2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

Endpoint summary

Additional information

The mutagenic potential of of 2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1 H-pyrrolo[1 ,2-b][1 ,2,4]triazole-7-carboxylic acid-2,6-di-tert-butyl-4-methyl-cyclohexyl ester (UC-141) was assessed in three in vitro tests.

An in vitro bacterial reverse mutation assay (Ames test) was performed using UC-141 according to OECD guideline 471 (Buskens, 2003). The preincubation method was applied using S. typhimurium strain TA 100 and E. coli WP2 uvrA at concentrations up to the limit value of 5000 µg/plate with and without metabolic activation; and S. typhimurium strains TA 1535, TA 1537 and TA 98 at concentrations up to the precipitation limit of 1000 μg/plate,with and without metabolic activation. The test substance did not induce mutations in any of the S. typhimurium strains or in E. coli WP2 uvrA with or without metabolic activation. No cytotoxic effects were observed up to and including the limit value of 5000 μg/plate, with and without metabolic activation. The positive controls were shown to be valid.

The cytogenetic effects of UC-141 were tested in a chromosome aberration assay with cultured human peripheral lymphocytes, performed according to OECD guideline 473 and in compliance with GLP (Buskens, 2004). The possible clastogenicity of UC-141 was determined in two independent experiments. 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). In the first experiment UC-141was tested at concentrations of 10, 33 and 100 μg/mL for a 3 h exposure time with a 24 h harvest time with and without metabolic activation. Precipitation of the test substance in the culture medium was observed at 100 µg/mL.

In the second experiment the cultures were treated with the test substance at concentrations of 10, 18 and 24 µg/mL for 24 h (harvest time 24 h) and with 3, 10 and 24 µg/mL for 48 h (harvest time 48 h) in the absence of S9 mix, respectively. Cytotoxicity (defined as a more than 50% reduction in mitotic index) was reached at the highest dose level each. In the presence of S9 mix UC-141 was tested at concentrations of 10, 33 and 100 µg/mL for a 3 h exposure time with a 48 h harvest time. At 100 µg/mL the substance precipitated in the culture medium.

There were no increases in the number of chromosome aberrations at any dose level in any of the experiments. The positive controls induced a significant increase in the number of chromosomal aberrations. Thus, based on these results UC-141 is considered not to be clastogenic in human lymphocytes.

Gene mutation of UC-141 in mammalian cells was investigated using a HPRT test according to OECD guideline 476 and GLP (Bednáriková, 2013). Based on the results of a cytotoxicity range-finding study two main mutation experiments were conducted with duplicate cultures in the absence and presence of metabolic activation. Chinese hamster lung fibroblasts (V79) were incubated for 3 hours with test substance concentrations of 31.25, 62.5, 125, 250 and 500 µg/mL culture medium in the absence and presence of metabolic activation. A statistically significant increase (p<0.01) in mean mutant frequency was observed in both cultures with metabolic activation at 125 µg/mL. Since this increase in mean mutant frequency was not greater than 3-fold above that of the concurrent vehicle control and there was no dose-response-relationship, the statistical significance was considered to be biologically irrelevant.The upper dose range was limited by precipitation of the test substance in the culture medium from 600 µg/mL as determined in a solubility test. No precipitation was observed in the main experiments with concentrations up to 500 µg/mL. The test substance did not cause any cytotoxic effect up to the highest concentration tested. The positive controls were valid and induced significant increases in mutation frequency. The values of the solvent control were within the range of the historical control. In conclusion, UC-141 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.

UC-141 showed no evidence of clastogenic and 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
Chromosome aberration assay (OECD 473): negative with and without metabolic activation in human peripheral lymphocytes
Mammalian cell gene mutation test, HPRT test (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 the mutagenic 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.