Dibismuth trisulphide

Endpoint summary

Additional information

Ames test

In compliance with the OECD Guideline No. 417 and EU Method B.13/14, five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Dibismuth trisulfide in two independent experiments,

in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls (were within the corresponding historical control data ranges). The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

No substantial increases or decreases (or any sign of cytotoxic effect of the test item) were observed in revertant colony numbers of any of the five test strains following treatment with Dibismuth trisulfide at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations (with and without metabolic activation) by frameshift or base-pair substitution in the genome of the strains used. Therefore, the Dibismuth trisulfide is considered non-mutagenic in this bacterial reverse mutation assay.

Mouse Lymphoma

In the mutation assay according to OECD guideline 476 the cells were treated with a range of the test item concentrations for 3 hours in absence and also in presence of S9 Mix and for 24 hours in absence of S9 Mix. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. Cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

In the Assay 1 as well as in the Assay 2 the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with the historical data.The parallel tested positive control chemicals induced a statistically significant increase in the mutant frequency (2 Sample t-Test, α = 0.01). Additionally, the induced mutation frequency (IMF: number of mutants over the control value per 106 viable cells) exceeded the global evaluation factor (GEF), which is equal to 126.

When compared to the vehicle control dose–related and statistically significant changes in mutation frequencies were observed in the Assay 1 in absence and presence of exogenous metabolic activation and in the Assay 2 in the presence of metabolic activation. The obtained mutation frequencies remained unequivocally below the GEF criterion for positive response in the Assay 1. For Assay 2 the mutation frequencies were found to be above the GEF criterion at concentrations ≥ 2500 μg/mL. Heavy precipitate was noted at the concentration levels where statistical significances were noticed (except the concentration level of 1580 μg/mL, in Assay 2, in presence of S9, where the statistical significance was not attained by precipitate). The precipitate character allowed unequivocal identification, therefore it can be stated that the precipitate did not disturb the evaluation itself.

However, testing compounds in the precipitating range is problematical with respect to defining the exposure periods for assays where the cells grow in suspension. After the defined exposure period, the cells are normally pelleted by centrifugation and are then re-suspended in fresh medium without the test compound. If a precipitate is present, the compound is carried over through to the later stages of the assay, and the exact control of exposure is not possible; therefore the obtained mutagenicity results were treated with extra care and a third confirmatory Assay 3 was considered as necessary. In Assay 3 mutation frequencies remained unequivocally below the GEF criterion for positive response, hence the equivocal positive results of the Assay 2 were not confirmed.

The examined concentrations covered the cytotoxicity range from the 90-80 % cytotoxicity to no toxicity in Assay 1 and Assay 2. To fulfil the cytotoxicity criterion of the assay, the test item was investigated beyond its limit of solubility under culture conditions. The chosen concentrations in Assay 3 cover the critical concentration range in precipitation as well as in high mutation frequencies points of view.

The final conclusion was drawn from the results of the statistical and biological evaluation.The Assays with Dibismuth trisulfide in Mouse Lymphoma L5178Y TK +/- 3.7.2 C cells were considered valid and met acceptance criteria.

Under the conditions of this study, the test item dibismuth trisulfide does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Chromosome Aberrations test (in vitro):

A chromosome aberration test of dibismuth trisulfide is not available. Consequently, read-across from the inorganic bismuth citrate was performed. Bismuth citrate did not induce chromosome aberrations up to the highest tested concentration of 250 µM. Bismuth citrate did not show any genotoxic effect. In contrast to inorganic bismuth compounds, organic bismuth compounds might show genotoxic potential. Therefore, dibismuth trisulfide was considered to be non-genotoxic in this test system.

Justification for selection of genetic toxicity endpoint
Most reliable study in mammalian cells.

Short description of key information:
The test item is considered non-mutagenic in the bacterial reverse mutation assay. Dibismuth trisulphide induced no gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used. Due to read-across from bismuth citrate dibismuth trisulfide is considered to be non-genotoxic effect in the chromosome aberration test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained in the in vitro studies dibismuth trisulphide is considered to be non-genotoxic, non-mutagenic or non-clastogenic. Therefore it has not to be classified according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.