4-chloro-o-xylene

Endpoint summary

Additional information

In vitro

Bacterial reverse mutation assay

In the key study, the potential mutagenicity of the test material was evaluated in a bacterial reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA, in both the presence and absence of S-9 mix. The study was performed in accordance with OECD 471 under GLP conditions.

Based on the findings of a preliminary toxicity assay, the maximum doses tested in the mutagenicity assay were 5000 µg/plate in the presence of S9 with all tester strains, 1000 µg/plate in the absence of S9 with Salmonella tester strains and 5000 µg/plate in the absence of S9 with E. coli. Positive controls appropriate for each strain, in the presence and absence of S9-mix, were included. Due to excessive toxicity in some test conditions, portions of the assay were repeated. An independent repeat assay was also performed up to a maximum dose of 5000 µg/plate.

The test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control materials. It was concluded that, under the conditions of this assay, the test material gave a negative, i.e. non-mutagenic response in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA in both the presence and absence of S9 mix.

 

Chromosome aberration assay

In the key study, an in vitro chromosome aberration test was performed to evaluate the potential of the test material to cause gene mutation or clastogenic effects in mammalian cells. The study was performed to GLP and was designed to the requirements of OECD 473 and the ICH technical requirements for registration of pharmaceuticals for human use (1996, 1997).

Based on the findings of a preliminary toxicity test, the chromosome aberration test doses ranged from 7.5 – 120 µg/mL for all three exposure groups. In the chromosome aberration test, Chinese Hamster Ovary (CHO) cells were treated for 4 and 20 hours without S9 activation and for 4 hours with S9 activation. The percentage of cells with structural aberrations in the non-activated 4 h exposure group was significantly increased above that of the solvent control; however these were not considered to be biologically significant as they were within the historical solvent control range. The percentage of cells with numerical aberrations in the test material treated group was not significantly increased above that of the solvent control at any dose level. The percentage of cells with structural or numerical aberrations in the S9 activated 4 h exposure group was not significantly increased above that of the solvent control at any dose level. The percentage of cells with structural aberrations in the non-activated 20 h exposure group was significantly increased above that of the solvent control; however these were not considered to be biologically significant as they were within the historical solvent control range. The percentage of cells with numerical aberrations in the test material treated group, were not significantly increased above that of the solvent control at any dose level. Under the conditions of this study, the test material is negative for the induction of structural and chromosome aberrations in CHO cells.

 

Mouse lymphoma assay

In the key study, an in vitro cell mutation assay in L5178Y TK+/-cells was performed to evaluate the potential of the test material to cause gene mutation or clastogenic effects in mammalian cells. The study was performed to GLP and was designed to the requirements of OECD 476 and ICH S2A, S2B.

Based on the findings of a preliminary toxicity test, the mutagenicity doses ranged from 10 - 150 µg/mL for both non-activated and S9 activated 4 h exposures. No non-activated and six S9 activated cultures displayed mutant frequencies between 55 and 99 mutants per 1E06 clonable cells over that of the solvent control. One S9 activated cloned culture showed a mutant frequency ≥ 100 mutants per 1E06 clonable cells over that of the solvent control. There were no concentration-related increases in mutant frequency. Toxicity in the cloned cultures was observed at 80 µg/mL without activation and 125 and 150 µg/mL with S9 activation. For the extended treatment, based on the findings of the preliminary toxicity test, the mutagenicity doses ranged from 10 - 150 µg/mL for non-activated 24 h exposure. No cloned cultures displayed mutant frequencies between 55 and 99 mutants per 1E06 clonable cells over that of the solvent control. There was no concentration related increase in mutant frequency. Toxicity in the cloned cultures was observed at 70 µg/mL.

The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the method used to detect small colony mutants. Under the conditions of this study, the test material was concluded to be negative without S9 activation and equivocal with S9 activation in the L5178Y/TK+/- mouse lymphoma mutagenesis assay.

 

In the supporting study, an in vitro cell mutation assay in L5178Y TK+/-cells was performed to evaluate the potential of a structural analogue of the test material (the regioisomer 3-chloro-o-xylene) to cause gene mutation or clastogenic effects in mammalian cells. The study was performed to GLP and was designed to the requirements of OECD 476 and ICH S2A, S2B.

Based on the findings of a preliminary toxicity test, the mutagenicity doses ranged from 5.0 - 150 µg/mL for both non-activated and S9 activated 4 h exposures. No cloned cultures exhibited induced mutant frequencies of ≥ 90 per 1E06 clonable cells. There was no dose-response trend. Toxicity in the cloned cultures was observed at doses ≥ 52 µg/mL without activation and ≥100 µg/mL with S9 activation. For the extended treatment, based on the findings of the preliminary toxicity test, the mutagenicity doses ranged from 5.0 - 150 µg/mL for non-activated 24 h exposure. No cloned cultures exhibited induced mutant frequencies of ≥90 per 1E06 clonable cells. There was no dose-response trend. Toxicity in the cloned cultures was observed at doses ≥56 µg/mL.

The colony sizing for the MMS positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies. Under the conditions of this study, the structural analogue of the test material was concluded to be negative with and without S9 activation in the L5178Y/TK+/- mouse lymphoma mutagenesis assay.

Justification for selection of genetic toxicity endpoint

No single key study was selected on the basis that the available studies all address different aspects of genetic toxicity and the data should be considered as a whole. All studies were conducted in accordance with standardised guidelines under GLP conditions.

Short description of key information:

IN VITRO

Bacterial reverse mutation assay: negative OECD 471 (Wagner, 2004)

Chromosome aberration assay: negative, OECD 473 (Ramadevi, 2004)

Mouse lymphoma assay: negative without S9 activation, equivocal with S9 activation, OECD 476 (San, 2004)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to genetic toxicity.