Reaction mass of (3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1989-01-18 to 1989-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study, comparable to OECD 471 guideline study although not mentioned in the study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

EC Number:

229-861-2

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]-dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Cas Number:

6790-58-5

Molecular formula:

C16H28O

IUPAC Name:

(3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Constituent 2

Reference substance name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Cas Number:

234431-64-2

Molecular formula:

C16H28O

IUPAC Name:

(3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Constituent 3

Reference substance name:

(3aR,5aS,9aS,9bS)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan

Cas Number:

68365-89-9

Molecular formula:

C16H28O

IUPAC Name:

(3aR,5aS,9aS,9bS)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan

Constituent 4

Reference substance name:

(3aS,5aR,9aR,9bR)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Cas Number:

2006270-65-9

Molecular formula:

C16H28O

IUPAC Name:

(3aS,5aR,9aR,9bR)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan

Test material form:

solid

Details on test material:

- Physical state: pale yellow liquid or white solid
- Stability under test conditions: stable

Method

Target gene:

Histidine gene for Salmonella

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-beta-naphtoflavone-induced rat S9 liver homogenate fraction

Test concentrations with justification for top dose:

0, 1, 5, 10, 25, 50 and 75 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility in DMSO > 50 mg/mL

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: ICR 191

Remarks:

Without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

TEST 1: in agar (plate incorporation)
- Expression time (cells in growth medium): 48 hours at 37°C
- Number of replications: 4 replicate plate for the test material and negative control, two replicate plates for the positive control

TEST 2 : preincubation
- Preincubation period: 30 minutes at 37 °C
- Expression time (cells in growth medium): 48 hours at 37°C
- Number of replications: 4 replicate plate for the test material and negative control, two replicate plates for the positive control

DETERMINATION OF CYTOTOXICITY
- Method: growth of the background lawn and/or frequency of spontaneous revertants (toxicity prescreen on TA100)

Evaluation criteria:

Reproducible, dose-related increase in the number of his+ revertant. This increase should reach at least a doubling of the number of spontaneous revertants for S. typhimurium strains TA1535, TA1537, TA1538 and TA98. For strains TA97, TA100 and TA102, a 1.5-fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control date;

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA97, TA98, TA100 & TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See "Tables of result" in "Attached background material"

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium:
- Water solubility: Test substance was solubilized in DMSO to improve solubility
- Precipitation: milky suspension at 75 µg/plate
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: was done in TA100, but results were not reported

COMPARISON WITH HISTORICAL CONTROL DATA: Not done

ADDITIONAL INFORMATION ON CYTOTOXICITY: distinct toxic effect were detected in different strains. See "Tables of result" in "Attached background material"

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA1538, TA97, TA98, TA100 & TA102.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA1538, TA97, TA 98, TA100 and TA 102 were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (phenobarbital / beta-naphtoflavone rat liver homogenate fraction - S9). Plate incorporation assay was used in the first experiment, whereas liquid preincubation assay was performed for the second experiment. The dose range for the main test was determined in a preliminary toxicity assay and was 1 to 75 µg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused visible reduction in the growth of the bacterial background lawn in the prescreen test and was therefore, tested up to cytotoxicity.

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.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA97 TA98, TA100 & TA102.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.