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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genetic toxicity studies have been conducted on MTDID 15670. The results of the studies are:

 

Bacterial Reverse Mutation Assay: Negative when tested according to OECD 471 (1997).

 

Bacterial Reverse Mutation Screening Assay: Negative when tested according to OECD 471.

 

Mouse Lymphoma Assay: Negative when tested according to OECD 490.

Additional information

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Phenobarbital and B-napthoflavone). The study was performed in compliance with OECD GLP (1997). The test method was based on OECD No. 471 (1997) and EC No. 440/2008 Part B (2008). The test article was dissolved in dimethyl sulfoxide. A dose range-finding test was performed with concentration up to 5000µg/plate in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was dosed in experiment 1 (direct plate assay) at concentrations of 1.7, 5.4, 17, 52, 164, and 512µg/plate in the absence of S9 and at 5.4, 17, 52, 164, 512, and 1600µg/plate in the presence of S9 in the TA1535, TA1537 and TA98 strains. In order to obtain more information, experiment 2 (pre-incubation assay) exposed TA 1535, TA1537, TA98, and TA100 to concentrations of 10, 25, 50, 100, 200, and 400 μg/plate in the presence and absence of S9. E. coli WP2uvrA was exposed to concentrations of 100, 200, 400, 750, and 1500 μg/plate in the presence and absence of S9. A follow-up third experiment (experiment 3; pre-incubation assay) was performed to evaluate lower concentrations in the absence of S9-mix; TA1535, TA1537, TA98 and TA100 were exposed to concentrations of 0.01, 0.1, 1, 10, and 100 μg/plate in the absence of S9. Strain specific positive controls and negative (vehicle) controls were tested in parallel. All treatments were performed in triplicate. Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the S. typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the E. coli strain contained 15 μg/plate tryptophan. Top agar consisted of Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C. All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.8 – 38.8°C). After this period, revertant colonies (His+) for S. typhimurium and (Trp+) for E. coli were counted. In experiment 1 (TA1535, TA1537 and TA98 strains via direct plate assay), precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate. A reduction of the bacterial background lawn and a biologically significant decrease in the number of revertants were observed in all tester strains. No increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 2 (TA 1535, TA1537, TA98, and TA100 and WP2uvrA via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate to extreme toxicity was observed at all tested concentrations in the absence of S9-mix. Moderate to extreme toxicity was also observed at test item concentrations including or above 50 μg/plate with S9-mix; experiment 3 was performed to evaluate lower concentrations in the absence of S9-mix. In the pre-incubation test, no increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 3 (TA1535, TA1537, TA98 and TA100 without S9 via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate toxicity was observed at concentrations ≥10 μg/plate. In the pre-incubation test, no increase in the number of revertants was observed upon treatment without metabolic activation. All criteria for a valid test were met as described in the protocol. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the E.coli reverse mutation assay with and without metabolic activation (S9-mix).  

 

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 in the presence and absence of a metabolic activation system (S9-mix from rats).  The GLP status of this study was not specified. The test method was based on the protocol of Ames et al. (1973) and OECD 471. The test article was dissolved in ethanol. A dose range-finding test was performed with concentration up to 5000 µg/mL in the absence and presence of metabolic activation. Based on the results of the dose range finding test, the test article was dosed at concentrations of 0, 1.5, 5, 15, 50, and 150 µg/mL in the presence and absence of S9; the test article was toxic towards the tester strains at the top dose levels, therefore, 150µg/mL was chosen as the top dose level in the mutation test. Each culture was run in duplicate and the experiment was run twice. The following procedure was carried out for each strain. Aliquots of bacterial suspension (0.1 mL) and sterile 0.1 M sodium phosphate buffer (pH 7.4; 0.5 mL) were added to each of one set of sterile bijou bottles. The test compound (0.1 mL) was added to the cultures at five concentrations; negative control (solvent) and positive controls were included. Histidine-deficient agar (2.0 mL) was added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing minimal agar (15 mL). Plates were incubated for 72 hours at 37°C. For cultures with metabolic activity, S9 mix (0.5 mL) was added to the bottle in place of sterile buffer. Colonies were counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group were assessed. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test article at any dose level with and without metabolic activation. Negative (solvent) and positive controls responded as expected. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay (Ames assay) with and without metabolic activation (S9-mix).  

 

 

The potential mutagenicity of the test article was evaluated in L5178Y TK+/- cells in the presence or absence of metabolic activation (S9 phenobarbitol and beta-naphthoflavone induced rat livers) in an in vitro mammalian cell gene mutation test. The study was performed in accordance with GLP (OECD 1997). The test method was based on OECD Guideline 490 (2015). The study was performed using test material dissolved in DMSO in the presence of S9 for 3 hours (5, 10, 25, 50, 65, 80, 100, 115, 130 and 150 μg/ml) and in the absence of S9 for 3 hours and 24 hours (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 60 μg/ml) based on range finding studies. Due to the number of small colonies in the positive control group at 24 hours in the absence of S9, acceptability criteria were not met and this portion of the experiment was rejected; this experiment was repeated and the positive control performed as expected. The following concentrations were selected to measure mutation frequencies: 5, 10, 15, 25, 30, 35, 40 and 45 μg/ml in the absence of S9; 10, 25, 50, 80, 100, 115, 130 and 150 μg/ml in the presence of S9. In a second mutagenicity test, the following concentrations in the absence of S9 (24 hour exposure) were chosen to obtain more information on the possible mutagenicity of the test article: 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50 and 65 μg/ml. To measure mutation frequencies, the following concentrations were chosen based on the dose range finding study and experiment 1: 0.1, 0.5, 1, 5, 10, 20, 30 and 40 μg/ml. Following the mutation assays, the number of mutation frequencies at the TK locus in the presence and absence of S9 were not increased in the exposed group when compared to controls in any experiment. Based on the results of the study, the test material was not mutagenic in the mouse lymphoma assay using L5178Y TK+/- cells in the presence or absence of metabolic activation.   

Justification for classification or non-classification

Criteria for classifying the test article as mutagenic are not met.