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Additional information

The potential for 2,2,4,4-tetramethyl-1,3-cyclobutanediol to induce genetic toxicity (mutations/aberrations) was thoroughly characterized in three different in vitro assays that followed OECD guidelines 471, 473, and 476. All studies were also conducted under GLP assurances. None of the studies showed evidence of genotoxicity  

 

In a study following OECD 471, the test article was assessed for its potential to induce gene mutations using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate up to 5000 ug/plate. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Appropriate reference mutagens were used as positive controls and they showed a distinct increase of induced revertant colonies. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 mix).

 

A second study (OECD 476) assessed the potential of 2,2,4,4-tetramethyl-1,3-cyclobutanediol to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. The highest applied concentration (1500 μg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

 

The third study was conducted following OECD test guideline 473 assessed for the potential of 2,2,4,4-tetramethyl-1,3-cyclobutanediol to induce structural chromosome aberrations in Chinese Hamster V79 cell line in two independent experiments. In each experimental group two parallel cultures were set up. Dose selection for the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations were 45.0, 90.0, 180.0, 360.0, 720.0, and 1440.0 ug/ml +/- S9. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in Experiment II with metabolic activation, where only 50 metaphase plates per culture were scored. In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. Appropriate mutagens used as positive controls induced statistically significant increases in cells with structural chromosome aberrations. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No evidence of genotoxicity was observed in any study, accordingly no classification is warranted.