3-Pyridinecarbonitrile, 4-methyl-2,6-bis[(4-methylphenyl)amino]-5-[2-[2-(trifluoromethyl)phenyl]diazenyl]-

Endpoint summary

Additional information

Ames-test:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD guideline No. 471. The assay was performed in two independent experiments both with and without rat liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants, were observed at the highest concentration in strain TA 100 without S9 mix in experiment I, and in strain TA 1537 with S9 mix in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. As the substance contains an azo group, the use of the Prival modification of the Ames test would have been preferred.

Chromosome aberration in vitro:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix according to OECD guideline No. 473.

Two independent experiments were performed. In experiment I. the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

In the pre-experiment. precipitation of the test item in culture medium was observed after treatment with 2.3 µg/mL and above in the absence of S9 mix and with 4.7 µg/mL and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed. In both cytogenetic experiments, in absence and the presence of S9 mix precipitation of the test item in culture medium 4 hrs after start of treatment was observed at 5 µg/mL and above.

In this study, in the absence and the presence of S9 mix, no relevant cytotoxicity indicated by reduced cell numbers or mitotic indices was observed after treatment with the test item.

In both experiments, in the absence and the presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.5 % aberrant cells, exclusive gaps) were within the range of the solvent control values (0.0 - 3.5 % aberrant cells, exclusive gaps) and within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Two significant increases were observed in experiment I in the absence of S9 mix at preparation interval 18 hrs after 4 hrs treatment with 2.5 and 5.0 µg/mL. Although these increases of 2.0 % and 2.5 % aberrant cells, respectively, were statistically significant compared to the low response (0.0 % aberrant cells) in the solvent control data, the responses were cleariy within the historical control data range (0.0 - 4.0 % aberrant cells). In addition, doserelated increases in the number of aberrant cells were observed at 1.3 to 5.0 µg/mL in experiment I after 4 hrs treatment in the absence of S9 mix (0.5 %. 2.0 %. and 2.5 % aberrant cells) and at 1.3 to 5.0 µg/mL in experiment II in the presence of S9 mix at preparation interval 28 hrs (0.5 %, 1.0 %. and 2.5 % aberrant cells). Also these values were cleariy within the range of the historical control data. Therefore, the statistical significances and the dose-dependencies have to be regarded as being biologically irrelevant. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4 - 3.2 %) as compared to the rates of the solvent controls (1.5-5.6%). In both experiments. EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

The test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to clearly precipitating test item concentrations.

Short description of key information:
The test item as described in section 1.2 was found to be non- mutagenic and non-classtogenic in vitro (OECD 471, OECD 473, GLP).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, the test item was not classifiied or labelled according to Directive 67/548/EEC (DSD) and 1272/2008 EC (CLP) for genetic toxicity.