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EC number: 616-145-3 | CAS number: 74852-61-2
- Bacterial reverse mutation assay
In a K2 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100 and TA102, performed according to a method similar to OECD Guideline 471, it was concluded that T001325 has no mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of S9-mix under the test conditions described in the report, up to the maximum test concentration of 5000 μg/plate. An expert statement was also added to justify the fact that no further testing is necessary.
- Chromosome aberration study
In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T001325 was considered to be non-clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.
- In vitro gene mutation study in mammalian cells
In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that T001325 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.
Evaluation of the mutagenicity (first experiment):
An increase above the 95% upper control limit was observed at the mid dose of 0.47 μg/ml in the presence of S9-mix. Although this increase is above the 95% upper control limit, no dose-related response is observed and the increase in the mutation frequency (147 per 10^6 survivors) is below the MF(controls) + 126 (208 per 10^6 survivors), therefore this increase is considered to be not biologically relevant.
Bacterial reverse mutation assay
A key study (K2, Thompson and Bowles, 2004) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Salmonella typhimurium strains TA98, TA100 and TA102 were treated with solutions of the test item using the Ames plate incorporation method at 9 dose levels, in triplicate, both with and without the addition of 10% liver S9 in standard co-factors. The dose range was 0.5 to 5000 µg/plate in the main experiment.
The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test item was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour with an associated precipitate was observed at and above 1500 μg/plate. These observations did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Expert statement in addition of Ames test
According to Annex VII, section 8.4, in vitro gene mutation study using bacteria (Ames test) is a standard information requirement at the present tonnage level. The registrant is aware of the version of the EU Test Method B.13/14/OECD TG 471 in force since 1997 which indicates that at least five strains of bacteria should be tested. The Ames test, currently included in the dossier, is performed following OECD 471 but included testing on three S. typhimurium strains (TA 98, TA 100, TA 102) only.
The selection of these three strains is based oninternal historical data gathered during more than 25 years of testing. This is not a specific observation but is widely accepted in pharmaceutical companies. In general, most compound screening strategies are based on this limited set of 2 strains (TA 98 and TA 100). Moreover, the introduction of plasmid pKM101 in strains TA1535 and TA1538 resulted in the corresponding isogenic strains TA100 and TA98. Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone recombinational DNA repair pathway. Thus TA100 and TA98 are believed to be more sensitive than their plasmid-free counterparts. Therefore the number of compounds that is exclusively positive in TA1535 and/or TA1537 (the 2 strains that are not tested) is extremely small (less than 2.3% or 90 on 3083 compounds tested). The relationship and historical aspects are clearly described in Mortelmans et al, Mutation Research 455 (2000). We therefore are convinced that the proposed 3 strains are sufficient for registration of intermediates in the production of active pharmaceutical ingredients. It should be noted however that, as TA102 is also tested, the potential to detect certain specific types of mutagens has been elaborated. This mutation is also reverted by mutagens that cause oxidative damage. In addition, this DNA repair proficient strain TA102 detects cross-linking agents such as bleomycin and mitomycin C. Therefore, no additional test is performed to evaluate five strains.
Wright (2004) performed an in vitro chromosome aberration study in human lymphocytes (method equivalent/similar to OECD Guideline 473).
The test item, dissolved in vehicle, 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 3.125 to 100 µg/mL in the two main experiments. 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 of 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 (Group 3) the exposure period was 24 hours without S9 mix.
A microscopic assessment of the slides showed that metaphase cells were present at up to 100 μg/mL in all exposure groups. The test item did not induce a dose-related inhibition of mitosis at any dose level in any exposure group. Dose selection for metaphase analysis was therefore based on the precipitating dose levels. 100 metaphases were scored per evaluated culture for the chromosome aberrations.
The test item did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.
The test item did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.
T001325 was therefore considered to be non-clastogenic to human lymphocytes in vitro.
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.
In vitro gene mutation study in mammalian cells:
Verspeek - Rip (2016) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. 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 15 µg/mL in the absence and presence of S9-mix. The treatment period was 3 hours. No significant toxicity was observed up to the concentration 15 µg/mL in the absence and presence of S9-mix. The test item precipitated in the culture medium at the concentration of 15 µg/mL.
In the second mutation experiment, the test item was tested up to concentrations of 7.5 µg/mL in the absence of S9-mix. The treatment period was 24 hours. No significant toxicity was observed up to the concentration 7.5 µg/mL. The test item precipitated in the culture medium at the concentration of 7.5 µg/mL.
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 the report.
Based on the results of positive control chemicals the test conditions were considered adequate and it was concluded that the metabolic activation system (S9 -mix) functioned properly. In addition, the acceptability criteria for the negative control substance were met and the mutation frequency was situated within the 95% control limits of the distribution of the historical solvent control database.
Based on negative results in all in vitro genetic toxicity tests with T001325 and the criteria of the CLP Regulation (EC) 1272/2008, T001325 should not be classified for mutagenicity.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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