[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-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:

2018-09-08 to 2018-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted by the Council on July 21, 1997

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:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

EC Number:

209-269-0

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Cas Number:

564-20-5

Molecular formula:

C16H26O2

IUPAC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Test material form:

solid

Method

Target gene:

His gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat Liver S9 Fraction
- Species & Sex: Wistar Rat, Male
- Inducer & Dose: Aroclor 1254 (Analab, USA), 500 mg/kg b.wt
- Method of preparation of S9 mix: The S9 fraction was buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form the “S9 mix”.
- Concentration of S9 mix in the final culture medium : 5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 system was tested in TA100 (using DimethI sulfoxide, Aflatoxin B1, 2-Aminoanthracene and Benzo(a) Pyrene) and TA98 (using DimethyI sulfoxide and MeIQ).

Test concentrations with justification for top dose:

The folllowing concentrations were tested in the Initial Toxicity-Mutation Test: 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg sclareolide /plate.

Based on the cytotoxicity result, the following concentrations were selected for the Confirmatory Mutation Test:
Strains TA1537, TA1535, TA98, and TA100: 46.88, 93.75, 187.5, 375, 750, and 1500 µg sclareolide /plate
Strain TA102: 156.25, 312.5, 625, 1250, 2500, and 5000 µg sclareolide/plate

Vehicle / solvent:

The test item was insoluble in distilled water (stock A, 50 mg/mL). The test item was found to be soluble in dimethyl sulfoxide (stock B, 50 mg/mL) after sonication. Hence, dimethyl sulfoxide was selected as the vehicle for treatment. A volume of 100 µL of the test item from stock A (50 µg/mL) was added to 2 mL of top agar, to assess the precipitation. Slight precipitation was observed at the tested concentration of 5 mg/plate, which may not affect enumeration of colonies. Hence, 5 mg/plate was selected as the highest concentration to be tested for initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Cell Viability Test:
Cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range and so they were used for the study.

Genotype Confirmation Test:
The genotype of the tester strain was confirmed for all the strains regularly (once in a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Sterility Check for the Operating System:
A sterility check for the operating system was performed along with mutagenicity test.

Initial Toxicity-Mutation Test:
Before commencing the confirmatory mutation test, test item was tested for initial toxicity-mutation test using all five tester strains of Salmonella typhimurium. The experiment was conducted both in the absence and presence of metabolic activation system (5% v/v S9 mix).
The first stock solution (stock A) of the sclareolide was prepared by suspending 500.02 mg ≈ 500 mg of sclareolide in dimethyl sulfoxide and volume was made up to 10 mL (50000 µg/mL). Lower solutions B to H, were prepared by serially dilution.
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 500 µL of 0.2 M phosphate buffer was added in the absence of metabolic activation system and 500 µL of 5% v/v S9 mix was added in the presence of metabolic activation system.
Volume of 100 µL of the relevant stock solution of test item, distilled water and relevant positive control were used for treatment, as a negative control and as a positive control, respectively. Finally, 100 µL of bacterial culture was added to the tubes and mixed.
Cultures used were checked for cell viability prior to testing. This treatment mixture was poured on MGA plates and allowed to solidify.
Duplicate sets of plates were maintained for each concentration of test item, positive control and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of background bacterial lawn inhibition and reduction in number of colonies.

Confirmatory Mutation Test:
Based on the result of initial toxicity mutation test, the confirmatory mutation test was performed both in absence and presence of the metabolic activation system (10% v/v S9 mix). The treatments were performed by plate incorporation technique using triplicate set of plates as described in the initial toxicity-mutation test.
Two different dilution series was prepared for treatment. The first dilution series was prepared by dissolving of 150 mg of sclareolide and the volume was made up to 10 mL (15000 µg/mL) in DMSO. Further stock solutions (stock I to M), were prepared by serially diluting the above stock using a factor of 2.
The second dilution series was prepared by dissolving of 500.01 mg (≈ 500 mg) of sclareolide in DMSO, and the volume was made up to 10 mL (Stock A, 50000 µg/mL) after sonication. Further, stock solutions (stock B to F), were prepared by serially diluting the corresponding stock solutions using a factor of 2.

Evaluation criteria:

Cytotoxicity Evaluation Criteria:
Five analysable doses were available to evaluate assay data. Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.

Assay Evaluation Criteria:
A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Biological relevance of the result was considered first.
Strains TA1535, TA1537: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strains TA98, TA100 and TA102: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

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 102

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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 5

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

Applicant's summary and conclusion

Conclusions:

Sclareolide is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100, and TA102 in the presence and absence of S9 metabolic activation system.

Executive summary:

An OECD 471 complaint bacterial reverse mutation assay was conducted on sclareolide under GLP conditions using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100 and TA102).

Sclareolide was tested in two independent experiments, in absence and presence of the metabolic activation. Bacterial cultures were exposed to thetest itemat 8 concentration levels (two plates/concentration) between1.5 and 5000mg/platein the initial toxicity-mutation test (trial-I). No increase in the number of revertant colonies (no mutagenic effect) was observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains.

To confirm the negative results obtained in the initial toxicity mutation test, confirmatory mutation test was conducted with an increased S9 concentration (i.e., 10% v/v S9 mix) andmodified concentration spacing (trial II). Bacterial cultures were exposed tosclareolideat 6 concentration levels (three plates/concentration) between 46.88 and 1500 µg/plate for tester strains TA1537, TA1535, TA98, TA100, and between 156.25 and 5000 µg/plate for tester strain TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were enumerated.

Sclareolide did not induce any significant increase in the number of revertant colonies, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met as described in the study plan.From the results of this study, under the specified experimental conditions,test itemisconcluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.