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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation study in bacteria (AMES): positive

Chromosome aberration study (two tests): negative

Gene mutation study in mammalian cells (Hprt and xprt genes): negative

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

Genetic toxicity in vivo

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

Additional information

GENETIC TOXICICTY IN VITRO

No experimental data from in vitro mutagencity tests on the substance are available. The following data were obtained for Similar Substance 02. It is expected that the Target Substance will present a similar mutagenicity profile. Justification for the use of a read-across approach is provided in Section 13 of IUCLID.

BACTERIAL GENE MUTATION ASSAY

Similar Substance 02 was evaluated for its ability to induce point mutations by base pair change or frameshift in an in vitro bacterial cell reverse mutation assay (Ames test), according to the plate incorporation method of the OECD Guideline 471 (1983). The assay used Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 in two independent experiments, using identical procedures, both with and without liver microsomal activation (S9). Each concentration, including the controls, was tested in triplicate. Five concentrations of Similar Substance 02 were performed, from 10.0 to 5000 µg/plate. No toxic effects occurred in any of the test groups either in the presence or absence of metabolic activation, as evidenced by a reduction in the number of spontaneous revertants. The plates incubated with the test item showed normal background growth in concentrations up to 5000 ug/plate with and without S9 mix in all strains used. Appropriate reference mutagens were used as positive controls.

Up to the highest investigated dose, a significant and reproducible dose-dependent increase in revertant colony numbers was obtained in S. typhimurium strains TA1537, TA1538, TA98, and TA100. The presence of liver microsomal activation did not influence these findings. However, the positive response in strain TA98 was not reproduced in the independent experiment in the presence of S9 mix. Positive controls showed a distinct increase of induced revertant colonies.

Therefore, the test item is considered to be mutagenic in this reverse mutation (Ames) assay.

 

CHROMOSOMAL ABERRATION ASSAY

Two in vitro Chromosomal Aberration tests on Similar Substance 02 are available, both according to the OECD 473 (1983):

In the first test, preparation of chromosomes was performed 18 h (low, medium and high concentration rate), and 28 h (high concentration rate) after start of treatment with Similar Substance 02 in distilled water. The treatment interval was 4 h. In each experimental group, two parallel cultures were used. 100 metaphases per culture were scored for structural chromosomal aberrations except for the positive control culture where 25 metaphases were scored. The test was performed without S9 mix for 18 h (0.30, 1.00 and 2.50 mg/ml) and for 28 h (2.50 mg/ml); and with S9 mix for 18 h (0.10, 0.30 and 1.00 mg/ml) and for 28 h (1.00 mg/ml).

The highest concentration of Similar Substance 02 used was 5.0 mg/ml. A plating efficiency assaywas performed in a former study as indicator for toxicity response. Precipitation of Similar Substance 02 occurred from the 0.10 mg/ml test and did not allow appropriate evaluation of the highest concentration used (5.0 mg/ml). In the absence of S9 mix, the mitotic index was reduced at both fixation intervals at the highest concentration scored (2.50 mg/ml). In presence of S9 mix at fixation interval 18 h, a concentration-dependent decrease in mitotic index was found at concentrations higher than 0.10 mg/ml. A steep increase in toxicity of the test item occured in the concentration range 1.00 - 2.50 mg/ml at both fixation intervals and in the presence of S9 mix, which prevented the evaluation of cultures treated with concentrations higher than 1.00 mg/ml. There was no relevant increase in cells with structural aberrations after treatment with the test item at each fixation interval, either without or with metabolic activation by S9 mix. Appropriate reference mutagens used as positive controls showed distinct increases in cells with structural chromosome aberrations.

In the second test, preparation of chromosomes was performed 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with Similar Substance 02. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. 100 metaphases per culture were scored for structural chromosomal aberrations. The test was performed without S9 mix for 7 h (30.0 µg/ml), for 18 h (0.8, 10.0 and 30.0 µg/ml) and for 28 h (30.0 µg/ml); and with S9 mix for 7 h (30.0 µg/ml), for 18 h (0.8, 10.0 and 30.0 µg/ml) and for 28 h (30.0 µg/ml). The concentration range of the test item applied was determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment with 30.0 µg/ml did not reduce the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration only at fixation interval of 7 hours, however, concentrations higher than 30.0 µg/ml could not be dissolved in the culture medium. There was no relevant increase in cells with structural aberrations after treatment with the test item at any fixation interval, either without or with metabolic activation by S9 mix. Appropriate reference mutagens used as positive controls showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, Similar Substance 02 does not induce structural chromosome aberrations in the V79 Chinese hamster cell line. Therefore, the test item is considered to be non-mutagenic in a chromosomal aberration test.

MAMMALIAN CELL GENE MUTATION ASSAY

In order to investigate the potential of Similar Substance 02 to induce gene mutations, a HPRT test was performed which evaluated the HGPRT locus of V79 cells of the Chinese hamster in vitro, according to the OECD Guideline 476 (1984). The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. The test was performed without S9 mix at 5.0, 10.0, 25.0 and 50.0 µg/ml, and with S9 mix at 5.0, 10.0, 25.0 and 50.0 µg/ml.

According to the pre-experiment for toxicity, the concentration range was selected to yield concentration-related toxic effects. The highest concentration applied produced no distinct decrease in plating efficiency, however, concentrations higher than 50.0 µg/ml precipitated in the culture medium. Up to the highest investigated dose, no relevant increase in mutant colony numbers was obtained in either of the two independent experiments. Appropriate reference mutagens were used as positive controls which showed a distinct increase in induced mutant colonies.

In conclusion, under the reported conditions, Similar Substance 02 did not induce point mutations at the HGPRT locus in V79 cells, therefore, it is considered non-mutagenic in this HGPRT assay.

Additionally, three in vivo mutagenicity studies were available for Similar Substance 02, and all three were found to be negative for mutagenicity. Specifically, two in vivo micronucleus assays (OECD 474, 1983) and an in vivo DNA repair synthesis assay were performed using the same batch of the in vitro tests.

GENETIC TOXICITY IN VIVO

Three in vivo genotoxicity studies on SS02 (two mammalian erythrocyte micronucleus tests and one unscheduled DNA synthesis (UDS) test with mammalian liver cells) confirm the absence of mutagenic potential.

Justification for classification or non-classification

According to the CLP Regulation (EC) No. 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories depending on whether they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Based on the avilable experimental data on genetic toxicity, the substance is not classified for genetic toxicity according to the CLP Regulation (EC) No. 1272/2008.