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

In Vitro Bacterial Reverse Mutation Assay

The mutagenic potential of for 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 an in vitro bacterial reverse mutation assay (Ames test), which was performed according to OECD 471 (Buskens, 2003).

The plate incorporation method was applied using S. typhimurium strain TA 100 and E. coli WP2 uvr A 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 uvr A 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.

In Vitro Chromosome Aberration Assay

The potential clastogenic activity of the test material was determined in vitro in a chromosome aberration assay using Chinese hamster lung (V79) cells (Hargitai, 2014). The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473 and EU Method B.10. The study included three concentration selection cytotoxicity assays and two chromosome aberration assays.

In the main test, cell cultures were exposed to the test material formulated in DMSO up to cytotoxic concentrations and/or the solubility limit according to the OECD guideline recommendations. The two independent assays used duplicate cultures and at least 200 well-spread metaphase cells (or until a clear positive response was detected) for analysis of each test concentration, negative (vehicle) and positive control sample.

In chromosome aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 2000, 1000, 500, 250, 125 and 62.5 μg/mL.

In chromosome aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 40, 20, 10, 5, 2.5, 1.25, 0.625 and 0.313 μg/mL without metabolic activation; and 2000, 1000, 500, 250, 125 and 62.5 μg/mL with metabolic activation.

None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay with and without metabolic activation when compared with the appropriate negative (vehicle) control values.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1 - 4) were found in some cases in the negative (vehicle) control, positive control or test material treated samples in the performed experiments. No endoreduplicated metaphases were found in the main tests.

Under the conditions of the test, the test material did not induce a significant level of chromosome aberrations and is therefore considered to be not clastogenic in this test system.

In Vitro Mammalian Cell Gene Mutation Assay

The potential for the test material to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (6-thioguanine (6TG) resistance) was assessed using Chinese hamster lung V79 cells in an in vitro mammalian cell gene mutation assay (Bednáriková, 2013). The study was performed under GLP conditions and in accordance with the standardised guideline OECD 476.

The study consisted of a cytotoxicity range-finder experiment followed by two main mutation experiments, conducted with duplicate cultures in the absence and presence of metabolic activation. The activation system consisted of 20-methylcholanthrene-induced rat liver post-mitochondrial fraction (S9-mix). The test material was dissolved in acetone:ethanol (1:1) and dosed at a final concentration of 1 % v/v.

The test material was dosed at five concentrations ranging from 31.25 to 500 µg/mL in both the presence and absence of metabolic activation for a 3 hour exposure period. The maximal concentration tested was limited by solubility and precipitation of the test material in the treatment medium. Concurrent negative, vehicle and positive controls were carried out. Following an expression period of 7 days, plating took place to determine survival, viability and 6TG resistance.

Under the conditions of the test, exposure to the test material did not induce mutation at the HPRT locus of V79 Chinese Hamster lung cells up to a concentration of 500 µg/mL in both the absence and presence of metabolic activation.

Justification for selection of genetic toxicity endpoint
One single study could not be selected at key, since all three studies are necessary in addressing this endpoint. The three studies assess different types of in vitro genetic toxicity; bacterial gene mutation, mammalian chromosome aberrations and mammalian gene mutation. All three studies were performed under GLP conditions and in accordance with standardised guidelines. Accordingly these studies have been assigned a reliability score of 1 in line with Klimisch et al. (1997).

Short description of key information:
In vitro:
Bacterial reverse mutation assay, Ames test (OECD 471): negative
Chromosome aberration assay (OECD 473): negative
Mammalian cell gene mutation assay (OECD 476): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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