3,3,5-trimethylcyclohexyl methacrylate

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:

February / April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3,5-trimethylcyclohexyl methacrylate

EC Number:

231-927-0

EC Name:

3,3,5-trimethylcyclohexyl methacrylate

Cas Number:

7779-31-9

Molecular formula:

C13H22O2

IUPAC Name:

3,3,5-trimethylcyclohexyl 2-methylprop-2-enoate

Test material form:

liquid

Method

Target gene:

n/a

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver

Test concentrations with justification for top dose:

Pre-experiment : 0.0031-0.01-0.3016-0.1-0.316-1.0-2.5-5.0 µl/plate
Main experiments : 0.0316-0.1-0.316-1.0-2.5-5.0 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine : TA97a + TA98 (without S9) ; 2-aminoanthracene : all strains with S9

Details on test system and experimental conditions:

-method of application : in agar plate incorporation (experiment 1) ; preincubation (experiment 3)
-triplicate (3 plates) by concentration in the main experiments
-Pre-experiment for toxicity: The toxicity of the test item was determined with strains TA98 and TA100. 8 concentrations were tested for toxicity and induction of mutations with 3 plates each, with plate incorporation method.
Toxicity may be detected by a reduction in the number of revertants, a clearing or diminution of the background lawn or by the degree of survival of treated cultures.
-triplicate (3 plates) by concentration in the main experiments
-Experiment 1 : For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate : test solution at each dose levels, solvent, S9 mix or S9 mix substitution buffer, bacteria suspension and overlay agar.
-Experiment 2: For pre-incubation method, the test item solution was preincubated with the tester strains and sterila buffer or the S9 for 60 minutes at 37 °C prior adding the overlay agar and pouring onto the surface of a minimal agar plate.
In the experiment 1 and 2, after the solidification the plates were inverted and incubated at 37°C for at least 48 hours in the dark.

Evaluation criteria:

A test is considered acceptable if for each strain :
-the bacteria demonstrate their typical responses to crystal violet and ampicillin,
-the control plates without S9 are within the historical control data ranges,
-corresponding background growth on both negtaive control and test plate is observed,
-the positive controls show a distinct enhancement over te control plate.

A test item is considered as mutagenic if :
-a dose-related increase in the number of revertants occurs and/or
-a reproductible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
A biologically relevant increase is described as follow :
-if in strains TA97a, TA100 and TA102 the number of reversions is at least twice as high,
-if in strains TA1535 and TA98, the number of reversions is at least 3 times higher as compared ti the spontaneous reversion rate.

Statistics:

no

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA98 and TA102 strains with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the experiment 1, no toxicity of the test item was observed in the tester strains TA1535, TA97a and TA102. In the tester strain TA98 a reduction of revertant colony numbers was noted at the highest dose group with S9. In tester strain TA100 a reduction of the background lawn was observed at the highest dose groups (with S9). In the experiment 2, no toxic effects of the test item were found in any tester strain used up to the highest dose group evaluated (+/-S9).
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3,3,5 -trimethyl cyclohexyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 3,3,5 -trimethyl cyclohexyl methacrylate for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) were performed with the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102. The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.

In the experiment 1, no toxicity of the test item was observed in the tester strains TA1535, TA97a and TA102. In the tester strain TA98 a reduction of revertant colony numbers was noted at the highest dose group with S9. In tester strain TA100 a reduction of the background lawn was observed at the highest dose groups (with S9). In the experiment 2, no toxic effects of the test item were found in any tester strain used up to the highest dose group evaluated (+/-S9).

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3,3,5 -trimethyl cyclohexyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

In conclusion, it cas be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.