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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-01-21 to 2000-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21 st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31st July 1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254-induced enzymatic systems contained in rat liver microsomal fraction)
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix; for strains TA 1535, TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix; for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix; for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix; for strain TA 102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine for all strains
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Preliminary toxicity test:
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in number of revertant colonies and/or a thinning of the bacterial lawn.

Main test:
In two experiments, five dose-levels of the test item (three plates/dose-level) were tested on each strain, with and without S9 mix.
In each experiment, the following controls were included using triplicate plates:
- vehicle controls: each bacterial tester strain treated with the vehicle,
- positive controls: each bacterial tester strain treated with appropriate reference mutagens.

The experiments were performed according to :
- direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test items solution (0.1 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relvant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
- preincubation method (second experiment with S9 mix): test substance solution (0.1 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of inhibition at 37 °C, revertants were scored with an automatic counter.
Evaluation criteria:
Treatment of results:
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle).

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with our historical data
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data.

Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test substance was freely soluble in vehicle (distelled water) at 50 mg/mL.
Consequently, with a maximum dose volume of 100 µL/plate, the dose-levels were: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A slight emulsion was observed in the Petri plates when scoring the revertants at the highest dose-level.
No toxicity was noted towards the three strains used, with and without S9 mix.

Mutagenicity experiments:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test substance was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.
The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.
A slight or moderate emulsion was observed in the Petri plates when scoring the revertants, at the highest dose-level, in both mutagenicity experiments with and without S9 mix.

Experiments without S9 mix:
Except for a slight or moderate toxicity, noted in the first experiment in the TA 1535, TA 98 and TA 100 strains at dose-levels >= 1250 µg/plate, no noteworthy toxicity was observed in both experiments.
No noteworthy increase in the number of revertants was observed in both experiment, in any of the five strains.

Experiments with S9 mix:
No noteworthy toxicity was noted all strains used, in the both experiment.
No noteworthy increase in the number of revertants was observed in both experiment, in any of the five tester strains.
Conclusions:
The test item cyclohexylidenebis[tert-butyl]peroxide does not show mutagenic acitivity in bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

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 eyperiments 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 distelled 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 th acceptance criteria. The study was therefore considered valid.

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-01-16 to 2001-01-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted on 21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
8 June 2000
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- first experiment: 0, 18.7, 37.5, 73.8, 148.7, 297.4, 597.1, 890.9, 1188.3 µg/mL
- second experiment: 0, 56.25, 112.5, 225, 450, 720, 900 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:

A range of dose-levels of the test item (two cultures/dose-level) were tested, with and without metabolic activation. In case of treatment with S9 mix, the final concentration of S9 fraction in the culture medium was 1.5 %.

In each experiment, the following controls were included using at least duplicate cultures:
- vehicle controls: cutures treated with the vehicle,
- positive controls: cultures treated

First experiment:
Lymphocyte cultures were then exposed to the test or control substances, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatement, corresponding to approximately 1.5 normal cell cycles.
As this experiment gave positive results, no other experiments were undertaken.

Second experiment:
- without S9 mix, cells were exposed continuously to the test or control substances.
- with S9 mix, cells were exposed to the test or control substances for 3 hours and then rinsed.
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. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding 1.5 normal cell cycles and 24 hours later.
Evaluation criteria:
A reproducible and statistically significant increase in frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparision was performed using the x2test, in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No preliminary cytotoxicity test were performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the experiments and dose-levels for scoring of chromosomal aberrations were selected on the basis of the cytotoxicity indicated by reduction of mitotic index (MI).
Conclusions:
The test item cyclohexylidenebis[tert-butyl]peroxide does not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

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 cuture 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 contraols 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-16 to 2011-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment without S9 Mix:
5, 10, 20, 40, 80, 85, 90*, 95* µg/mL

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

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

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

*These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: suitable solvent
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 5 and 20 hours
- Expression time (cells in growth medium): 1, 3, and 6 days
- Selection time: 5 days

SELECTION AGENT: Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500000

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: None observed


RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. A dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 106 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence (9 concentrations) of S9 mix (50 μL/mL) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (9 concentrations). 24 hours after the beginning of treatment, the cultures were washed with Ham's F12 medium covered with trypsin-EDTA solution and counted and the cell concentration was adjusted to 40 cells/mL with Ham's F12 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37oC in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at beginning and end of the treatments.
In addition, pH and osmolality were considered for dose level selection. Results of the Pre-test on cell toxicity were used for dose selection of the test item used in the Main Mutation Assays. In Experiment 1 eight and nine and in Experiment 2 nine and nine concentrations were selected for the treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:
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. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

Conclusions:
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.
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.