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Additional information

Eight reports were reviewed. These were four bacterial in vitro test reports, three non-bacterial in vitro test reports and one genotoxicity in vivo test report.

Bacterial in vitro tests:

Four studies were reviewed. The studies by MHW Japan (1997) and BASF AG (2006a) were conducted according to the guidelines for screening mutagenicity of testing of chemicals and reverse mutation assay (OECD TG 471 and TG 472) in compliance with GLP. The third study was performed in the course of the United States National Toxicology Program (Zeiger et al., 1987) and was judged to be reliable with restrictions (E. coli strain missing). All three studies were identified as key studies. The fourth study was a reduced Ames screening test with the S. typhimurium strain TA98 preceding the above-mentioned OECD guideline study by BASF AG (2006b).

1) BASF AG (2006 a+b):

According to the results of the studies by BASF AG (2006a+b), the test substance was not mutagenic in any of S. typhimurium strains TA98, TA100, TA1535 and TA1537, and in E. coli strain WP2 uvrA. A bacteriotoxic effect was observed in the standard plate test as well as in the preincubation assay depending on the strain and test conditions from about 2750 μg/plate onward.

2) MHW, Japan (1997):

The test was conducted twice for all cells with and without rat S9, and the results were negative except for a marginal response in S. typhimurium strain TA 98 in one out of two tests with metabolic activation. In the following confirmation test the positive result could be reproduced. Toxic effects were observed at 1250 µg/plate (TA98, TA1537), 2500 µg/plate (TA1535), and 5000 µg/plate (TA100, WP2 uvrA) without metabolic activation, and 2500 µg/plate (TA1535), 5000 µg/plate (TA100, TA98, TA1537) with metabolic activation. Toxic effects were not observed for WP2 uvrA with metabolic activation.

3) Zeiger et al. (1987):

Zeiger et al. (1987) reported that the test substance was negative in any of S. typhimurium strains TA98, TA100, TA1535 and TA1537 at doses of 10 to 10000 µg/plate with hamster S9, with rat S9, and without metabolic activation. Toxic effects were observed at 3333 µg/plate (TA98, TA100), 1000 µg/plate (TA1535, TA1537) with and without metabolic activation.

Non-bacterial in vitro tests:

Three studies were reviewed. Two studies were chromosomal aberration tests in vitro according to OECD TG 473 with cultured Chinese hamster lung cells (CHL/IU) (MHW Japan, 1997) and human lymphocytes (Atochem, 1991), respectively. The study by MHW Japan (1997) was conducted in compliance with GLP and was identified as the key study. Based on the results of this study, the test substance is considered to induce chromosomal aberrations, which were mainly chromatid exchanges, and polyploidy with and without metabolic activation. However, the aberrations are weak even at the highest concentrations. Supportingly, the study of Atochem (1991) showed a weak but statistically significant clastogenic activity of the test substance at high concentrations in both the presence and absence of metabolic activation.

The third study was an in vitro gene mutation test in CHO cells (HPRT locus assay) by BASF AG (2006). Based on the results of this study, the test substance did not cause any increase in the mutant frequencies either without metabolic activation or after adding a metabolising system in two experiments performed independently of each other. Thus, the test substance is considered to have no mutagenic activity in vitro in mammalian cells.

In vivo genotoxicity studies:

One in vivo micronucleus test in mice was reviewed. The study was conducted according to OECD TG 474 in compliance with GLP and was identified as the key study (Atochem, 1993). OF1 mice (5 males and 5 females per group) received two intraperitoneal administrations of the test substance separated by 24 hours at a dose level of 75 mg/kg, the maximum tolerated dose. Cyclophosphamide at a dose level of 25 mg/kg (two times i.p. injection) served as the positive control. The test animals were killed at 24 and 48 hours after the second administration and bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse and for the polychromatic-to-normochromatic erythrocytes (PCE/NCE) ratio. The number of micronucleated polychromatic cells in the dosed animals was not significantly different from that of the animals in the control group. A decrease in the PCE/NCE ratio was observed indicating that the test substance had actually reached the bone marrow.

In conclusion, the test substance did not induce cytogenetic damage to the bone marrow cells of mice in this test.


Short description of key information:
In three Ames tests the test substance was not mutagenic in any bacterial strain tested (BASF AG, 2006 a+b, Zeiger et al., 1987). In one Ames test the test substance did not induce gene mutations in 3 strains of S. typhimurium and in E. coli but did induce gene mutations in S. typhimurium strain TA98 with metabolic activation in one out of two replicates (MHW Japan, 1997). In mammalian CHO cells, the test substance was not mutagenic in vitro (BASF AG, 2006). Furthermore, the test substance showed only a weak but statistically significant clastogenic activity at the highest doses in CHL lung cells (MHW Japan, 1997) and human lymphocytes (Atochem, 1991) with and without metabolic activation. In vivo, however, the test substance was negative when administered intraperitoneally at the maximum tolerated dose in a single dose micronucleus test in mice (Atochem, 1993). Based on these results, and taking into account data of structurally related substances, it is unlikely that this substance is mutagenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was mutagenic in various in vitro test systems (weakly mutagenic in one strain in one Ames test; clastogenic in two in vitro chromosomal aberration tests); however these results could not be confirmed in vivo in a test with mammals.

EU classification according to Annex VI of the Directive 67/548/EEC:

- No classification required.

GHS classification according toAnnex I 1272/2008 CLP (EU GHS):

- No classification required.

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