6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Endpoint summary

Additional information

In vitro:

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 one E. Coli strain (WP2 uvrA pKM101) were used to test the mutagenic potential of the test substance (313, 625, 1250, 2500, and 5000 µg per plate), both with and without metabolic activation (RTC 1995). The test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation (RTC 1995). In the first main assay a statistically significant increase in the number of cells bearing aberrations including gaps, compared with the relevant control values, was observed following treatment with the test substance at the highest dose-level (500 µg/mL) selected for scoring at the 12 hour sampling time in the presence of S9 metabolism. In the second main assay at the 12 hour sampling time, statistically significant increases in the number of cells bearing aberrations excluding gaps, compared with the relevant control values were observed at the highest dose-level (5000 µg/mL) selected for scoring and at the following dose of 2320 µg/mL. For the lowest dose of 1080 µg/mL, marked increases in the number of cells bearing aberrations excluding gaps, not reaching statistical significance, were also observed. Although the test substance did not meet all criteria to be considered positive (eg. reproducibility at the same dose level for both experiments), nevertheless a clear clastogenic effect was observed.

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: 10, 25, 50, 100, 250, 500, 750 and 1000µg/mL; Experiment I with metabolic activation: 25, 50, 100, 250, 500, 1000, 2000, 3000, 4000 and 5000µg/mL; Experiment II without metabolic activation: 10, 25, 50, 100, 250, 500, 1000, 1500, 2000 and 2500µg/mL; Experiment II with metabolic activation: 316, 1000, 1800, 2400, 3000, 3400, 3800, 4200, 4600 and 5000µg/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 15.7% for 1000 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 16.7%. In experiment II without metabolic activation the relative growth was 11.1% for the highest concentration (2500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 28.5%. 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.

In vivo:

In a GLP-compliant micronucleus test, performed according to OECD guideline 474, Swiss CD-1 mice (5/sex/treatment group) were treated once by oral gavage with the test substance (0, 500, 1000, 2000 mg/kg bw) dissolved in 0.5% carboxymethylcellulose followed by a 24 and 48 hours post exposure period (RTC 1995). Following treatment with the test substance, no marked increases in the incidences of micronucleated polychromatic erythrocytes (PCE’s) were observed for combined sexes at any sampling time. Following treatment with the test substance slight increases in the ratio of normochromatic erythrocytes's to PCE's over the control value were observed at the 48 hour sampling time indicating that the test substance exerted a mild toxic effect on bone marrow erythropoietic cells. In conclusion, the test substance is not considered to be mutagenic in the micronucleus test.

Short description of key information:
The test substance is considered to be mutagenic in the in vitro chromosomal aberration test, but not in the Ames test and HPRT mutation assay. The in vivo erythrocyte micronucleus test did not show any genetic toxicity. Therefore it can be concluded that the substance is not genotoxic.

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.