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In Vitro Gene Mutation Study in Bacteria

In the key study, the mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.

 

The study was both performed and reported to a high standard. As such it was deemed acceptable to assign the study a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997).

 

Supporting information is available on the structural analogue dioctyltin oxide (DOTO). In the supporting study, the mutagenicity of DOTO was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with the OECD guideline 471 and EPA OPPTS 870.5100 and to GLP. The cultures were exposed to 0, 62, 185, 556, 1667 and 5000 µg/plate. Precipitation of the test material was observed at a number of the concentrations tested. In some of the plates, a slight dose-dependant increase in the number of revertants was noticed; however this was accompanied by an increased background lawn and precipitation. This was therefore not attributed to the mutagenic potential of the test material. Under the conditions of the study, the results obtained with the test material in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, in both the absence and presence of metabolic activation (S9-mix), the test material was not mutagenic. As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997).

 

In vitro Gene Mutation Study

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L5178Y TK+/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxiliary metabolic system (S9 mix). Large and small mutant colonies were scored for all cultures in each experiment. Initially, two independent experiments were performed. In Experiment 1, cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation. However, due to a marked difference in toxicity in the 4-hour exposure groups in the presence of metabolic activation between Experiment 1 and 2, and an apparent maximum exposure being achieved at the penultimate dose level in Experiment 2, a confirmatory Experiment 3 was performed using a 4-hour exposure group at ten dose levels in the presence of metabolic activation (1 % S9) only.

 

The dose range, selected following a preliminary toxicity test, was 3.5 to 112 µg/mL in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 20 µg/mL in the absence of metabolic activation and 20 to 90 µg/mL in the presence of metabolic activation. In Experiment 3 the dose range was 20 to 110 µg/mL in the presence of metabolic activation only.

 

Under the conditions of the test, the maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. Overall, precipitate of test material was observed at and above 28 µg/mL in the mutagenicity test. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK+/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first, second or third experiment. The test material is therefore considered to be non-mutagenic to L5178Y cells under the conditions of this assay.

 

As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.  

 

In vivo Cytogenicity Study

The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of Swiss male mice was assessed in vivo in a study conducted in accordance with OECD 474 and to GLP. At oral doses up to 2000 mg/kg bw (the limit dose recommended by the guideline) no chromosomal damage or damage to the mitotic spindle apparatus was noted. Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.

 

As the study was performed on a read-across substance, the study was assigned a reliability score of 2 (reliable with acceptable restrictions) in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.


Justification for selection of genetic toxicity endpoint
As multiple studies are presented to address genetic toxicity, no one study was selected as the key study as they represent different types of genetic toxicity and are therefore not comparable.

Short description of key information:
In vitro Gene Mutation Study in Bacteria
Negative, OECD 471, EU Method B.13/14, Thompson 2012

In vitro Gene Mutation
Negative, dioctyltin oxide, L5178Y TK+/-, OECD 476, EU Method B.17, EPA OPPTS 870.5300, Flanders 2012

In vivo Clastogenicity Study
Negative, dioctyltin oxide, OECD 474, de Vogel 2004

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification and labelling as defined in Regulation (EC) No. 1272/2008 (CLP) and Directive 67/548/EEC (DSD), the test material does not require classification for genetic toxicity.