4-(1,1-dimethylethyl)cyclohexyl 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:

Initiation date: 03-10-'84 Completion date: 05-10-'84

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(1,1-dimethylethyl)cyclohexyl methacrylate

EC Number:

256-277-5

EC Name:

4-(1,1-dimethylethyl)cyclohexyl methacrylate

Cas Number:

46729-07-1

Molecular formula:

C14H24O2

IUPAC Name:

4-tert-butylcyclohexyl 2-methylprop-2-enoate

Specific details on test material used for the study:

Chemical name (IUPAC): t-Butyl cyclohexyl methacrylate
Trade name/code: Nourycryl NC 110
Appearance: Clear mobile liquid
Storage: At ambient temperature in the dark
Purity > 98%

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0, 3333.3 and 5000.0 µg/plate

In the preliminary test survival was reduced at test concentrations from 33.3 µg/plate, therefore, in the main test concentrations up to 24 µg/plate were used

Main test (with and without metabolic activation): 2.4, 4.2, 7.5, 13.0, 24.0 µg/plate

Vehicle / solvent:

Ethanol was used due to solubility of the substance in this vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA 1535

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA 100

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strains TA 98 and TA 1538

Positive control substance:

other: 4-nitro-o-phenyl-enediamine

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA 1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PRINCIPLE OF THE TEST METHOD
Bacteria, plated onto minimal medium agar plates, are exposed to the test chemical with and without metabolic activation (S9 mix). After 48h of incubation, revertant colonies showing histidine independent growth are counted and compared to the number of spontaneous revertants in solvent-treated control cultures.

Preparations
Bacterial cultures
Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No.2) and incubated in a shaking water bath (37°C , 150 spm) until the cultures reach an O. D. of 0.4 at 700 nm (10^9 cells/ml). Freshly grown cultures of each strain are used for a test.

Test procedure
Standard plate test
Top agar in top agar tubes is melted and heated to 45 ºC. The following solutions are successively added to 3 ml of top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml)of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol, and in case of activation assays 0. 5 ml of S9 mix. The ingredients are mixed on a Vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidification of the top agar, the plates are turned and incubated in the dark at 37 ºC for 48 h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
Eleven serial half-log dilutions of the test substance were plated with a diluted TA100 culture on non-selective agar (viability counting). For viability determinations, equal numbers of bacterial cells were seeded on each plate in the presence of the test substance. The percentage survival of an appropriately diluted TA100 culture on non-selective agar is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. The survival of strain TA100 is reduced at test substance concentrations from 33.3 μg/plate upwards.

Main test
All bacterial strains showed negative responses over the entire dose range of the test substance. Strain-specific positive control chemicals showed that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considered as nonmutagenic in the Ames Salmonella/microsome assay.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range of the test substance. Based on these results, the test substance can be considered as non mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

A sample of Nourycryl MC 110 was tested in the Ames Salmonella/microsome test up to the limit of toxicity (24 μg/plate).

The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester stnins (TA1535; TA1537; TA1538; TA98 and TAl00). The test substance can, therefore, be considered as non mutagenic in this test system.