Butanoic acid, 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-indenyl ester

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:

7 November 2001 - 7 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

441-420-8

EC Name:

-

Cas Number:

113889-23-9

Molecular formula:

C14H20O2

IUPAC Name:

tricyclo[5.2.1.0²,⁶]dec-3-en-8-yl butanoate; tricyclo[5.2.1.0²,⁶]dec-4-en-8-yl butanoate

Test material form:

liquid

Method

Target gene:

His-gene and Trp-gene

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY STUDY
- Strains TA100 and -WP2uvrA‾ were tested
-Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested (+/-S9): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate


MUTATION STUDY - EXPERIMENT 1 (RANGE-FINDING STUDY)
-TA98, TA100, TA1535, TA1537 (-S9): 1.5, 5, 15, 50, 150, 500 µg/plate
-TA100, TA1535, TA98 (+S9): 5, 15, 50, 150, 500, 1500 µg/plate
-TA1537 ( +S9): 15, 50, 150, 500, 1500, 5000 µg/plate
-WP2uvrA‾ (+/-S9): 50, 150, 500, 1500, 5000 µg/plate

MUTATION STUDY - EXPERIMENT 2 (MAIN STUDY)
-TA98, TA100, TA1535, TA1537 (-S9): 1.5 to 500 µg/plate
-TA98, TA100, TA1535, TA1537 (-S9): 15 to 5000 µg/plate
-WP2uvrA‾ (+/-S9): 50 to 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide, prior to use dried using molecular sieves (sodium alumino-silicicate)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

benzo(a)pyrene and 2-Aminoanthracene are tested with S9, the other three substances without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine and Tryptophan

NUMBER OF REPLICATIONS: Triplate for each bacterial strain and for each concentration of the test material both with and without S9.

DETERMINATION OF GENOTOXICITY
-number of revertants

OTHER:
- Temperature 37 °C

VALIDITY:
The reverse mutation assay was considered valid if the following criteria were met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls
- The appropriate characteristics for each tester strain have been confirmed
- Each mean positive control value should be at least two times the respective vehicle control value for each strain
- There should be a minimum of four non-toxic test material dose levels
- There should not be excessive loss of plates due to contamination

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: The test material should have induced a reproducible, dose related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

No data

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY STUDY
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella tester strains. The toxic responses were greater on plates dosed in the absence of S9-mix with weakened lawns initially observed at 150 µg/plate. Plates dosed in the presence of S9 exhibited variable toxicity at higher test material dose levels (1500 µg/plate). No toxicity was exhibited in Escherichia coli strain WP2uvrA‾ either with or without S9-mix.

MUTATION STUDY
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. All of the positive control chemicals used in this test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of bacterial strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

To determine the mutagenic potential of Cyclobutanate, a bacterial reverse mutation assays (Ames test) was performed according to OECD 471 using Salmonella thyphimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA. The bacterial cultures were treated according to the Ames plate incorporation method at up to six dose level, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9). The dose range was determined in a prelimitary toxicity assay and ranges between 1.5 and 5000 µg/plate, depending on bacterial tester strain type and presence or absence of S9 -mix.

The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. All test strains demonstrated appropriate phenotypic characteristics.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Therefore, the testing material was considered to be non-mutagenic under the conditions of this test. According to the criteria outlined in Annex VI of 67/548/ECC and Annex I of 1272/2008/EC, Cyclobutanate does not have to be classified as mutagenic.