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Key value for chemical safety assessment

Additional information

In vitro:

Ames test:

In a reverse gene mutation assay (CCR GmbH, 1992) the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and the strains WP2 and WP2 uvrA of E. coli were exposed to the test item at concentrations of 0.0, 3.3, 100.0, 333.3, 666.6, 1000.0 and 5000.0 µg/plate (3 plates/dose) in the presence and absence of mammalian metabolic activation applying the plate incorporation test. The positive controls induced the appropriate responses in the corresponding strains. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation.

CA assay:

An in vitro assay according to OECD Guideline 473 was performed to assess the potential of the test item to induce structural chromosome aberrations in the Chinese hamster cell line V79 (CCR GmbH, 1993). Preparation of chromosomes was done 18 hours (low, medium and high concentration range), and 28 hours (high concentration range) after start of treatment with the test item which was dissolved in DMSO. The treatment interval was 4 hours. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control cultures where 25 metaphases were scored. The concentration range (10 - 100 µg/mL) of the test item administered was limited by its cytotoxicity and its solubility. In both experiments, the mitotic indices were at least slightly reduced with the highest concentration scored at each fixation interval (with and without S9 mix) except in Exp. 2 at interval 18 hours (with S9 mix). In both experiments, in the presence of S9 mix, there were statistically relevant increases in cells with structural aberrations after treatment with the test item at both fixation intervals. In conclusion, under the experimental conditions reported, the test item induced reproducibly structural chromosome aberrations in the V79 Chinese hamster cell line only in the presence of the extrinsic metabolic activation system.

In vivo:

UDS test:

The test item was assessed in the in vivo/ in vitro UDS assay according to the Draft OECD Guideline 486 for its potential to induce DNA repair (UDS) in the hepatocytes of male Wistar rats (CCR GmbH, 1993). The test item was tested at the following dose levels: 2 hour treatment period: 2000 mg/kg bw and 16 hour treatment period: 200 and 2000 mg/kg bw.The volume administered orally was 10 mL/kg bw. After a treatment period of 2 or 16 hours, the animals were narcotised and sacrificed. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR. For each dose level, including the controls, hepatocytes from four treated animals were assessed for the occurrence of UDS. No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of the hepatocytes was not substantially affected by the in vivo pre-treatment with the test item. No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent vehicle controls. Treatment with 2 -AAF as positive substance revealed distinct increases in the number of nuclear and net grain counts. Thus, under the experimental conditions reported, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Micronucleus assay:

An in vivo toxicity test was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (CCR GmbH, 1993). At 24 hours (150, 500 and 1500 mg/kg bw ) and 48 hours (1500 mg/kg bw) after a single application 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. A total of 1000 PCE per animal were scored for micronuclei. The animals expressed toxic reactions. In the micronucleus assay, 3/ 6 males treated with 1500 mg/kg bw for 48 hours died. After treatment with the test item the number of normochromatic erythrocytes (NCE) was not substantially increased as compared to the corresponding negative controls, thus indicating that the test item had no cytotoxic effectiveness. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test item and with any dose level used. A dose of 30 mg/kg bw cyclophosphamide administered as positive control showed a distinct increase of induced micronuleus frequency. In conclusion, 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:
The test item induced increases in chromosome aberrations in presence of a metabolic activation system using Chinese hamster V79 cells (OECD 473). The test item was not mutagenic in the Ames test (OECD 473) and in two vivo test systems (UDS (OECD 486), micronucleus assay (OECD 474)). Alls tudies were performed under GLP.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained in both the in vitro and in vivo genetic toxicity studies and taking into account the provisions laid down in Council Directive 67/548/EEC and Regulation 1272/2008/EC, a classification has not to be done with respect to genetic toxicity.