2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

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 28 July to 13 August 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

08 April 2015.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

EC Number:

815-500-1

Cas Number:

1853175-99-1

Molecular formula:

C15 H26 O2

IUPAC Name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature, darkness.

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

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:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GieBen (Batch nos. 3424, 3366, 3393)
- method of preparation of S9 mix : produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration of S9 in the final culture medium: S9-fraction 10% v/v

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 50, 150, 500, 1500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 with and without S9 under the direct plate incorporation method.
Confirmation test: 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 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 determined in demineralised water, dimethyl sulfoxide and ethanol. The test item is only soluble in ethanol in a concentration of 50 g/L. Therefore, a test item solution containing 50 g/L in ethanol was prepared for both experiments.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

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 +/-1°C
- Exposure duration: 37 +/- 1°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 TA97a, TA98, TA100, TA102 and TA1535.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate in TA98, TA100 and TA1535 (second experiment).

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 S. typhimurium TA97a, TA98, TA100, TA102 and TA1535.

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 (TA97a, TA98, TA100, TA102 and TA1535) 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: 50, 150, 500, 1500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA153 with and without S9 under the direct plate incorporation method

Confirmation test: 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 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 5000 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 TA97a, TA98, TA100, TA102 and TA1535.

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