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EC number: 221-111-2 | CAS number: 3006-86-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 000
- Report date:
- 2000
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- Reference substance name:
- Cyclohexylidenebis[tert-butyl] peroxide
- EC Number:
- 221-111-2
- EC Name:
- Cyclohexylidenebis[tert-butyl] peroxide
- Cas Number:
- 3006-86-8
- Molecular formula:
- C14H28O4
- IUPAC Name:
- 1,1-bis(tert-butylperoxy)cyclohexane
Constituent 1
Method
Species / strainopen allclose all
- 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
Controlsopen allclose all
- 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.
Results and discussion
Test resultsopen allclose all
- 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.
Applicant's summary and conclusion
- 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.
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