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Additional information

In vitro data

Several Ames test results for MBT have been reported. Although the study results are reliable the test design of the studies does not comply with the current guideline with regard to the number and kind of tester strains. However, several assays are comparable to a guideline study (CMA 1984, Monsanto Co. 1976, Hinderer 1984). In these studies no biologically relevant and dose dependent increases in revertants was evaluated in any of the tester strains evaluated with and without metabolic activation.

This finding was confirmed in a HGPRT assay with CHO cells (CMA 1984). The test substance was evaluated up to 50 µg/ml without and up to 300 µg/ml with metabolic activation. Cytotoxicity was indicated with and without metabolic activation (-S9: 33.33 µg/ml 59 % rel. survival; +S9: 333.33 µg/ml 17 % rel. survival). The test substance MBT was negative with and without metabolic activation (CMA 1984).

The mutagenic potential of MBT was also evaluated in several mouse lymphoma assays. The test substance MBT was negative in a guideline study (OECD TG 476) done under GLP conditions (WTR 1997). Some earlier mouse lymphoma assay tests showed negative or weak positive responses. However, the findings of these assays are questionable because of relevant methodological deficiencies.

In a chromosome aberration assay with CHL cells an induction of polyploidy cells and endoreduplications were noted after treatment with 0.2µg/ml MBT. The frequency of polyploidy cells and endoreduplications was 3.6 % and 6.2 % without metabolic activation and 2 % and 0.4 % with metabolic activation, respectively. No endoreduplications were noted in the solvent control (Matsuoka 2005). Furthermore, in a limited chromosome aberration assay with CHO cells an increase in aberrant cells were noted (NTP 1988). However, the findings are questionable because of significant chemically induced cell cycle delay, which presumably indicated high toxicity. In addition, in a limited sister chromatid exchange (SCE) assay a relative increase in SCE's was noted in presence of metabolic activation (NTP 1988). However, the relevance of this finding is questionable because of presumed high toxicity indicated by a significant chemically induced cell cycle delay and the lacking of a dose-response relationship.

In vivo data

The genotoxic potential of MBT was evaluated in an in vivo micronucleus assay with CD-1 mice (4 males and 4 females per group). Single dose group animals received 300 mg/kg of MBT and multiple dose group animals received MBT in a split dose regimen with two doses of 300 mg/kg each, separated by 24 hours. The positive control article triethylenemelamine, was administered intraperitonealy to a separate group of mice (4 males and 4 females) at a dose of 0.5 mg/kg. Thirty hours after treatment the positive control animals were sacrificed. The negative control animals were given two doses of corn oil separated by 24 hours and sacrificed 48 hours after the first dose. Single Dose Group I and Single Dose Group II were sacrificed at 30 and 48 hours, respectively after a single injection. Multiple Dose Group I and Multiple Dose Group II were given two doses of the test article separated by 24 hours and sacrificed at 48 and 72 hours, respectively after the initial injection.

Systemic availability of the test substance was indicated by occurrence of clinical signs after application. The following signs were observed at the first dose: prostration, hypoactivity, hypernea, ptosis, tremors upon stimulation and an occasional animal exhibited a loss of righting. Observations at 4 and 24 hours following the first dose included ptosis with no other visible signs in all treated animals. No mortality occurred in the study.

The results for test article MBT were negative in the micronucleus test at a dose level of 300 mg/kg in the single dose groups and with a second dose of 300 mg/kg in the multiple dose group administered in a split dose regimen. The test material did not produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all animals administered MBT were within the normal historical range of spontaneous micronuclei incidence.

The test substance MBT was evaluated in a dominant lethal test in Sprague-Dawley rats. Male rats were treated with 0, 2500, 8750, or 15000 ppm MBT in the diet. Following a 13 week treatment each male was housed with two virgin female rats per week for two weeks. All females with evidence of mating from each group were sacrificed on gestation day 13 for determination of dominant lethal effects. MBT produced dose-related toxicity in male rats treated with 8750 and 15000 ppm in the diet. Toxicity was limited to reduced body weights and food consumption. When the rats were serially mated over two weeks after 13 weeks of treatment, no dominant lethal effect was observed

Short description of key information:
The test substance MBT was investigated for its potency to induce gene mutation in bacteria and mammalian cells. MBT was negative in several bacterial gene mutation studies and did not induce gene mutations at the hgprt locus and was negative in a mouse lymphoma guideline study. However, an increase in polyploidy cells and endoreduplications was noted in an in vitro chromosome aberration assay in CHL cells. Moreover an increase in chromosome aberrations and SCE's was noted in CHO cells. However the biologically relevance of these findings is questionable.
No genotoxic effects of MBT were noted in an in vivo micronucleus assay with CD-1 mice. In addition, in a dominant lethal test with Sprague-Dawley rats no dominant lethal effects of the test substance were noted.
In summary, no genotoxic potential of MBT was noted in bacterial and mammalian cells mutation assays. However, the genotoxic effects noted in two in vitro chromosomal aberration assays and SCE assay are questionable. Moreover, no genotoxicity in vivo was indicated. Thus based on the findings discussed above, no genotoxic potential of MBT was revealed.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is required according to the classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).