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Bacterial mutagenesis

The test article (1,4 -cyclohexane dicarboxylic acid; CHDA) was evaluated for mutagenic activity in the Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay. The assay utilized Salmonella typhimurium tester strains TA98, TAlOO, TA1535, and TA1537, and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted in both the presence and absence of S9 mix with six doses of test article along with concurrent positive and vehicle controls. The assay was plated using three plates per dose. The doses of test article tested in the mutagenicity assay were 5,000, 3,330, 1,000, 667, 333, and 100 ug per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. The test article was determined to be non-mutagenic in the absence or presence of metabolic activation.

Mammalian cell mutagenesis

The potential to induce mammalian cell mutations at the mouse lymphoma thymidine kinase locus was evaluated 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 (1800 μg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. 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. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The potential to induce mammalian cell mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was evaluated in Chinese hamster ovary cells under conditions with and without metabolic activation. Preliminary cytotoxicity testing showed the test material to be noncytotoxic to CHO cells without activation up to 1000 ug/ml and, at most, moderately cytotoxic up to 1000 ug/ml in the presence of metabolic activation. The mutation assays were initiated with concentrations up to 5000 ug/ml because of the lack of appreciable cytotoxicity at 1000 ug/ml. Since the test material was insoluble in DMSO at 300 mg/ml and stocks up to 500 mg/ml would be needed for the mutation assay, the vehicle was changed to F12 culture medium. In addition, the use of media would aid in neutralization required at the higher concentrations. The test article (CHDA; EC 94 -0212) was soluble in all stocks prepared in F12 medium (up to 10,000 ug/ml). Two trials of the nonactivation assay were initiated but the first trial had incomplete data because of loss due to contamination, and a second trial was performed to confirm the results. In both of the nonactivation assays presented in this report, six treatments from 500 ug/ml to 5000 ug/ml were analyzed for mutant induction. No toxicity was induced at any of the concentrations analyzed. The mutant frequencies of treated cultures varied randomly with dose. All treatments in both trials were within the range acceptable for background mutant frequencies which is 0 to 15 x 10-6. CHDA was therefore considered negative without activation in this assay. In the presence of metabolic activation, two trials were also performed. The second trial was initiated because the two negative controls and three dose levels including the high dose had excessive contamination in Trial 1. Six treatments from 500 ug/ml to 5000 ug/ml were analyzed in both trials and low to moderate cytotoxicities were observed. Sporadic small increases were observed, but the increases were not dose related and were still within the acceptable range for mutant frequency variation in the negative controls. The test material was therefore nonmutagenic with activation in this assay.

Chromosome aberration studies

The potential to induce chromosomal aberrations was evaluated in Chinese hamster ovary (CHO) cells with and without metabolic activation. The test article was dissolved in dimethylsulfoxide at a stock concentration of 225 mg/ml for the dose rangefinding assay. Concentrations of 0.0750, 0.225, 0.750, 2.25, 7.50, 22.5, 75.0, 225, 750, and 2250 ug/ml were tested in rangefinding assays with and without metabolic activation. Due to the presence of a precipitate after dosing, higher dose levels could not be tested. Severe toxicity was observed in the cultures dosed with 2250 ug/ml. No appreciable reductions in the mitotic indices were observed in the other test cultures analyzed. Based on these data, replicate cultures of CHO cells were incubated with 250, 500, 750, 1000, 1500, and 2000 ug/ml in 20.0 hour assays with and without metabolic activation. Cultures treated with 750, 1000, 1500, and 2000 ug/ml from the assays with and without metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed. A confirmatory trial was conducted using concentrations up to 2250 ug/ml, the highest dose possible for this test material. Replicate cultures of CHO cells were incubated with 500, 1000, 1500, 2000, and 2250 ug/ml and harvested after 20.0 and 44.0 hours with and without metabolic activation. Complete toxicity was observed in the cultures dosed with 2250 ug/ml without metabolic activation. Cultures dosed with 500, 1000, 1500, and 2000 ug/ml without metabolic activation and 1000, 1500, 2000, and 2250 ug/ml with metabolic activation were analyzed for chromosomal aberrations. No significant increases in cells with chromosomal aberrations were observed in the 20.0 hour nonactivation assay and 44.0 hour assay with metabolic activation. Significant increases in the numbers of cells with chromosomal aberrations were observed at a single dose level of 2000 ug/ml from the 44.0 hour nonactivation assay (although only in one culture) and 2250 ug/ml from the 20.0 hour assay with metabolic activation. A significant increase in percent polyploidy was observed at a single dose level, 2000 ug/ml, from the 44.0-hour assay with metabolic activation. The test article induced a statistically significant increase in chromosomal aberrations in CHO cells at a single toxic dose level in the extended assay without metabolic activation, and only at 20.0 hours and not in the extended assay with metabolic activation. The response observed in the activation assay did not persist in the next generation of cells. No dose relationship was observed in the assays where a positive response was observed, and the positive response was observed mainly in only one of the replicate cultures that displayed severe toxicity. The response observed is possibly induced by the severe toxicity observed in these cultures and may not have any biological relevance or significance.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

1,4-cyclohexanedicarboxylic acid (CHDA) was shown to be non-mutagenic is both bacterial and mammalian cell assays with and without metabolic activation. Based upon these findings, CHDA does not satisfy the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).