Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

The bacterial reverse mutation test is able to identify substances that cause point mutations, by substitution, addition or deletion of one or a few
DNA base-pairs. Mutagenic substances can induce reversion in histidine-(for Salmonella typhimurium) or tryptophan-(for Escherichia coli)
deficient strains which are then able to grow and form colonies in a histidine- or tryptophan limited medium, while non-reverted strains cannot.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 7-8 March 2007

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl methacrylate

EC Number:

231-403-1

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl methacrylate

Cas Number:

7534-94-3

Molecular formula:

C14H22O2

IUPAC Name:

1,7,7-trimethylbicyclo[2.2.1]hept-2-yl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

- Supplier : Rhodia Inc. (Cranbury, NJ USA)
- Name of test material: Isobornyl Methacrylate (IBOMA)
- CAS Registry No. : 7534-94-3
- Molecular weight : 222 g/mol
- Substance type: organic
- Physical state at room temperature: liquid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix fraction of Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Main experiment:
+/-S9 mix; 0, 156.3 (2nd experiment only), 312.5, 625, 1250, 2500 and 5000 µg/plate
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 µg/plate. The concentration range include two logarithmic decades. Five to six adequately spaced
concentrations were tested. Two independent experiments were performed.


Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in
the preliminary toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity for the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent control: DMSO were performed

Negative solvent / vehicle controls:

yes

Remarks:

vehicle: DMSO

True negative controls:

no

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

for TA 1535, TA 100

Positive control substance:

sodium azide

Remarks:

sodium acide concentration: 1 µg/plate Migrated to IUCLID6: without metabolic activation

Controls 3

Positive controls:

yes

Remarks:

for TA 1537

Positive control substance:

9-aminoacridine

Remarks:

9-aminoacridine concentration: 50 µg/plate Migrated to IUCLID6: without metabolic activation

Controls 4

Positive controls:

yes

Remarks:

for TA 98

Positive control substance:

2-nitrofluorene

Remarks:

2-nitrofluorene concentration: 0.5 µg/plate Migrated to IUCLID6: without metabolic activation

Controls 5

Positive controls:

yes

Remarks:

for WP2 uvrA strain

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4-nitroquinoline-N-oxide concentration: 2.0 µg/plate Migrated to IUCLID6: without metabolic activation

Controls 6

Positive controls:

yes

Remarks:

for TA 1535, TA 1537 and TA 98

Positive control substance:

other: 2-Anthramine; with metabolic activation

Remarks:

2-Anthramine concentration: 2 µg/plate

Controls 7

Positive controls:

yes

Remarks:

for TA 100

Positive control substance:

benzo(a)pyrene

Remarks:

benzo(a)pyrene concentration: 5 µg/plate Migrated to IUCLID6: with metabolic activation

Controls 8

Positive controls:

yes

Remarks:

for WP2 uvrA strain

Positive control substance:

other: 2-Anthramine; with metabolic activation

Remarks:

2-Anthramine concentration: 10 µg/plate

Details on test system and experimental conditions:

EXPERIMENTAL DESIGN
Treatment
The test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test and the first experiment were performed
according to the direct plate incorporation method. The second experiment with and without S9 mix was performed according to the preincubation
method.

Salmonella typhimurium and E. coli strains were treated with Isobornyl methacrylate using the direct plate incorporation method with at least five dose levels, plated in triplicate, for the preliminary test and the first mutagenicity experiment with and without S9. The second experiment tested six dose levels with and without S9 mix and was performed according to the preincubation method. The dose range was determined in a preliminary toxicity assay in which the highest dose tested was 5000 µg/plate. Moderate toxicity was observed in the TA 100 stran at 5000 µg/plate dose level with and without S9 mix. For the plate incorporation method, 0.1 mL bacterial suspension and 2 mL of overlay agar were mixed with 0.05 mL S9 mix (when required) or phosphate buffer (pH 7.4). Following homogenization, the mixture was on minimal medium. Once solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2 °C.
In the preincubation method, 0.05 mL of IBOMA solution, vehicle control or positive control solution and 0.05 mL S9 mix (when required) or phosphate buffer (pH 7.4) were mixed with 0.1 mL bacterial suspension and added to a glass culture tube. The mixture was incubated for 60 ± 3 minutes at 37 ± 2 °C under shaking. After incubation, 2 mL of overlay agar were added and the mixture was poured onto minimum agar plate. Once solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2°C.

Evaluation criteria:

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 and TA 98 and WP2 uvrA the number of reversions is at least twice as high and in strains TA 1535 and TA 1537 it is at least three times higher as compared to the vehicle controls. Also, a dose-dependent and reproducible
increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or not. 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

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 1535

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 1537

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 98

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 2500 µg/plate for TA 100

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for WP2 uvrA

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Isobornyl methacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, the test substance has to be judged as nonmutagenic up to 5000 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Executive summary:

In a reverse gene mutation assay (according to OECD 471) in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to Isobornyl methacrylate (99.07 % stabilised with 143 ppm MEHQ) at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

 

With the direct plate incorporation method, moderate to marked toxicity was noted in the TA 1535 strain at 5000 µg/plate and in the TA 100 strtain at dose levels of >= 2500 µg/plate with metabolic activation. Moderate toxicity was noted for TA 100 at 5000 µg/plate with metabolic activation. Exposure to graded doses of Isobornyl methacrylate in either the presence or the absence of metabolic activation. Exposure to graded doses of the test substance in either the presence or the absence of metabolic activation did not increase the reversion rates in any of the tester strains in either experiment.

Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

Conclusion:

Under the conditions of this study Isobornyl Methacrylate (IBOMA; CAS: 7534-94-3), did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli at the concentrations tested with and without metabolic activation.