. The ratio between polychromatic and normochromatic erythrocytes did not indicate any treatment-related cytotoxicity in the bone marrow of the animals. The positive control substance cyclophosphamide was administered i.p. at a dose level of 40 mg/kg bw and significantly increased the number of polychromatic erythrocytes with micronuclei after 44 h exposure, thus confirming the sensitivity of the assay. Under the conditions of this micronucleus assay, the test substance did neither induce structural and/or numerical chromosome aberrations nor aneugenicity in male and female NMRI mice.


Justification for selection of genetic toxicity endpoint
No study was selected, since the available genetic toxicity studies were negative.

Short description of key information:
In vitro:
Negative Ames test (OECD 471) with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA 102, with and without metabolic activation
Negative result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, with metabolic activation
Positive result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, without metabolic activation

In vivo:
Negative results in an in vivo erythrocyte micronucleus test (OECD 474) in mice

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data on genetic toxicity in vitro and in vivo, the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC.