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Additional information

Two in vitro tests and two in vivo tests were performed according to OECD guidelines with GLP.

The Ames test was performed with the Salmonella typhimurium and Escheria coli (TA 98,TA 100, TA 1535, TA 1537, WP2 uvrA and WP2) at six dose levels of 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 μg/plate, both in the presence and absence of metabolic activation, to check the potential of mutagenicity according to OECD 471 and 472. No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation. No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no reproducible tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The in vitro chromosome aberration test was performed for the potential of the test substance to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in two independent experiments. In the absence of S9 mix, in both experiments the mitotic index was reduced after treatment with the highest evaluated concentration at each fixation interval. In the presence of S9 mix the mitotic index was strongly reduced after treatment with 100.0 µg/ml at interval 28 h. In the presence of S9 mix, at fixation interval 28 h, there were statistically significant and biologically relevant increases in cells carrying structural chromosome aberrations after treatment with the test article. At the 18 h interval and in the absence of S9 mix, no biologically relevant increase was observed. Thus, the test article did induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 cells (Chinese hamster cell line) in vitro under the test condition.

To investigate the positive in vitro finding, a micronucleus test in vivo was performed to assess the mutagenic properties of the test article by means of the micronucleus test in bone marrow cells of the mouse according to OECD 474. The mice were exposed at dose levels of 2000 mg/kg b.w. in the preliminary test and 200, 670 and 2000 mg/kg b.w. in a main test, respectively. There was no significantly difference between the test groups and negative control group for the ratio of polychromatic to normochromatic erythrocytes, while the positive control shows obvious decreased for the ratio and statistically significantly increase in the number of micronucleated cells. Based on the result of this in vivo micronucleus test, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

The UDS test was performed in the in vivo/in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats with doses of 1000 mg/kg bw and 2000 mg/kg bw (3 h and 16 h preparation interval) according to OECD TG 486. No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 3 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. In addition, no substantial shift to higher values was obtained in the percentage distribution of the nuclear grain counts. Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg bw) were used as positive controls. In vivo treatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts. Thus, we conclude the test article does not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Both of the two in vivo tests indicated the negative results although the in vitro chromosome aberration test indicated a positive result. The in vivo test result is more reliable for its higher similarity to the human metabolic environment. In addition, the Ames test got the negative result as well. Thus, we can conclude that the test substance indicates no genetic toxicity under the experiment condition.

Justification for selection of genetic toxicity endpoint
all in vitro and in vivo studies used in the weight of evidence approach were OECD guideline tests with GLP

Short description of key information:
No genotoxicity of the test substance found in in-vivo studies.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results of the in vivo and in vitro tests, the substance is not classified for mutagenicity according to CLP (Regulation (EC) No 1272/2008) or DSD (Directive 67/548/EEC) respectively.