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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

non-mutagenic (gene mutation in bacteria, mammalian chromosome aberration, gene mutation in mammalian cells, host-mediated)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

non-mutagenic (mammalian chromosome aberration, dominant lethal assay)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenicity potential of the substance (in-vitro and in-vivo) was evaluated by considering available data on Similar Substance 01, due to the absence of data on the target substance. Justification for Read Across is given in Section 13 of IUCLID.

In vitro studies

The mutagenic potential of the substance was evaluated in the in-vitro gene mutation study in bacteria according to the OECD Guideline 471 and EU Method B.13/14. Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains were exposed to the substance in a range of 1.6 - 1000 µg/plate (experiment 1) and 62.5- 1000 µg/plate (experiment 2) both with and without metabolic activation. Positive control substances were tested in parallel. Precipitation was noted at 1000 µg/plate (experiment 1) and at 500 and 1000 µg/plate (experiment 2). No evidence of cytotoxicity was observed. No substance treatment of any of the tester strains, either in the absence or presence of S-9 mix, resulted in a statistically significant (at the 1 % level using Dunnett's test) and dose-related increase in revertant numbers. The substance was found negative with and without metabolic activation under the test conditions.

The in-vitro mutagenicity potential of Similar Substance 01 was further evaluated in a mammalian chromosome aberration studies. The cytogenicity potential of the test material was assessed in the in vitro chromosome aberration test according to the OECD Guideline 473 and EU Method B.10. Chinese hamster lung fibroblasts (V79) were exposed to the substance at 29.4, 42 and 60 µg/ml (experiment I) and 60, 92.3, and 142 µg/ml (experiment II). The cells were exposed for 2 h with metabolic activation -cells were then washed twice with sterile saline and re-fed with fresh medium, cultures were then incubated for a further 18 or 42 hours before harvesting- and 20 and 44 hours without metabolic activation. After the exposure period the cells were treated with colchicine, were harvested, fixed and stained. 100 metaphases of each culture were analysed for aberrations while cytotoxicity was determined by the mitotic index. Positive and negative substances controls run in parallel. No cytotoxicity was noted while precipitation was observed at 39 µg/ml and above. The substance is considered to be non-mutagenic in this chromosome aberration test both with and without metabolic activation.

The mutagenicity potential of Similar Substance 01 was also evaluated in the in-vitro mammalian cell gene mutation assay according to the OECD Guideline 476. Mouse lymphoma L5178Y cells were exposed to the substance at 31.25, 62.5, 125, and 250 µg/ml with and without metabolic activation for 3 hours. After 2 days in growth medium cells were incubated with a selection agent (TFT) for 12 days. The cytotoxicity was determined by the cloning efficiency method. Concurrent positive and solvent controls were also included. Precipitation: at 125 and 250 µg/ml was noted. The relative survival and mutant frequency was determined for the test material, positive and negative control. The substance was found negative with and without metabolic activation.

The analogue substance was also tested in the host-mediated assay against Salmonella (TA-1530, G-46) and Saccharomyces cerevisiae strains (D-3) on plates. The test substance was inoculated orally (50, 500, 5000 mg/kg of substance) to ten mice in two trials: in the acute trial animals were treated once, in the subacute trial animals were treated on 5 consecutive days. The indicator organisms (yeast and bacteria) were inoculated intraperitoneally into mice afterwards. Dilutions of each peritoneal exudate were plated and incubated at 37 °C for 18 (yeast) or 40 (bacteria) hours and mutant frequency was determined. No significant reversion or recombinant increases were produced in Salmonella or Saccharomyces strain. The results from tests using Salmonella strain G-46 indicated that the test compound induced reversion in both the acute and subacute trials. A slight dose response was observed in the acute trials but not in the subacute trials. Repeat tests of the subacute trials indicated that the compound induced reversion, although the results were dose independent. All in vitro tests, when Similar Substance 01 was directly tested against yeast and bacteria on plates, were negative.

in vivo studies 

The genetic toxicity potential of Similar Substance 01 was evaluated in two in vivo chromosome aberration assays performed similar to the OECD Guideline 475. Male rats were orally exposed to the substance (at 50, 500 and 5000 mg/kg bw/day) for 5 consecutive days in one study and only once in the second study. Prior to euthanasia, animals were treated with colcemid intraperitoneally in order to arrest the bone marrow cells in C-mitosis. Chromosome preparations were made from bone marrow cells and were stained, and metaphase cells were analysed for chromosomal aberrations. The preparations were examined using microscopes with brightfield optics and xenon light sources. The chromosomes for each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and any other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index. Negative controls (vehicle) run in parallel while a single administration of Triethylene Melamine (TEM) was performed in the same study, yielding the expected results.

Mitotic indices were normal in both studies. In the first study, the only aberrations observed were 3 % breaks in the negative controls and 2 % breaks in the high dose level. These were within normal limits. The substance was found to be negative in the in vivo mammalian bone marrow chromosomal aberration test

In a dominant lethal assay, performed similar to OECD-guideline 478, 10 male rats were treated with Similar Substance 01 by gavage at concentrations of 50, 500 and 5000 mg/kg bw, singly or on 5 consecutive days. Saline was used as vehicle control and triethylenmelamine (0.3 mg/kg bw, applied by intraperitoneal injection) as positive control. Both yielded the expected results. Dominant lethal effects were not observed.

Justification for classification or non-classification

The available experimental test data is used for the assessment of the classification of the substance.

The substance is found to be negative both with and without metabolic activation in the in vitro studies and also negative in the in vivo studies performed.

The available experimental test data is reliable and suitable for classification purposes. On the basis of the experimental results, the substance is not classified for genotoxicity under the CLP Regulation (EC) No. 1272/2008.