1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

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:

June 21, 2005 - August 26, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

EC Number:

251-649-3

EC Name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

Cas Number:

33704-61-9

Molecular formula:

C14H22O

IUPAC Name:

1,1,2,3,3-pentamethyl-2,3,4,5,6,7-hexahydro-1H-inden-4-one

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble at 50 mg/mL in distilled water and dissolved in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL with DMSO was considered to be stable and therefore preferably selected as a solvent in this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2uvrA); sodium azide (TA1535); Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HC1 (TA1537). +S9: 2-aminoanthracene (all test strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min.
- Selection time (if incubation with a selection agent): 48 hours

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

NUMBER OF REPLICATIONS: Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups.

NUMBER OF CELLS EVALUATED: S. typhimurium and E. coli strain were in the range of 2.3-2.7x109 cells/mL and 3.8-4.2 x 109 cells/mL resp.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibiton

OTHER EXAMINATIONS:
- Other: RANGE-FINDING/SCREENING STUDIES: Dose finding Test-1 and Test-2: Two dose-finding tests were performed to determine test concentrations in the main test. Based on observed growth inhibition at more than 78.1 µg/plate without S9 mix and at more than 313 µg/plate with S9 mix in all test strains, these concentrations were used as highest doses in the main test.
- Other: The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

The test compound was judged positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner and a reproducibility of the test results was also assured. In all other cases, it was judged negative.

Statistics:

No statistical methods were used.

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: no confounding effects found.

RANGE-FINDING/SCREENING STUDIES:
DOSE FINDING TEST-1: Bacterial growth inhibition was observed at more than 78.1 µg/plate without S9 mix, and at more than 313 µg/plate with S9 mix in all test strains. The test compound precipitates were observed at 5,000 µg/plate without S9 mix.
DOSE FINDING TEST-2: Bacterial growth inhibition was observed at 78.1 µg/plate without S9 mix in all test strains, and at more than 156 µg/plate in TA100, TA1535 and TA1537, 313 µg/plate in WP2 uvrA and TA98 with S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacterial growth inhibition was observed at 78.1 µg/plate without S9 mix in all test strains, and at more than 156 µg/plate in TA100, TA1535 and TA1537, and at 313 µg/plate in WP2uvrA and TA98 with S9 mix.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The ability of the test substance to induce mutations was investigated in the Bacterial Reverse Mutation Assay (Ames, OECD 471, GLP) by using 4 histidine-requiring strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537), and a tryptophan-requiring strain of Escherichia coli (WP2 uvrA) with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The study procedures described in this reverse mutation assay were equivalent to OECD guideline 471. Test concentrations used in the main test were as follows: - S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate; + S9 mix: 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate. The mutagenicity of the test compound was judged negative because the number of revertant colonies in the test compound treatment groups was less than twice that of each negative control in all test strains. The number of revertant colonies in the positive control groups were more than twice that of negative control groups. The test results showed that the number of revertant colonies for the negative control and the positive controls were within the range of the historical data at Hita laboratory, indicating that the test can be considered valid. It is concluded that the test substance has no ability to induce mutations in bacteria under the present test conditions.