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Ames test

Cyclohexylidenebis[tert-butyl]peroxide was tested in the Ames test with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 according to EU method B14 and OECD guideline No.471.

A preliminary toxicity was performed to define the dose-levels of cyclohexylidenebis[tert-butyl]peroxide to be used for mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9-mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were preformed according to the direct plate incorporation method except for the second test with S9-mix, which was performed according to the preincubation method (60 minutes, 37 °C). Five strains of bacteria Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in distilled water.

Since the test item was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate.

The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9-mix.

Except for slight to moderate toxicity noted in the first experiment without S9-mix in the TA 98, TA 1535 and TA 100 strains, no noteworthy toxicity was observed. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of five strains.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The test item cyclohexylidenebis[tert-butyl]peroxide does not show mutagenic activity in bacterial reverse mutation test with Salmonella typhimurium.

 

In vitro chromosome aberration

Cyclohexylidenebis[tert-butyl]peroxide was tested in the chromosome aberration test in cultured human lymphocytes according to OECD guideline no. 473 and EU method B.10.The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

In the first experiment, lymphocyte cultures were exposed to the test or control substances, with or without S9-mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hour before harvest, each culture was treated with a colcemid solution to block cells at the metaphase-stage of mitosis.

As this first experiment was negative, the study was continued with a second experiment.

In the second experiment, cells were exposed continuously to the test or control substances (without S9-mix) and cells were exposed to the test or control substances for 3 hours and then rinsed (with S9-mix). Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding 1.5 normal cell cycles and 24 hours later, respectively. One and a half hour before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis.

The test item was dissolved in ethanol.

Without S9-mix, a 54 -75 % decrease in the mitotic index, without any clear dose-relationship, was noted in the first experiment. In the second experiment, no noteworthy decrease in the mitotic index was induced.

With S9-mix, a 40-81 % decrease in the mitotic index was noted at dose-levels >= 597.1 µg/mL, at the 20-hour harvest time. At the 44-hour harvest time, a 71-76 % decrease in the mitotic index was induced at dose-levels >= 720 µg/mL.

Both with and without S9 mix, the test item does not induce any significant increase in the frequency cells with chromosome aberrations, in both experiments and at both harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

The test item cyclohexylidenebis[tert-butyl]peroxide does not induce chromosome aberrations in cultured human lymphocytes.

 

HPRT test

The test item, cyclohexylidenebis[tert-butyl] peroxide was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-dimethylformamide and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9-mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below: Experiment 1, 5-hour treatment period without S9-mix: 5, 10, 20, 40, 80, 85, 90* and 95* μg/mL

Experiment 1, 5-hour treatment period with S9-mix: 5, 10 20, 40, 80, 100, 110, 120, and 130 μg/mL

Experiment 2, 20-hour treatment period without S9-mix: 5, 10, 20, 40, 80, 85*, 90*, 95* and 100* μg/mL

Experiment 2, 5-hour treatment period with S9-mix: 5, 10 20, 40, 80, 100, 110, 120, and 130 μg/mL

*These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically differences between treatment and control groups and no dose-response relationships were noted. In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation. As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9-mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Cyclohexylidenebis[tert-butyl] peroxide tested both without and with metabolic activation (S9-mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Cyclohexylidenebis[tert-butyl] peroxide was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Justification for selection of genetic toxicity endpoint
One bacterial reverse mutation assay and one in vitro mutation assay with mammalian cells are available. Potential of induction of chromosome aberration was assessed in an in vitro chromosome aberration test in mammalian cells. All tests were performed in accordance to GLP and OECD/EU guideline.

Short description of key information:
Ames test:
Under the experimental conditions of a bacterial reverse mutation assay (Ames test), the test item did induce point mutations by base pair changes or frameshifts in the genome of five Salmonella typhimurium strains. Therefore, the test item is considered to be mutagenic in the bacterial reverse mutation assay.

HPRT Test:
The test item was not mutagenic in this in vitro cell gene mutation test performed with CHO-K1 (Chinese hamster ovary) cells.

In vitro chromosome abberation test:
The test item did not show a clastogenic potential in the in vitro chromosome abberation test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro data the test item is not classified for genotoxicity according to Regulation (EC) No 1272/2008 (CLP) .