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Genetic toxicity in vitro

Description of key information

AMES test (OECD 471, GLP): negative (CCR 651508, 1997; CCR 704428, 1998)

Chromosomal Abberation test (OECD 473, GLP): negative (RCC 597700; 1998)

Genetic toxicity in vivo

Description of key information

Unscheduled DNA Synthesis test (OECD 486, GLP): negative (Institut Pasteur De Lille FSR-IPL 040012; 2004)

Micronucleus test (OECD 474, GLP): negative (CIT 25608MAS; 2003)

Additional information

Mutagenicity in bacteria

Bemotrizinol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in two reverse mutation assays:

Study 1 (CCR 651508; 1997):

This study was performed to investigate the potential of Bemotrizinol to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ug/plate. No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with Bemotrizinol 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.

Study 2 (CCR 704428; 1998):

This study was performed to investigate the potential of Bemotrizinol to induce gene mutations using the Escherichia coli strain WP2 uvrA. The test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 ug/plate. No substantial increase in revertant colony numbers was observed following treatment with Bemotrizinol at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no reproducible tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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

Therefore in a weight of evidence approach, Bemotrizinol is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

Cytogenicity in mammalian cells

CA (in vitro):

In the chosen key study for cytogenicity in mammalian cells acc. to OECD TG 473, Bemotrizinol, dissolved in acetone, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in two independent experiments (RCC 597700; 1998). The exposure period was 4 h with and without metabolic activation or 18 h and 28 h without S9 mix. In the cytogenetic experiments, Bemotrizinol concentrations within a range of 6.5 - 210 µg/ml (without S9 mix) and 3.3 - 210 µg/ml (with S9 mix) were applied for the investigation of the potential to induce cytogenetic damage. No biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with Bemotrizinol. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with Bemotrizinol as compared to the frequencies of the controls.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, Bemotrizinol did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, Bemotrizinol is considered to be non-mutagenic in this chromosome aberration test.

 

 

Genetic toxicity in vivo

UDS (in vivo):

Bemotrizinol was investigated at a dose level of 1000 mg/kg bw and 2000 mg/kg bw and at two expression times (2 -4 hours and 12 -16 hours) in the in vivo unscheduled DNA Synthesis (UDS) test in hepatocytes from 3 male Fischer rats in a study performed in accordance with OECD TG 486 under GLP (Institut Pasteur De Lille FSR-IPL 040012; 2004). Bemotrizinol did not reveal any genotoxic activity under the test conditions and did not induce unscheduled DNA synthesis in hepatocytes.

 

MNT (in vivo):

The ability of Bemotrizinol to cause chromosomal damage in vivo was investigated in a OECD 474 micronucleus test (CIT 25608MAS; 2003). Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Groups of 5 male and female Swiss mice were dosed twice intraperitoneally with vehicle only or with 500 mg/kg bw/ day 1000 mg/kg bw/ day and 2000 mg/kg bw/day Bemotrizinol. Cyclophosphamide served as positive control (oral, 50 mg/kg bw). The animals of the treated and vehicle control groups were killed 24 hours after the last treatment.  

For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with Bemotrizinol, were equivalent to those of the vehicle group. Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

In conclusion, Bemotrizinol did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, with a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day under the chosen testing conditions. Bemotrizinol is therefore not clastogenic or aneuploidic in mice under conditions of this study.

Justification for classification or non-classification

The present data on genetic toxicity do not fulfill the criteria laid down in regulation (EU) 1272/2008 and therefore, a non-classification is warranted.

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