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Additional information

The mutagenic potential of 2 -methyl-N-(4 -sulfamoylphenyl) prop-2-enamide (SPM-N) was assessed in three in vitro tests.

A bacterial reverse mutation assay (Ames test) was performed according to OECD Guideline 471 and in compliance with GLP (Verspeek-Rip, 2007). The plate incorporation method was applied using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2uvrA at concentrations from 3 - 5000 µg/plate, with and without metabolic activation. The test substance did not induce mutations in any of the tested strains, with or without metabolic activation. No cytotoxic effects were observed up to and including the limit value of 5000 μg/plate. Precipitation was noted from 3330 µg/plate, with and without metabolic activation. The positive controls were shown to be valid.

 

The potential of SPM-N to induce chromosomal aberrations was tested in cultured human peripheral lymphocytes according to OECD Guideline 473 and in compliance with GLP (Buskens, 2007). The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h exposure time) or on toxicity (24 and 48 h continuous exposure time), defined as inhibition of the mitotic index of 50% or greater. In the first experiment, short-term treatment (3 h) was performed with a 24 h harvest time, at concentrations of 33, 100 and 333 µg/mL in the presence and absence of metabolic activation. In the second experiment, a 24 h treatment with 24 h harvest time at concentrations of 100, 333 and 500 µg/mL without metabolic activation and a 48 h treatment with 48 h harvest time at concentrations of 33, 100 and 200 µg/mL without metabolic activation were performed. Cytotoxicity was noted at the highest dose levels in the continuous exposure time experiments (24 h and 48 h). In the presence of metabolic activation SPM-N was tested at concentrations of 33, 100 and 333 µg/mL for a 3 h exposure time with a 48 h fixation time. Precipitation of the test substance in the culture medium was observed at a concentration of 333 µg/mL and above. There were no increases in the number of chromosome aberrations at any dose level. Positive controls induced a significant increase in the number of chromosomal aberrations, indicating the sensitivity of the assay. Based on these results SPM-N is considered not to be clastogenic in human lymphocytes.

SPM-N was tested in a mammalian cell gene mutation assay under GLP according to OECD Guideline 476 (Lazová, 2013). Based on the results of a cytotoxicity range-finding study two main mutation experiments were performed, each conducted with duplicate cultures (A and B) in the absence and presence of metabolic activation. Chinese hamster lung V79 cells were exposed to SPM-N at concentrations ranging from 25 to 400 µg/mL in experiment 1 and from 6.25 to 200 µg/mL in experiment 2. The upper dose concentration was limited by solubility; the test substance precipitated in the treatment level at concentrations >250 µg/mL. The treatment period for all cultures was 3 hours. In one culture each of experiment 1 with and without metabolic activation statistically significant increases (p < 0.01) in mean mutant frequency were observed. Since these increases were not dose-dependent and since no increase in mutant mutation frequency was identified neither in the second culture nor in the independent experiment 2, the statistical significances were considered to be biologically irrelevant. In experiment 2, for one culture without S9 mix a statistically significant increase in mutant frequency was observed at a concentration of 12.5 µg/mL only. This increase in MMF was not greater than 3-fold above the concurrent vehicle control and with no evidence of an increase in mutant frequency at any of the other test concentrations. No cytotoxicity was observed in any of the experiments. The positive controls clearly increased mutant frequency, demonstrating the validity of the test system. In conclusion, SPM-N did not induce gene mutations in vitro at the HPRT locus of V79 Chinese hamster lung cells when tested under the conditions of this study.

In conclusion, SPM-N showed no evidence of clastogenic or mutagenic potential with and without metabolic activation in 3 in vitro test systems.


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

Short description of key information:
In vitro:
Bacterial reverse mutation assay, Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in E. coli WP2uvrA
Mammalian cytogenetic test, chromosome aberrations (OECD 473): negative with and without metabolic activation in human peripheral lymphocytes
Gene mutation in mammalian cells, HPRT (OECD 476): negative with and without metabolic activation in V79 cells

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.