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

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:

2012-07-10 - 2012-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

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

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 acrylate

EC Number:

227-561-6

EC Name:

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

Cas Number:

5888-33-5

Molecular formula:

C13H20O2

IUPAC Name:

(1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobornyl acrylate
- 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

Additional strain / cell type characteristics:

other:

Remarks:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix fraction of Aroclor 1254-induced, male Sprague-Dawley rats and male Syrian hamster livers

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II; Salmonella typhimurium
without S9 mix: 0.3; 1; 3, 10, 33; 100; 333; and 1000 µg/plate
with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Escherichia coli
with and without S9 mix: 33: 100; 333; 1000; 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. 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, Merck, Darmstadt, purity > 99 %)
- 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 with 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 azide (purity: >= 99.0%, supplier: Serva, D-69042 Heidelberg, Germany) dissolved in aqua dest.; concentration: 10 µg/plate Migrated to IUCLID6: without metabolic activation

Controls 3

Positive controls:

yes

Remarks:

for TA 1537, TA 98

Positive control substance:

other: 4-nitro-o-phenylenediamine, without metabolic activation

Remarks:

4-nitro-o-phenylenediamine (purity: > 99.9%, supplier: Fluka (Sigma Aldrich), 82024 Taufkirchen/Germany, dissolved in DMSO; concentration: 10 µg/plate

Controls 4

Positive controls:

yes

Remarks:

for E. coli WP2 uvr A

Positive control substance:

methylmethanesulfonate

Remarks:

methylmethanesulfonate, MMS (purity: > 99.0%, Supplier: Sigma Aldrich, 82024 Taufkirchen/Germany Migrated to IUCLID6: without metabolic activation

Controls 5

Positive controls:

yes

Remarks:

for TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvr A

Positive control substance:

other: 2-aminoanthracene, with metabolic activation

Remarks:

2-aminoanthracene (purity: 97.5%, supplier: Sigma-Aldrich, D-82024 Taufkirchen, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate in TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation assay as described by Haworth et al. 1983, with some differences.

- Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley, U.S.A.) and were stored as recommended (Maron and Ames, 1983).
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analyszed prior to their use for mutagenicity assays.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone.
Administration:
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation

Evaluation criteria:

Validity of the data is confirmed by the laboratory´s historical control data from January 2011 until December 2011 representing approx.550 experiments (WP2 uvrA the historical data are based on approx. 200 experiments).

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

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

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the decribed mutagenicity test with Salmonella typhimurium and Escherichia coli and under the experimental conditions reported, Isobornyl acrylate did not induce gene mutations by base pair changes or frame shifts 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.

Executive summary:

The study was performed to investigate the potential of Isobornyl acrylate to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment /Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II; Salmonella typhimurium

without S9 mix: 0.3; 1; 3, 10, 33; 100; 333; and 1000 µg/plate

with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Escherichia coli

with and without S9 mix: 33: 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at higher concentrations with and without

metabolic activation in strains TA 1535, TA 1537, TA 98, and TA 100 in both independent

experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication

factor of 0.5), were observed at higher concentrations in strains TA 1535, TA 1537, TA 98,

and TA 100 with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Isobornyl acrylate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Isobornyl acrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia colireverse mutation assay.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.