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In vitro bacterial reverse mutation assay

The potential of the test material to cause genetic toxicity was investigated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to the test material in DMSO via the plate incorporation method at dose levels of 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and without S9 activation in a preliminary toxicity and initial mutagenicity assay. The strains were then exposed to 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate in an independent repeat/confirmatory mutagenicity assay.

Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.

 

In vitro mammalian cell gene mutation assay

The genotoxicity potential of the test material was investigated in a mammalian cell gene mutation assay conducted in accordance with the standardised guideline OECD 476 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Mouse lymphoma L5178Y TK+/- cells were exposed to the test material in DMSO at the following concentrations: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3000 µg/mL with and without S9 activation in a preliminary assay; 5, 7.5, 10, 12.5, 15 and 17.5 µg/mL in the 4 hour mutagenicity assay without S9 activation; and 15, 20, 25, 30 35 and 40 µg/mL in the 4 hour mutagenicity assay with S9 activation.

Under the conditions of this study, the test material was considered to be negative with and without metabolic activation in the L5178Y TK+/- mouse lymphoma mutagenesis assay.

 

In vitro mammalian chromosome aberration test

The genotoxicity potential of the test material was investigated in an in vitro mammalian chromosome aberration test conducted in accordance with the standardised guideline OECD 473 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Chinese hamster ovary cells (CHO-K1) were exposed to the test material in DMSO at the following concentrations: 0.296, 0.888, 2.96, 8.88, 29.6, 88.8, 296, 888 and 2960 µg/mL in the preliminary toxicity assay; 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay without S9 activation; and 1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay with S9 activation and 20-hour assay without S9 activation.

Toxicity of DMBPC in CHO cells treated for 4-hours without metabolic (S9) activation was 61 % at 25 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (25 µg/mL) was 10 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 25 µg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control.

Toxicity of DMBPC in CHO cells when treated for 4-hours with S9 activation was 55 % at 15 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (15 µg/mL) was 69 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 5.0 and 15 µg/mL. The percentage of cells with structural aberrations in the test article-treated group was statistically increased above that of the solvent control at all concentrations evaluated. However, the percentage of cells with structural aberrations (5.5 %) in the DMBPC-treated groups at 2.5 µg/mL was within the historic solvent control range of 0.0 to 6.5 %. Therefore, this would not be considered biologically significant. The percentage of structurally aberrant cells at concentrations of 5 and 15 µg/mL, 9.0 and 8.5 %, respectively, fall just outside the historical solvent control range and this is considered to be positive. The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control.

In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20-hour exposure group were evaluated for chromosome aberrations. Toxicity of DMBPC in CHO cells was 31 % at 17.5 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure. The mitotic index at the highest test concentration evaluated for chromosome aberrations (17.5 µg/mL) was 68 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 17.5 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at 17.5 µg/mL. The percentage of cells with structural aberrations in the DMBPC-treated at 17.5 µg/mL group (8.0 %) was outside the historical solvent control range of 0.0 to 6.0 % and would be considered positive for the induction of structural chromosome aberrations. The percentage of cells with numerical aberrations in the DMBPC-treated groups was not statistically significantly increased above that of the solvent controls at any dose level. 

Based on the findings of this study, DMBPC was concluded to be positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group. DMBPC was concluded to be negative for the induction of numerical chromosome aberrations in CHO cells.

 

In vivo micronucleus assay

The potential of the test material to cause in vivo genotoxicity was investigated in a micronucleus assay conducted in accordance with the standardised guideline OECD 474 under GLP conditions.

Male and female ICR mice were exposed to the test material via the intraperitoneal route in corn oil. Following a pilot study and toxicity assay, for the definitive micronucleus assay mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Mice were observed after dose administration for clinical signs of toxicity.

Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow collected and processed. Two slides for examination were prepared from each mouse.

Under the conditions of this study, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.

 

Discussion

DMBPC was negative for mutagenicity in the in vitro bacterial reverse mutation assay (BioReliance, 2001a), as well as the in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay (BioReliance, 2001b). The overall weight-of-evidence demonstrates that DMBPC is not mutagenic.

In the in vitro chromosome aberration assay, DMBPC was weakly positive for induction of structural chromosome aberrations in CHO cells, but it was negative for numerical chromosome aberrations in CHO cells (BioReliance, 2001c). In the in vivo micronucleus assay, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg bw did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow (BioReliance, 2001d). Therefore, DMBPC was concluded to be negative in the in vivo micronucleus test.

The in vitro chromosomal aberration assay can detect large chromosomal damage and aneuploidy, while the in vivo micronucleus assay can detect small chromosomal damage, large chromosomal damage, and aneuploidy (GLP/OECD Guideline 473 and 474). Considering that the in vitro chromosomal aberration assay and the in vivo micronucleus assay detect similar types of chromosomal damage, and that the weakly positive results in the in vitro chromosome aberration assay were not confirmed by the negative results of the in vivo micronucleus test, coupled with the fact that in vivo studies are considered to be the most biologically relevant, the weight-of-evidence demonstrates that DMBPC is not mutagenic.


Justification for selection of genetic toxicity endpoint
No single study was selected as key on the basis that the available data all address different aspects of genetic toxicity.

Short description of key information:
IN VITRO
Negative with and without metabolic activation in S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA (bacterial reverse mutation assay); OECD 471
Negative with and without metabolic activation in L5178Y TK+/- mouse lymphoma cells (mammalian cell gene mutation assay); OECD 476
Positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group (mammalian chromosome aberration test); OECD 473

IN VIVO
Negative in male and female ICR mice (micronucleus assay); OECD 474

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.