A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Endpoint summary

Additional information

IN VITRO

The genotoxic potential of the test substance was assessed in the Ames test with different strains of Salmonella typhimurium, in presence and absence of S9-mix, according to OECD guideline 471 (1983), as plate incorporation test (CCR 124312). The used strains were TA 1535, TA 1537, TA 1538, TA 98 and TA 100. A preliminary toxicity test was carried out with concentrations of 1, 3.3, 10, 33.3, 100, 333.3, 1000, and 5000 µg/plate with strains TA 98 and TA 100. Since no effects on background growth were seen, the top dose level was chosen as highest concentration in the main experiments:

Experiment I: 10, 33.3, 100, 333.3, 1000, 5000 µg/plate

Experiment II: 10, 100, 333.3, 1000, 3333.3, 5000 µg/plate

A significant and reproducible dose-dependent increase in revertant colony numbers was seen in the Salmonella typhimurium strain TA 1538 with and without S9-mix and TA 98 without S9 mix. The background growth was not effected by the test substance. Thus, the test substance is considered to be mutagenic in the Salmonella typhimurium reverse mutation assay under conditions used in this study.

The genotoxic potential of the test substance in mammalian cells was assessed in the HGPRT test with Chinese Hamster V79 cells according to OECD guideline 476 (CCR 152908). The mutant frequency was evaluated after treatment with 6, 15, 30 and 60 µg test substance/ml by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. Mutant frequencies were calculated from the number of mutant colonies corrected for cell survival. The plating efficiency was not reduced after treatment. The mutation frequency of the negative controls was 7.1 to 31.3 mutants/1E+6 cells and 2.2 to 38.1 mutants/1E+6 cells in the groups treated with the test substance. Since no biologically relevant increased mutant frequency was seen upon treatment, the test substance had no genotoxic potential under conditions used in this study.

The test article 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 the OECD guideline 473 (CCR 124323). Preparation of chromosomes was done 7, 18 and 28 h after start of the treatment with the test article. The treatment interval was 4 h. The dose levels retained for evaluation were 60 µg/ml (7 h and 28 h), and 3, 30 and 60 µg/ml (18 h), both in absence and presence of S9-mix. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosome aberrations. In the main experiment, at fixation intervals 7 h (with S9 mix) and 18 h (without S9 mix), the mitotic index was reduced after treatment with the highest dose level ( 60 µg/ml) in the absence and presence of S9-mix, indicating that the test material had cytotoxic properties. Higher concentrations than 60 µg/ml precipitated in the culture medium.

Both, in the absence and presence of S9-mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.25 % - 2.50 %) were in the range of the control values: 0.50 % - 2.50 %. Both positive control substances, Ethylmethanesulfonate (0.72 mg/ml, without S9-mix) and Cyclophosphamide (CPA, 1.40 µg/ml, with S9-mix) showed distinct increases of cells with structural chromosome aberrations. Thus, under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese Hamster cell line.

IN VIVO

The test article was assessed in the micronucleus assay (MNT) according to the OECD TG 474 for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (CCR 130307). NMRI mice of both sexes were used; each group comprised 5 animals per sex. The test article was suspended in 4 % carboxymethylcellulose (CMC) and applied by single gavage at a dose level of 5000 mg/kg bw. Selection of the dose level was based on the results of a pretest. CMC was used as negative control (solvent control) and 30 mg/kg bw cyclophosphamide administered per single gavage served for positive control. After 24h, 48h and 72h following treatment, the animals were sacrificed and the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei, 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. Cytotoxicity was indicated by the ratio between polychromatic and normochromatic erythrocytes (NCE) in the same sample, which was reported as number of NCE per 1000 PCE.

The animals of the MNT expressed slight toxic reactions. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls indicating that the test article had no cytotoxic properties. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with test article were in the same range as compared to the negative control groups. Cyclophosphamide used as positive control induced as expected a distinct increase in micronucleus frequency. Thus, under the experimental conditions used, the test article did not induce micronuclei in NMRI mice as determined by the micronucleus test.

Short description of key information:
IN VITRO
The genotoxic potential of the test substance was assessed in the Ames test with different strains of Salmonella typhimurium, in presence and absence of S9-mix, according to OECD guideline 471 (1983), as plate incorporation test (CCR 124312). The test substance was mutagenic in the Salmonella typhimurium reverse mutation assay under conditions used in this study.
The genotoxic potential of the test substance in mammalian cells was assessed in the HGPRT test with Chinese Hamster V79 cells according to OECD guideline 476 (CCR 152908). No genotoxic potential could be evidenced.
The test article 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 the OECD guideline 473 (CCR 124323). Under the experimental conditions used, the test article did not induce structural chromosome aberrations in the V79 Chinese Hamster cell line and thus, was not cytogenic.

IN VIVO
The test article was assessed in the micronucleus assay (MNT) according to the OECD TG 474 for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (CCR 130307). Under the experimental conditions used, the test article did not induce micronuclei in NMRI mice as determined by the MNT.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Several valid in vitro studies and an in vivo study are available for assessment of the genotoxic potential of the present test article. The in vitro studies comprised an Ames Test, a HGPRT test with Chinese Hamster V79 cells and a chromosome aberrations assay with V79 cells of the Chinese Hamster; in vivo genotoxicity was assessed using the micronucleus test with NMRI mice. Except for the Ames test, all studies were negative. Thus, no classification of the test article for genotoxicity according to the EU Directive 67/548/EEC and the CLP Regulation (EC)/1271/2008 is needed.