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In a study provided by ECHA in a NONS file, PR 254 was found to be non-mutagenic with and without metabolic activation

in a bacterial reverse mutation assay.

The clastogenicity and aneugenicity potential of the registered substance was determined by a Micronucleus Test in vitro (OECD test guideline 487). The GLP study was carried out in human peripheral blood lymphocytes both with and without metabolic activation system in two separate assays. Seven concentrations were used in the Preliminary test: 0.05, 0.1, 0.2, 0.5, 1, 3 and 5 mg/ml of final culture. There was slight cytotoxic effect in all of the tested concentrations with and without metabolic activation.

Three concentrations of the Test item were selected for the Main test: 0.05, 0.1 and 0.2 mg/ml of final culture. These concentrations were chosen on the basis of low solubility of the Test item under physiological conditions. Concentrations used in the Main test were the highest which could be properly evaluated. The cells were treated with a cytokinesis-blocker (Cytochalasin B) and, therefore, arrested in binucleate state, harvested, and the slides were stained. The binucleate cells were analyzed microscopically for the presence of micronuclei. A total of 2000 binucleate cells were examined per one concentration value on coded slides. Concurrent positive (Colchicine, Cyclophosphamide) and negative (Dimethylsulfoxide) controls were included in each experiment. No significant increase in the percentage of binucleate cells with micronuclei over the exposed cell cultures was observed in any of the doses tested in the Main test. Therefore, the Repeated test was performed. Also in the Repeated test no increase of incidence of percentage of aberrant cells was observed. Again, no significant increase in the percentage of binucleate cells was observed. As the registered substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes, it is considered negative.

The genetic toxicity of the registered substance PR254 was investigated in a Mouse Lymphoma assay using L5178Y TK+/-

3.7.2 C cells according to OECD Test Guideline 476 under GLP conditions with and without metabolic activation (±S9 Mix). Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the handling, PR 254 was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control.

The following concentrations were investigated in the Assay 1: 3-hour treatment (±S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL; The following concentrations were investigated in the Assay 2: 24-hour treatment (-S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL;3-hour treatment (+S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL.

The performed Assays fulfilled the validity criteria regarding the negative control and positive controls as well as in connection with the number of analysable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, did not exceed the relevant GEF thresholds for positive call and remained within the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control at the 3-hour treatments (±S9 Mix) and the obtained statistical significances at the 24-hour treatment at the concentration levels of 316 and 1000 μg/mL were not considered as biologically relevant.Therefore, PR254 is considered to be not mutagenic in this test according to OECD 476.

This result is supported by negative Mouse Lymphoma assays of the structural analogues PR264 and PO73.

Short description of key information:

Bacterial reverse mutation assay (Ames): negative

Micronucleus test (in vitro): negative

In vitro mammalian cell gene mutation test (L5178Y/TK+/- mouse lymphoma assay): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on in vitro data from three different genetic toxicity tests, the registered substance does not need to be classified for genetic toxicity and mutagenicity.