Cyclohexylidenebis[tert-amyl] peroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylidenebis[tert-amyl] peroxide

EC Number:

239-741-1

EC Name:

Cyclohexylidenebis[tert-amyl] peroxide

Cas Number:

15667-10-4

Molecular formula:

C16H32O4

IUPAC Name:

1,1-bis[(2-methylbutan-2-yl)peroxy]cyclohexane

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C)

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-levels . 625 ƒÊg/plate.
No noteworthy toxicity was noted towards all the strains used, with and without S9 mix.
In the second experiment without S9 mix, a slight increase in the number of revertants was noted in the TA 1537 strain (up to 3.3-fold the vehicle control value).
Since this slight increase was neither dose-related, nor reproducible (not observed in the first experiment performed under the same experimental conditions), and since the threshold of 3-fold the vehicle control value seems to have been reached only due to the low value of the vehicle control (near the lowest value of the vehicle control historical data range), it was considered not to be relevant.

Applicant's summary and conclusion

Conclusions:

1,1-DI(t-AMYLPEROXY)CYCLOHEXANE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

LUPEROX 531 M60 was evaluated for induction of reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations. A preliminary toxicity test was performed to define the dose-levels of LUPEROX 531 M60 to be used for the 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 performed 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 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (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. LUPEROX 531 M60 was dissolved in ethanol. All the concentrations and dose-levels were expressed as active item, 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE, taking into account the active material content of 60.5%. 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 item was freely soluble and non-toxic in the preliminary assay, the highest dose-level was 5000µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-levels=625µg/plate. No noteworthy toxicity was noted towards all the strains used, with and without S9 mix. In the second experiment without S9 mix, a slight increase in the number of revertants was noted in the TA 1537 strain (up to 3.3-fold the vehicle control value). Since this slight increase was neither dose-related, nor reproducible (not observed in the first experiment performed under the same experimental conditions), and since the threshold of 3-fold the vehicle control value seems to have been reached only due to the low value of the vehicle control (near the lowest value of the vehicle control historical data range), it was considered not to be relevant. The test item did not induce any other noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. LUPEROX 531 M60 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.