Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Endpoint summary

Additional information

The genotoxic potential of Triafamone has been investigated in a series of in-vitro and in-vivo studies. All the studies complied with OECD guidelines and GLP requirements.

In-vitro studies

According to OECD 471 (1997) and under GLP conditions, two Ames tests were performed to investigate the potential of Triafamone to induce gene mutations according to the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Both assays were performed in two independent experiments both with and without liver microsomal activation.

In the first study (M-464325-01-1), Triafamone was tested in the plate incorporation test in concentrations ranging from 3 to 5000 µg/plate and in the pre-incubation test from 33 to 5000 µg/plate. The test substance precipitated in the overlay agar in the test tubes at 2500 and 5000 µg/plate with S9 -mix in the plate incorporation test and at 5000 µg/plate with and without S9 -mix in the pre-incubation test. However, the undissolved particles had no influence on the data recording. The plates incubated with the test substance showed normal background growth up to 5000 μg/plate both with and without S9 mix in all tester strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of S9 -mix. All positive controls were valid. Therefore, Triafamone is considered to be non-mutagenic in the Ames test.

In the second Ames test (M-370265-01-1), Triafamone was investigated in the plate incorporation method in concentrations from 16 to 5000 µg/plate. An independent repeat was performed as preincubation. There was no evidence mutagenic activity following incubation with Triafamone. The positive controls had marked mutagenic effects. Therefore, Triafamone is considered to be non-mutagenic without and with S9-mix in the plate incorporation as well as in the pre-incubation modification of the Salmonella/microsome test.

The clastogenic potential of Triafamone was evaluated in a chromosome aberration test in Chinese hamster V79 cells (M387133-01-1). According to OECD 473 (1997) and under GLP conditions, V79 cells were exposed for 4 h to Triafamone concentrations of 125, 250, 500, 600 and 700 µg/mL with and without metabolic activation. Cultures of all concentrations were harvested 18 h after the beginning of the treatment. In addition, cells treated with 500, 600 and 700 µg/mL were harvested 30 h after the beginning of the treatment. Without S9 mix an additional experiment was performed using continuous treatment for 18 h, harvest at the same time, and concentrations of 50, 100, 200, 300 and 400 µg/mL. Based on cytotoxicity and precipitation in the medium, concentrations of 125, 250 and 600 µg/mL Triafamone (4 h treatment) and 50, 100 and 300 µg/mL (18 h treatment) were chosen for reading in the absence of S9 mix. In the presence of S9 mix 125, 250 and 700 µg/mL Triafamone were used. None of the cultures treated with Triafamone in the absence and in the presence of S9 mix showed biologically relevant increased numbers of aberrant metaphases. The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the used S9 mix. Under the experimental test conditions, Triafamone is considered not to be clastogenic for mammalian cellsin-vitro.

In accordance with OECD 476 (1997) and under GLP conditions, Triafamone was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay; HPRT test) in V79 cells in concentrations ranging from 25 µg/mL to 800 µg/mL without and with S9 mix (M-387854-01-1). Under both activation conditions, no cytotoxic effects of 80% to 90% were induced. However, the test substance was tested up to at least its solubility limit in the medium. The test substance induced no biologically relevant increases in mutant frequencies. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies when compared to the corresponding negative controls. This demonstrated the sensitivity of the test system and the activity of the used S9 mix. Neither with nor without S9 mix indications of mutagenic effects of the test substance could be found in the HPRT forward mutation assay. Therefore, Triafamone was evaluated as non-mutagenic in V79 cells under the experimental conditions.

In-vivo studies

The micronucleus (MN) test was used to investigate Triafamone in mice for a clastogenic effect on the chromosomes of bone-marrow erythroblasts (M-391169-01-1). In accordance with OECD 474 (1997) and under GLP conditions, male and female mice treated with Triafamone received two intraperitoneal administrations of 500, 1000 and 2000 mg/kg bw in 0.5% aqueous Cremophor, separated by 24 h. The femoral marrow of all groups was prepared 24 hours after the last administration.

Males and female Crl: NMRI BR mice treated twice with Triafamone in doses up to 2000 mg/kg bw showed symptoms of toxicity after administration, starting at 500 mg/kg bw. All animals survived until the end of the test. An altered ratio between polychromatic and normochromatic erythrocytes was seen in both male and female animals. Together with the observation of symptoms of toxicity, this finding demonstrates relevant systemic exposure of the animals to Triafamone.

After treatments of males and females with doses up to and including 2000 mg/kg bw, no indications of a biologically relevant clastogenic effect of Triafamone were found. The slight increase in micronucleated polychromatic erythrocytes observed in male animals, which reached statistical significance (p < 0.05) at the lowest tested dose of 500 mg/kg bw, was considered to reflect biological variability, as the effect did not show a dose-response relationship and could not be reproduced in an independent second evaluation. Cyclophosphamide, the positive control, had clear clastogenic effects. For both sexes the ratio of polychromatic to normochromatic erythrocytes was not altered. Under the experimental test conditions, Triafamone did not induce a clastogenic effect on the chromosomes of bone-marrow in mice.

Triafamone was assessed in thein-vivoUDS (unscheduled DNA synthesis) assay for its potential to induce DNA repair in the hepatocytes of male and female rats according to OECD 486 (1997) and under GLP conditions (M-447199-01-1). The test substance was dissolved in 30% DMSO and 70% PEG 400. After a single oral treatment of 1000 and 2000 mg/kg bw and a post-treatment period of 4 and 16 h, respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 h to 3HTdR (methyl-3H-thymidine), which is incorporated if UDS occurs.

Overall, the viability of the hepatocytes was not substantially affected by thein-vivotreatment with Triafamone. Except the cell viability of the hepatocytes isolated from one male of the 1000 mg/kg bw 16 h, one female of the 1000 mg/kg bw 4 h and one female of the 2000 mg/kg bw 4 h treatment group which were below 60% and therefore the results of these animals were scored but not reported. Ruffled fur, reduction in spontaneous activity, abdominal posture, salivation and diarrhoea were signs of systemic toxicity observed in males. None of the tested dose levels revealed biologically relevant UDS induction in the hepatocytes of the treated animals as compared to the corresponding vehicle controls. The net grain counts were not distinctly increased after treatment of the animals with the test substance at 4 h or 16 h, respectively. Net grain counts obtained after treatment with the test substance remained consistently negative at all tested doses. In addition, no substantial shift to higher values of percentage of cells in repair was reported. Treatment with positive controls revealed distinct increases in the number of nuclear and net grain counts.In conclusion, it can be stated that under the experimental conditions reported up to the maximal tolerated dose of 2000 mg/kg bw Triafamone did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Overall, the in-vitro and in-vivo results clearly showed that Triafamone is devoid of a genotoxic potential.

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in-vitro and in-vivo genetic toxicity studies were negative.

Short description of key information:
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102 negative with and without metabolic activation (according to OECD 471)
Chromosome aberration in V79 cells (chromosome aberration test): negative with and without metabolic activation (according to OECD 473; -S9 mix: concentrations of 125, 250 and 600 µg/mL (4 h treatment) and 50, 100 and 300 µg/mL (18 h treatment); +S9 mix: concentrations of 125, 250 and 700 µg/mL (4 h treatment))
Gene mutation in V79 cells (HPRT test): negative with and without metabolic activation (according to OECD 476; concentrations of 25, 50, 100, 200, 400, 600,800 µg/mL with and without S9 mix)

In vivo:
Chromosome aberration (micronucleus assay): negative (according to OECD 474; 3 dose levels (2x500, 2x1000, 2x2000 mg/kg bw; preparation 24 h after the last treatment) intraperitoneally injected to mice)
DNA repair (unscheduled DNA synthesis): negative (according to OECD 486; 2 dose levels (1000 and 2000 mg/kg bw; 4 and 16 h preparation intervals) orally administered to rats)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.