[1R-(1α,4β,4aα,6β,8aα)]-octahydro-4,8a,9,9-tetramethyl-1,6-methano-1(2H)-naphthol

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:

19 June 2014 - 29 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

[1R-(1α,4β,4aα,6β,8aα)]-octahydro-4,8a,9,9-tetramethyl-1,6-methano-1(2H)-naphthol

EC Number:

227-807-2

EC Name:

[1R-(1α,4β,4aα,6β,8aα)]-octahydro-4,8a,9,9-tetramethyl-1,6-methano-1(2H)-naphthol

Cas Number:

5986-55-0

Molecular formula:

C15H26O

IUPAC Name:

[1R-(1α,4β,4aα,6β,8aα)]-octahydro-4,8a,9,9-tetramethyl-1,6-methano-1(2H)-naphthol

Test material form:

solid: crystalline

Details on test material:

- Storage condition of test material: Store in cool dark place at room temperature

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

- Experiment 1:
All five strains (without and with S9): 5, 16, 50, 160, 500, 1600 and 5000 μg/plate
- Experiment 2:
TA 1535, TA 1537, TA 98 and TA 100 (without S9): 1.6, 5, 16, 50, 160 and 500 μg/plate
WP2uvrA (without and with S9): 16, 50, 160, 500, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: In a solubility test using the vehicle selected for this study, DMSO, the test article formed a non-viscous, transparent, colorless solution at 222.4 mg/mL.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

100 μL/plate DMSO

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1 and 2: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 hours at 37 ± 2°C

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

NUMBER OF CELLS EVALUATED: ≥ 1x10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.

Evaluation criteria:

A test article is considered to have produced a positive response if it induces a dosedependent increase in revertant frequency that is ≥ 2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.
A test article is considered to have produced a negative response if no dose-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.

Statistics:

Not performed.

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:

Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.

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:

Experiment 1: at ≥ 160 μg/plate without and with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.

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:

Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.

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:

Experiment 1: at ≥ 160 μg/plate without S9 and at ≥ 500 μg/plate with S9. Experiment 2: at ≥160 μg/plate without S9 and at 500 μg/plate with S9.

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:

at 5000 μg/plate without and with S9.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was observed at 5000 μg/plate with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met.
- Negative (solvent/vehicle) historical control data:
TA98: 8 - 60
TA100: 60 - 240
TA1535: 4 - 45
TA1537: 2 - 25
WP2uvrA: 5 - 40

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent direct plate experiments in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate in the first experiment. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or absence or reduction of bacterial background lawn, was observed at ≥ 160 μg/plate in all tester strains without S9 and at ≥ 500 μg/plate with S9 except TA1537 and WP2uvrA, where toxicity was observed at ≥ 160 and 5000 μg/plate, respectively. In the second experiment, the substance was tested in the four Salmonella strains in the absence and presence of S9-mix up to and including 500 μg/plate and in strain WP2uvrA in the absence and presence of S9-mix up to and including the precipitating concentration of 5000 μg/plate. Toxicity, as evident by the absence or reduction in the mean number of revertant colonies and/or reduced bacterial background lawn, was observed at ≥ 160 μg/plate in all tester strains without S9 and at 500 μg/plate with S9 except WP2uvrA, where toxicity was observed at

5000 μg/plate.

No increase in the mean number of revertant colonies was observed at any tested concentration in any tester strain in the presence or absence of S9 in the initial or the confirmatory assay. All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.