Sodium 5-({4-chloro-6-[ethyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-(1-sulfonato-2-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Endpoint summary

Additional information

Genetic toxicity in vitro, Ames test:

In a GLP compliant Ames test, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and 1 Escherichia coli strain WP2 uvrA, were used to the test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, 5000 µg per plate), both with and without metabolic activation, in the plate incorporation test and the pre-incubation test (RCC 2004). 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. No toxic effects occurred in the test groups with and without metabolic activation, and no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutationrates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimuriumand Escherichia coli reverse mutation assay.

Genetic toxicity in vitro, chromosomal aberration test:

In a GLP compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells (in vitro), were exposed to the test substance, with and without metabolic activation by S9 mix (RCC 2004). Two independent experiments were performed. The exposure period was 4 hrs with and without metabolic activation (exp. I) and 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix (exp. II). 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. The highest applied concentration in the pre-test on toxicity (5360 µg/mL; approx. 5000 µg/mL of the active ingredient) was chosen with regard to the purity of the test item with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data. In both experiments, clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. However, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage, except in experiment I in the presence of S9 mix. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The slight increase in the number of cells carrying exchanges after treatment with 800 µg/mL (3.0 %) in experiment I in the presence of S9 mix has to be regarded as biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells when tested up to cytotoxic test item concentrations.

Genetic toxicity in vitro, mammalian cell gene mutation assay:

No studies on the mutagenicity in mammalian cells of FAT40817 were available. However, a HPRT gene mutation test is available for the structural analogue FAT 40348.

In a GLP-compliant mammalian cell gene mutation assay, tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed (BSL Bioservice 2013). The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4h short-term exposure assay. Experiment II without metabolic activation was performed as 20h long time exposure assay. The following concentrations were used. Experiment I without metabolic activation: 2.5, 5, 10, 25, 50, 100, 200, 300, and 400µg/mL; Experiment I with metabolic activation: 0.25, 0.5, 1, 10, 20, 40, 60, and 80µg/mL; Experiment II without metabolic activation: 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200µg/mL; Experiment II with metabolic activation: 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100µg/mL. No precipitation was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.9% for the highest concentration (400 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8%. In experiment II without metabolic activation the relative growth was 15.0% for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100µg/mL with a relative growth of 16.7%. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. The positive controls showed distinct biologically relevant effects in mutation frequency. In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chines hamster.

Short description of key information:
The test substance is considered to be non-mutagenic in both the Salmonella typhimurium and Escherichia coli reverse mutation assay as in the in vitro chromosomal aberration test. The is substance is also considered to be non-mutagenic in the HPRT mutation assay with a structural analogue.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008