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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay 

In a K2 bacterial reverse mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100, performed according to OECD Guideline 471 (Vanparys et al., 2004), it was concluded that T002488 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of Aroclor 1254 induced rat liver enzymes. This well-documented study is used as key study for in vitro gene mutation in bacteria.

 

 - Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473 (Wright, 2005), T002488 was considered to be non-clastogenic to human lymphocytes in vitro in the absence and presence of rat liver enzyme metabolising system.

-  Gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, performed according to the OECD Guideline 490 (Verspeek-Rip, 2016), it was concluded that T002488 is not mutagenic in the mouse lymphoma L5178Y test system in the presence and absence of rat liver metabolic activation system under the experimental conditions described in the report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A key study (K2, Vanparys et al, 2004) was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Five strains of Salmonella typhimurium, TA1535, TA1537, TA102, TA98 and TA100, were treated with solutions of the test item using the Ames plate incorporation method at 7 dose levels, in triplicate, in the absence and in the presence of a rat liver metabolic activation system. The dose range was 9.77 to 625 µg/plate in the main experiment. DMSO was selected as vehicle.
In the absence and in the presence (20 and 50
μl S9 homogenate/plate) of a metabolic activation system, T002488 did not cause any biologically significant increase in the number of revertant colonies above the vehicle control incidence with all of the strains.

No toxic effects were observed. At concentrations of 39.07 or 78.13 or 156.25 onwards, a dose related increase in precipitation was observed. At the top dose level of 625μg/plate, no bacterial background could be observed due to heavy precipitation.

The vehicle (DMSO) and positive control counts of all strains fell within the required norms and the genotypes could be confirmed for all the strains validating the results from this study.

It can be concluded that T002488 has no mutagenic properties towards the various S. typhimurium strains under the test conditions.

Chromosome aberration:

Wright (K2, 2005) performed an in vitro chromosome aberration study in human lymphocytes (method equivalent/similar to OECD Guideline 473: In Vitro Mammalian Chromosome Aberration Test). The test item, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and the presence of metabolic activation by S9 mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test item, ranging from 18.03 to 576.75 µg/mL. A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Except where there was the need to clarify an equivocal response only one of the duplicate cultures was assessed for the presence chromosome aberrations. Two independent experiments were performed. In the first experiment (Groups 1 and 2) the exposure periods were 4 hours with and without S9 mix, with a 20 hour recovery period. In the second experiment the exposure period was 24 hours without S9 mix. 100 metaphases were scored per concentration for the chromosome aberrations.

The data for the 4-hour exposure groups show some modest reductions in mitotic index but no clear dose-response relationship. They were therefore considered not to be toxicity-related. In all exposures there were scorable metaphases cells up to 576.75 μg/ml but solubility was used as the criteria for the selection of the upper dose level for analysis.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

The test material did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.
The test material did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

In conclusion, T002488 is considered to be non-clastogenic to human lymphocytes in vitro.

In vitro gene mutation study in mammalian cells:

Verspeek - Rip (K1, 2016) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells according to the OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene).
The test item, dissolved in dimethyl sulfoxide, was assessed for its potential to induce forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3 and 24-hour treatment period.
In the first mutation experiment, the test item was tested up to concentrations of 52 μg/ml in the absence and presence of S9-mix. The treatment period was 3 hours. No toxicity was observed up to the concentration of 52 μg/ml in the absence and presence of S9-mix. The test item precipitated in the culture medium at the concentrations of 26 μg/ml and onwards.
In the second mutation experiment, the test item was tested up to concentrations of 20 μg/ml in the absence of S9-mix. The treatment period was 24 hours. No significant toxicity was observed up to the concentration of 20 μg/ml. The test item precipitated in the culture medium at the concentration of 20 μg/ml.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

 

 

Justification for classification or non-classification

Based on negative results in all available in vitro genetic toxicity tests with T002488 and the criteria of the CLP Regulation (EC) 1272/2008, the test item should not be classified for mutagenicity.