2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol

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:

From 13 February to 16 April 2012.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation. The substance is adequately identified, but some data on composition is missing. Therefore validation applies with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997.

Deviations:

yes

Remarks:

2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. No information on the characterisation of S9 with a mutagen that requires metabolic activation by microsomal enzymes has been included in the report.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2011-10-19,20&21 / Signed on 2011-11-17.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol

Cas Number:

1401065-88-0

Molecular formula:

C16 H26 O

IUPAC Name:

2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol

Test material form:

other: See specific details on each Endpoint Study Records

Details on test material:

- Storage condition of test material: Room temperature

Specific details on test material used for the study:

Physical state: colorless to pale yellow dense gel

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% v/v S9 fraction; rat liver microsome fraction (S9)

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 0.3, 0.8, 2.3, 6.9 and 20.6 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.3, 0.8, 2.3, 6.9 and 20.6 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: the solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 96%, DMSO
and corn oil). Observation of precipitation by the naked eye indicated that the test item is not soluble.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 48 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item was assayed by adding of 5 mg/plate to a minimal agar plate and incubating at 37°C for 48h. No growth was observed in the minimal agar plate after incubation with the test item.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.

Evaluation criteria:

Several criteria are used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains.
Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
A decrease in the number of revertant colonies >50% compared to solvent reference was observed, indicating cytotoxicity of the test item. The lowest cytotoxic concentration was 20.6 µg/plate and it was used as the highest formulation for Ames test.

MUTAGENICITY TEST:
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
− No dose response was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

OTHERS:
- Sterility test showed no contamination during the study.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test condition, the test mateiral is not mutagenic in presence and absence of metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test item diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Main test: 0.3, 0.8, 2.3, 6.9 and 20.6 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.3, 0.8, 2.3, 6.9 and 20.6 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

 

Negative and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls showed valid ratios (R) above 2.5.

 

Cytotoxic effect was observed at 20.6 µg/plate. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.