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Administrative data

Key value for chemical safety assessment

Additional information

In vitro:

Ames-test:

This study was performed to investigate the potential of the test substace to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and the Escherichia coli strain WP2 uvrA. The study was conducted according to OECD 471 guideline and GLP. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 ug/plate The plates incubated with the test item showed normal background growth up to 5000 ug/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in experiment I. In experiment II, toxic effects were observed at higher concentrations with and without metabolic actiyation in nearly all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Chromosome aberration test:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments according to OECD 473 guideline and GLP.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (650 ug/mL) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent. Dose selection ofthe cytogenetic,experiments was performed considering the toxicity data and the occurrence of precipitation.

In all experiments toxic effects indicated by reduced cell numbers or reduced mitotic indices of about and below 50 % of control were observed. However, in some experimental parts of the cytotoxic concentrations led to clearly reduced mitotic indices were not evaluable for cytogenetic damage. In experiment II and III, after 28 hrs continuous treatment in the absence of S9 mix reproduced statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. However, these effects were seen only at cytotoxic concentrations (cell viability 64% of control or below). The statistically significant increase in the number of cells carrying structural chromosomal aberrations observed in experiment II after 4 hrs treatment at 28 hrs preparation interval in the presence of S9 mix was not confirmed in experiment III. Therefore, the observation has to be regarded as biologically irrelevant. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix after 28 hrs continuous treatment only.

In vivo:

Micronucelus assay:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow ofthe mouse. The study was conducted according to OECD 474 guideline and GLP. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item 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. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.


Short description of key information:
Ames-Test: negative (RCC, 2003)
in vitro chromosome aberration test: positive
in vivo micronucleus assay: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, a classification and labeling for genetic toxicity is neither required according to DSD-DPD nor according to CLP.