Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

no mutagenic potential

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity potential of the substance was evaluated in the in vitro gene mutation study in bacteria, in the in vitro gene mutation study in mammalian cells and in the in vitro chromosome aberration study.

The test material was tested for mutagenic effects in vitro in histidine-requiring strains of S. typhimurium, according to an internal method similar to the OECD Guideline 471. The test was performing without and with metabolic activation in the range of concentration of 0.1 to 100 µg/20µl/plate, using strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA 1538). Each concentration and control were tested in triplicate. No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation. The test substance did not induce gene mutations in the strains of S. typhimurium used, both with and without metabolic activation.

The test item was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. The initial experiment had to be repeated for the strain TA1537 due to high colony numbers on the control plates. In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 μL/plate (maximum test concentration for soluble substances) in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. Thus, the test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in all evaluated strains, in the presence and the absence of metabolic activation. The experimental part for TA1537 was declared invalid due to high colony numbers on the control plates and was repeated in experiment 1b.

The Experiment 1b was performed for the strain TA1537 and exactly as in experiment 1. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed on the plates with TA1537. Thus, the test item showed no signs of toxicity towards the bacteria strain in both the presence and the absence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant or dose-related increase in the number of revertants in TA1537, in the presence and the absence of metabolic activation.

Based on the results of the first experiment, and since no cytotoxicity was observed up to the highest tested concentration, the test item was tested up to concentrations of 5 μL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method in a second experiment. The test item showed no signs of toxicity in all tested concentrations (5 μL/plate to 0.08 μL/plate).

The results of this experiment showed that the test item caused no significant or dose-related increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

The substance was assayed for its ability to induce chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation, according to OECD guideline 473. Three treatment series were included in the study. A short term treatment was performed where the cells were treated for 3 hours in the presence and absence of S9 metabolism. The harvest time of 24 hours corresponding to approximately 1.5 cell cycle was used. A long term(continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at 24 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). Dose levels of 1160, 580, 290, 145, 72.5, 36.3, 18.2, and 9.08 µg/ml) were used for all treatment series. Appropriate negative and positive control were included. Two replicate cell cultures were prepared at each test point. For all treatment series, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Where no toxicity was observed, the highest dose level was selected for scoring chromosomal aberrations.For each replicate culture, 150 well spread metaphases were scored to assess the frequency of aberrant cells.

No statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level and treatment condition. It is concluded that the substance does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

The substance was further examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment, according to OECD guideline 476. A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 1160 µg/ml (10 mM) and at a wide range of lower dose levels: 580, 290, 145, 72.5, 36.3, 18.1, 9.06 and 4.53 µg/ml. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. No precipitation was noted at any concentration tested. A main assay was performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. The following dose levels were used: 1160, 580, 290, 145, 72.5, 36.3 µg/ml. No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. It is concluded that the substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two following categories:

- 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.

The test substance did not show any reasons of concern in all in vitro tests performed.

Therefore, the substance is not classified for genetic toxicity according to the CLP Regulation (EC) No. 1272/2008.

Categories Display