Thioacetic acid

Endpoint summary

Additional information

Gene mutations

The potential of THIOACETIC ACID to induce reverse mutation was evaluated in Salmonella typhimurium.

A preliminary toxicity test was performed to define the dose-levels of THIOACETIC ACID to be used for the mutagenicity study. THIOACETIC ACID was then tested in three independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. The experiments were performed according to the direct plate incorporation method except the second and the third experiments with S9 mix, which were 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 substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. THIOACETIC ACID was dissolved in dimethylsulfoxide (DMSO).

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. Since the test substance was toxic, the highest dose-level was based on the level of toxicity, according to the criteria specified in the international regulations: reduction in the number of revertants and/or clearing of the bacterial lawn. The selected treatment-levels were for the first experiment: 3, 10, 30, 100, 300 µg/plate. As no toxicity was observed at 300 µg/plate, the dose-levels were increased for the second experiment: 62.5, 125, 250, 500, 1000 µg/plate. Moderate to important toxicity was noted without S9 mix in the five strains at dose-levels higher than 125 µg/plate and with S9 mix in the TA 1535, TA 1537 and TA 100 at dose-levels higher than 250 µg/plate. In the TA 98 and TA 102 strains with S9 mix, only slight toxicity was noted. Therefore as only 3/5 dose-levels were available, a third experiment was performed with the same treatment-levels as in the first experiment. Slight (TA 102) or severe (the four other strains) toxicity was observed at 300 µg/plate without S9 mix. No toxicity was observed with S9 mix in TA 1535, TA 1537 and TA 100 strains. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains.

THIOACETIC ACID did not show mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

Chromosomal aberrations

The potential of THIOACETIC AC1D to induce chromosome aberrations was evaluated in cultured human lymphocytes. The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37 °C in a humidified atmosphere of 5% CO2 / 95% air, for 48 hours. In the first experiment, lymphocyte cultures were exposed to the test or control substances, with or without S9 mix, for three hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hours before harvest, each culture was treated with a colcemid solution (10 pg/ml) to block cells at the metaphase-stage of mitosis. As this first experiment gave equivocal results, the study was continued with a second experiment. In the second experiment, without S9 mix, cells were exposed continuously to the test or control substances, with S9 mix, cells were exposed to the test or control substances for three hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. In order to check the reliability of the significant increase in the frequency of cells with chromosomal aberrations noted at the highest dose-level in the first experiment (without S9 mix after 3-hour treatment), a third experiment without S9 mix was performed, under the same experimental conditions, using a closer range of dose-levels. For all experiments, after hypotonic treatment (KC1 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The dose-levels of the positive controls were as follows:

. without S9 mix, mitomycin C: 3 p g/ml (3 hours of treatment) or 0.2 p g/ml (continuous treatment),

. with S9 mix, cyclophosphamide: 50 pg/ml.

At the dose-level of 10 mM, the pH and the osmolality were equivalent to those of the vehicle control.

Experiments without S9 mix:

With a treatment volume of 15 µl/5.5 ml culture medium, the treatment-levels were as follows:

. 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment,

• 0.156, 0.235, 0.3125, 0.469, 0.625, 0.9375 and 1.25 mM for the second experiment,

• 0.625, 0.9375, 1.25, 1.875, 2.5, 3.75 and 5mM for the third experiment.

No precipitate was observed at the end of the treatment period at 10 mM.

In the first experiment, a slight to complete toxicity was induced at dose-levels 0.625 mM. Chromosomal aberrations analysis were scored on the slides corresponding to the dose-levels of 0.625, 1.25 and 2.5 mM.

A slight and significant increase (p < 0.05) in the frequency of cells with chromosomal aberrations was noted at 2.5 mM. This increase was mainly related to the male donor for whom a strong toxicity was recorded and therefore this increase was not considered as biologically relevant.

In the second experiment, the test substance was moderately to strongly toxic for the 20-hour harvest time and slightly to strongly toxic for the 44-hour harvest time.

Analysis of chromosomal aberration was performed at the dose-levels of 0.235, 0.3125 and 0.469 mM, for the 20-hour harvest time and at 0.469 mM, for the 44-hour harvest time. No significant increase in the frequency of cells with chromosomal aberrations was noted at both harvest times.

In order to check the reliability of the significant increase of cells with chromosomal aberrations noted in the first experiment, a third experiment was performed under the same experimental conditions, using a closer range of dose-level.

In the third experiment, a slight to strong toxicity was induced at all dose-levels.

Analysis of chromosomal aberrations was performed at the dose-levels of 0.625 and 0.9375 mM. In this experiment, a slight but a non significant increase in the number of cells with chromosomal aberrations was noted at 0.625 and 0.9375 mM.

Considering that, after 3-hours treatment, the increase of the frequency of aberrant cells was neither reproducible between cultures (in both the first and third experiments) nor statistically significant (third experiment) and taking into account that no increase in the frequency of aberrant cells was noted when experimental conditions were changed (20-hours and 44-hours treatments in the second experiment) it was concluded that no clear evidence of a clastogenic effect of the test substance was demonstrated.

Experiment with S9 mix:

With a treatment volume of 15 µ1/5.5 ml culture medium, the treatment-levels were as follows:

. 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment,

. 0.156, 0.3125, 0.625, 1.25, 1.875 and 2.5 mM for the second experiment.

In the first experiment, a slight to complete toxicity was induced at all tested dose-levels.

In the second experiment, the test substance was slightly toxic for the 20-hour harvest time, at dose-levels>1.25 mM and moderately toxic for the 44-hour harvest time, at the highest dose-level.

The dose-levels selected for chromosomal analysis were:

• 0.625, 1.25 and 2.5 mM for the 20-hour harvest time in the first experiment,

• 1.25, 1.875 and 2.5 mM for the 20-hour harvest time in the second experiment,

• 2.5 mM for the 44-hour harvest time in the second experiment.

No significant increase in the frequency of cells with chromosomal aberrations was noted at both harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

No evidence of a clastogenic effect of THIOACETIC ACID in cultured human lymphocytes was demonstrated.

Short description of key information:
Thioacetic acid produced no genetic changes in standard in vitro tests  using bacterial and human cells and performed according to OECD  guidelines and GLP.  No gene mutations were induced in Salmonella typhimurium strain TA 1535,  TA 1537, TA 98, TA 100 and TA 102 and no evidence of a clastogenic  effect in cultured human lymphocytes was demonstrated.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to REGULATION (EC) No 1272-2008 and Annex VI of Commission Directive 2001/59/EC

: not classified

Based on the battery of genetic toxicology studies that have been conducted with thioacetic acid, this material is not a mutagen.