Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro data

The mutagenic potential of ZMBT was evaluated in a bacterial mutagenicity test (Monsanto Co. 1977). Although the study is reliable the test design of the study does not comply with the current guideline with regard to the kind of tester strains used. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. Treatment by the plate incorporation method was done in presence or absence of metabolic activation (S9-mix). A concentration range of 0.1 mg/plate to 500 µg/plate was evaluated. Toxicity was noted in tester strain TA 1538 at 500µg/plate without metabolic activation. No mutagenic response was noted in any of the tester strains used in presence or absence of metabolic activation (S9-mix). The authors concluded that the test substance did not induce a mutagenic response in any of the tester strains evaluated under the experimental conditions used.

There are no mammalian cell mutation assays available for ZMBT. A read-across across is performed with MBT (benzothiazole-2-thiol) (see discussion endpoint summary toxicokinetics).

The mutagenic potential of MBT was evaluated in a HGPRT assay with CHO cells (CMA 1984). A preliminary cytotoxicity assay was done with test substance concentrations of 0.03, 0.1, 0.33, 1.0, 3.3, 10.0, 33.33, 100.0, 333.3 and 1000 µg/ml with and without metabolic activation (S9-mix). Without metabolic activation cytotoxicity was indicated at 100, 333.3 and 1000 µg/ml; at 33.33µg/ml a survival of 58% was indicated. With metabolic activation cytotoxicity was noted at 1000 µg/ml; relative survival at 333.33µg/ml was 18 %. Based on the findings from the cytotoxicity assay a concentration range of 10 to 300µg/ml (with metabolic activation) and 1 to 50µg/ml (without metabolic activation) was evaluated in the mutation assay. No increase in the mutation frequency was observed. The authors concluded that the test substance MBT was negative under the experimental conditions used.

The negative finding in the HGPRT assay was confirmed in another mammalian cell mutation assay. No significant increase in mutation frequency was noted in a mouse lymphoma assay (WTR 1997).

In vivo data

There are only limited data available for in vivo genotoxicity. In vivo genotoxicity data from MBT was used for supporting reasons.

The genotoxic potential of ZMBT was evaluated in an in vivo bone marrow chromosome aberration assay (Mohanan 2000); however the publication is limited documented and the study design used in not in line with current guidelines. Swiss albino mice (4 animals per group) were administered with ca. 24, 43 and 96 mg/kg bw test substance once via gavage. Concurrent solvent control (cotton seed oil) and positive control (methyl methane sulphonate, 200 mg/kg) were also included in the study. No clinical signs or other observations were recorded in the publication. Colchicine was administered 90 minutes before scheduled sacrifice. All animals were sacrificed 36 hours after test sample application. Bone marrow cells from both femora were prepared, fixed and stained. 100 well-spread metaphases were microscopically analysed for chromosomal aberrations (only gaps and breaks, no detailed record). No cytotoxicity parameters (e.g. mitotic index) were recorded. The incidences of chromatid and chromosome gaps and breaks documented in all treatment groups were comparable to the solvent control. The authors concluded that ZMBT did not induce structural chromosomal aberrations in bone marrow cells of Swiss mice under the experimental conditions used.

Read across with MBT

The genotoxic potential of MBT was evaluated in an in vivo micronucleus assay with CD-1 mice (4 males and 4 females per group) (CMA 1984). 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 (CMA 1989). 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.

Based on the limited data from the ZMBT and the supporting data from the MBT no gentoxic potential of ZMBT was revealed.

In addition, data from the zinc ion Zn2+, the second component of ZMBT, did not give a clear evidence of in vivo genotoxicity (EU risk assessment 2004, MAK 2009).


Short description of key information:
The test substance ZMBT was investigated for its potency to induce gene mutation in bacteria. ZMBT was negative in a bacterial gene mutation study. There are no mammalian cell mutation data available for ZMBT. A read-across approach was performed with in vitro genotoxicity data from MBT. No mutagenic response was noted in a HGPRT assay, which was done with CHO cells (CMA 1984). The test substance was also negative in a mouse lymphoma assay (WTR 1997).
No genotoxic effects of ZMBT were noted in an in vivo bone marrow cell chromosomal aberration assay with Swiss albino mice (Mohanan 2000); however, the publication is limited documented and the study design used is not in line with current guidelines. However, supporting in vivo data from MBT also indicated a non-genotoxic potential. No in vivo gentoxic potential of MBT was observed in an in vivo micronucleus assay (CMA 1984) and in a dominant lethal assay (CMA 1989).

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).