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Bacterial reverse mutation assay /test (OECD 471):

The potential of 4 -methylthiosemicarbazide to induce reverse mutation was evaluated in Salmonella typhimurium.The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14)and in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of MTSC to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The test item MTSC was dissolved in dimethylsulfoxide (DMSO). The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level for the main test was 5000μg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000μg/plate, for both mutagenicity experiments with and without S9 mix. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Under these experimental conditions, the test item MTSC did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

 

 

In vitro mammalian cell gene mutation assay / Mouse lymphoma assay (OECD 476):

After a preliminary toxicity test, the test item 4-Methylthiosemicarbazide, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were mainly as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid. Since the test item was freely soluble and non-severely toxic in the preliminary test, the highest dose-level selected for the main test was 10 mM, according to the criteria specified in the international guidelines.

 Using a treatment volume of 0.5% in culture medium, the selected dose-levels for both mutagenicity experiments were 0.31, 0.63, 1.25, 2.5, 5 and 10 mM both with and without S9 mix.

In the experiments without S9 mix : Following the 3-hour treatment, a slight toxicity was induced at 10 mM, as shown by a 39% decrease in Adj. RTG.

Following the 24-hour treatment, a moderate to severe toxicity was induced at dose‑levels ≥ 2.5 mM, as shown by a 54 to 95% decrease in Adj. RTG.

No biologically relevant increase in the mutation frequency was noted in the presence of the test item following the 3- and 24-hour treatments.

In the experiment with S9 mix : In the first experiment, a moderate to marked toxicity was induced at dose-levels ≥ 0.31 mM, as shown by a 53 to 69% decrease in Adj. RTG. In the second experiment, a moderate to marked toxicity was induced at dose-levels ≥ 0.31 mM, as shown by a 42 to 70% decrease in Adj. RTG. Noteworthy, dose-related and reproducible increases in the mutation frequency were induced in the presence of the test item in two independent experiments.

In conclusion, the test item did not show any mutagenic activity in the mouse lymphoma assay in the absence of a rat metabolizing system, while it showed a mutagenic activity in this assay in the presence of a rat metabolising system.

 

 

In vivo micronucleus test in rats (OECD 474):

The objective of this study was to evaluate the potential of the test item, 4‑Methylthiosemicarbazide, to damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats (OECD 474).

Preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of 4-Methylthiosemicarbazide at dose-levels of 2.5, 5 and 10 mg/kg/day, at a 24-hour interval. For the high-dose group only, three supplementary males and three supplementary females were also treated with the test item in case of mortality.

One group of five males and five females received the vehicle (drinking water treated by reverse osmosis) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

In all groups treated with 4-Methylthiosemicarbazide, the mean values of the MPE and the PE/NE ratio were similar to those of their respective vehicle controls, and no statistically significant differences were noted. Therefore, the criteria for a positive response were not met.

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 2.5, 5 and 10 mg/kg/day.

 


Justification for selection of genetic toxicity endpoint
Mutagenicity evaluation concludes that MTSC is not mutagenic.

Short description of key information:
An Ames test and a mouse lymphoma assay were available on MTSC. Ames test (OECD 471) was negative, but the mouse lymphoma assay (OECD 476) was positive with metabolic activation (with a majority of small colonies).
To discard the clastogenic potential of MTSC, an in vivo micronucleus test (OECD 474) was performed on rat by oral route. This test was negative : The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 2.5, 5 and 10 mg/kg/day.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Proposed self-classification

- Regulation (EC) No 1272/2008 : Not classified.

- Directive 67/548/EEC: Not classified