Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

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:

23 February 2007 - 16 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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

1

Reference substance name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

EC Number:

916-331-7

Molecular formula:

C14H20O2

IUPAC Name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

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 TEST
With and without metabolic activation. Tested concentrations 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

MUTATION TEST EXPERIMENT 1 (Range-finding test)
With and without metabolic activation.
-Salmonella strains: 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
-E.coli strain WP2uvrA‾: 50, 150, 500, 1500, 5000 µg/plate

MUTATION TEST EXPERIMENT 2 (Main test)
-All Salmonella strains (-S9): 1.5, 5, 15, 50, 150, 500 µg/plate
-Salmonella strains TA98 & TA1537 (+S9): 15, 50, 150, 500, 1500, 5000 µg/plate
-Salmonella strains TA100 & TA1535 (+S9): 5, 15, 50, 150, 500, 1500 µg/plate
-E.coli strain WP2uvrA‾ (+/-S9): 50, 150, 500, 1500, 5000 µg/plate

For all experiments additional dose levels were included for the salmonella strains to allow for test material induced toxicity, ensuring that a minimum of four non-toxic doses were achieved.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully micible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.

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 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48hr (at 37°C)

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

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

VALIDITY: In accordance with OECD 471.
- Characteristic number of spontaneous revertants
- Confirmation of appropriate test strain characteristics
- All test strains in the approximate range of 1 to 9.9 * 10-9 bacteria per mL
- Mean positive control value at least twice vehicle control value
- Minimum of four non-toxic test material dose levels
- No excessive loss of plates due to contamination

Evaluation criteria:

In accordance with OECD 471. There are several criteria for determining a positive result, such as a dose-related increase in revertant frequencyover the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

No data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See text

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See text

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See text

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See text

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See text

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
A history profile of vehicle and positive control values is presented in appendix 2 of the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

PRELIMINARY STUDY
The test material was toxic to TA100 from 500 µg/plate and non-toxic to WP2uvrA‾. The test material formulation and S9 -mix were shown to be sterile.

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

CYTOTOXICITY
The test material caused a visible reduction in growth of the bacterial background lawn of the majority of the Salmonella strains, initially from 150 and 500 ug/plate with and without S9, respectively. No cytotoxicity was observed in Salmonella strain TA98 (main test, +S9) and in the E. coli strain.

Remarks on result:

other:

Applicant's summary and conclusion

Conclusions:

In a bacterial reverse mutation test in accordance with OECD 471, Cyclabute did not induce mutations and was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A bacterial reverse mutation assay (Ames test) was performed with bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA‾. The test was performed under GLP conditions and according to OECD 471. A preliminary test was performed to determine the test range. The test item was dissolved in DMSO and applied once at test initiation with concentrations ranging from 1.5 -5000 µg/plate. The validity criteria of the test results were fulfilled. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains. The test 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, cyclabute does not have to be classified as mutagenic.