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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

His gene

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.

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-013 to 2019-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted by the Council on 29 July, 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes cultured in vitro

Details on mammalian cell type (if applicable):

Cytotoxicity test: Blood was drawn from healthy, 35 years old male volunteer, non-smoker, non-alcoholic, free from drug, radiation and chemical exposure
Main study: Blood was drawn from healthy, 25 years old male volunteer non-smoker, non-alcoholic, free from drug, radiation and chemical exposure



- Heparin obtained from Biological E. Limited, Hyderabad
- The donor selected were non-smoker, non-alcoholic, free from drug, radiation and chemical exposure.
- A trained medical laboratory technician collected the blood by vein puncture using a 21 G needle attached to a 50 mL disposable syringe.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

The cells were treated with metaphase arresting substance (colchicine, 0.3 µg/mL) 3 h 30 minutes prior to harvesting.

Metabolic activation:

with and without

Metabolic activation system:

Metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used.
The S9 fraction was buffered and supplemented with the essential co-factors β-NADP, KCl, and D-Glucose-6-phosphate to form the “S9 mix”.
The S9 fraction was used at a concentration of 2% in the final culture medium.

Test concentrations with justification for top dose:

- Cytotoxicity test: 15.6, 31.3, 62.5, 125, and 250 µg sclareolide/mL with and without metabolic activation.

Based on the cytotoxicity test results, the following concentrations were selected for the Main Study:
Phase I: 3.13, 6.25, 12.5, 25, and 50 µg/mL of culture medium in the absence and 12.5, 25, 50, 80 and 100 µg/mL of culture medium in the presence of metabolic activation system.
Phase II: 3.13, 6.25, 12.5, 25, and 50 µg/mL of culture medium in the absence of metabolic activation system.

Vehicle / solvent:

Considering purity of the test item and molecular weight, the 10 mM concentration was 2547 µg/mL. As the 10 mM concentration was higher than 2000 µg/mL, 2000 µg/mL was selected as the highest applicable concentration, recommended by OECD guideline. Sclareolide was insoluble in distilled water at 200040.5 µg/mL, while formed suspension in dimethyl sulfoxide at 200040.5 µg/mL, which was found soluble at 100020.3 µg/mL upon sonication. Therefore, dimethyl sulfoxide (DMSO) was selected as the suitable vehicle in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cytotoxicity Test:
Before conducting the chromosomal aberration study, sclareolide was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (2% v/v S9 mix). The cytotoxicity test was performed with the test concentrations of 15.6, 31.3, 62.5, 125, and 250 µg sclareolide/mL of culture medium.The cultures were exposed for 3 h and 30 minutes both in the absence and presence of metabolic activation. The cells were treated with metaphase arresting substance (colchicine, 0.3 µg/mL) 3.5 h prior to harvesting. A concurrent negative control (dimethyl sulfoxide) was also maintained.

Main Study:
Based on the results of cytotoxicity test, sclareolide was evaluated for its potential to induce chromosomal aberration at the dose levels of 3.13, 6.25, 12.5, 25, and 50 µg/mL of culture medium in the absence and 12.5, 25, 50, 80 and 100 µg/mL of culture medium in the presence of metabolic activation system (2% v/v S9 mix).
The main study was conducted in two phases i.e., 4 hours without and with 2% v/v S9 mix in Phase I, and 24 hour and 30 minutes without S9 in Phase II. There were 2 replicates to each treatment concentration including positive and negative controls.

Phase I (Absence and presence (2% v/v S9) of metabolic activation and exposure for 4 h):
At the end of 4 h of treatment, cultures were centrifuged at 2000 rpm for 10 minutes and the treatment media was removed and replaced with fresh complete medium and incubated at 37 ± 1 °C and 5% CO2 in a humidified CO2 incubator until harvesting. The cells were harvested and processed for slide preparation approximately after 24 hour from the beginning of the treatment.

Phase II (Absence of metabolic activation and exposure for 24 h and 30 minutes):
In phase II the cultures were exposed with sclareolide at 3.13, 6.25, 12.5, 25, and 50 µg/mL of culture medium in the absence of metabolic activation for 24 h and 30 minutes at 37 ± 1 °C and 5% CO2 in a humidified CO2 incubator until harvesting.
The cells were harvested and processed for preparation of slides after the exposure period. As the phase II experiment was performed to confirm the negative results obtained in the absence of metabolic activation in Phase I, slide scoring data was evaluated after evaluating the data of phase I experiment.

Rationale for test conditions:

OECD test guideline

Evaluation criteria:

To assess the clastogenic activity of the test item following criteria were evaluated:
i. At least one of the test concentrations exhibits a statistically significant increase in % aberrant cells compared with the concurrent negative control.
ii. A dose related statistically significant (biologically relevant) increase in % aberrant cells in at least one concentration when evaluated with an appropriate trend test.
iii. Any of the results are outside the distribution of the historical negative control range.
To assess the non-clastogenic activity of the test item following criteria were evaluated:
i. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii. There is no concentration-related increase when evaluated with an appropriate trend test.
iii. All results should be within the distribution of the historical negative control data (i.e. Poisson based 95% control limits).
- Historical control data for this laboratory was also considered in the evaluation.
- An increase in the number of polyploid cells indicates that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. Biological relevance was considered first. The test item was considered non-mutagenic if the results did not meet the above mentioned criteria.

Statistics:

- Gaps and polyploidy were not included in the calculation of total aberration frequency.
- Data on percent aberrant cells and polyploidy were subjected to Shapiro-Wilk’s test for normality and Bartlett’s test to assess homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test.
- Where the data did not meet suitable homogeneity of variance, Student's t-test was performed to determine the level of significance between negative control, three selected test concentrations (selected based on the mitotic index data) and positive controls.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

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:

Precipitation, pH and Osmolality Tests:
A significant change in pH (±1 unit) or osmolality (≥ 50 mOsm/kg H2O) was not observed at 0 and 4 h in any tested concentration of 62.5, 125, 250, 500, 1000 µg sclareolide/mL of culture medium.
A significant change (≥ 50 mOsm/kg H2O) was observed at the highest concentration 2000 µg sclareolide/mL of culture medium with normal pH. Precipitation was observed at tested concentration levels 250, 500, 1000, and 2000 µg/mL at 0 hour and additionally at the test concentration level 125 µg/mL after 4 hour incubation. However, precipitation was not observed at the tested concentration 125 µg/mL at 0 hour and 62.5 µg/mL at 0 and 4 hour, respectively.
Therefore, the concentration 250 µg/mL of culture medium was selected, as the highest concentration for the cytotoxicity test.

Cytotoxicity Test:
The cytotoxicity was assessed based on results of solubility, precipitation, pH, and osmolality tests at concentrations 15.6, 31.3, 62.5, 125, and 250 µg sclareolide/mL of culture medium. Precipitation was not observed up to the tested concentration 125 µg/mL in both phases at 0 hours. Precipitation was observed at tested concentration 125 and 250 µg/mL after 4 hour incubation, in the absence and presence of the metabolic activation system.
The pH and osmolality in the beginning of the treatment at the concentration level 250 µg/mL were 7.28 and 435 mOsm/kg H2O, respectively (compared to 7.28 and 448 mOsm/kg H2O in the negative control), in the absence of the metabolic activation system. The pH and osmolality at the concentration of 250 µg/mL were 7.38 and 446 mOsm/kg H2O, respectively (compared to 7.32 and 443 mOsm/kg H2O in the negative control) in the presence of the metabolic activation system (2% v/v S9 mix).
Significant change in the pH or osmolality was not observed up to the highest concentration of 250 µg/mL, in the absence and presence of the metabolic activation.
Significant reduction in mitotic index was observed at concentration of 62.5 µg/mL as 76.21% and at 125 µg/mL as 86.62%, respectively in the absence and presence of the metabolic activation.
Percent reduction in mitotic index of 7.22, 27.79, 76.21, 86.10, and 88.86 was observed at tested concentrations of 15.6, 31.3, 62.5, 125, 250 µg/mL in culture medium, respectively, in the absence of metabolic activation. Percent reduction in mitotic index of 2.87, 24.11, 28.19, 86.62, and 90.91 was observed at tested concentrations 15.6, 31.3, 62.5, 125, and 250 µg/mL in culture medium respectively, in the presence of metabolic activation (2% v/v S9 mix). Hence, considering its cytotoxicity the concentration of 50 µg/mL and 100 µg/mL were selected as highest concentration, in the absence and presence of the metabolic activation for main study, respectively.

Main Study:
No relevant influence of the test item on pH or osmolality was observed in the absence (Phase I and II) and the presence of metabolic activation (Phase I). Precipitation was not observed up to the tested concentration of 50 µg/mL in absence and 100 µg/mL in presence of the metabolic activation system, respectively.

Phase I (Absence and presence (2% v/v S9 mix) of metabolic activation and exposure for 4 h):
Significant level of cytotoxicity (55 ± 5% reduction in mitotic index) was observed at 50.0 and 100 µg/mL, both in the absence and presence of the metabolic activation, respectively.
Percent reduction in the mitotic index 2.67, 4.94, 5.96, 27.13, and 50.20 was observed in the absence of the metabolic activation system treated at concentrations of 3.13, 6.25, 12.5, 25.0, and 50.0 µg/mL in culture medium, respectively.
Percent reduction in the mitotic index 5.77, 6.99, 8.57, 19.60, and 51.38 was observed in the presence of the metabolic activation (2% v/v S9 mix) tested at concentrations of 12.5, 25, 50, 80, and 100 µg/mL in culture medium, respectively.
Statistically significant reduction in mitotic index was observed at 50 µg/mL and 100 µg/mL, in absence and presence of the metabolic activation, respectively. Therefore, test concentrations selected for scoring of chromosomal aberrations in the absence of the metabolic activation were: 12.5, 25.0, and 50.0 µg/mL, while 50, 80, and 100 in the presence of the metabolic activation.
Sclareolide did not induce statistically significant or biologically relevant increase in the percent aberrant cells up to the concentration of 50 µg/mL in absence and 100 µg/mL, in the presence of the metabolic activation, respectively
Sclareolide did not induce an increase in the number of polyploid cells in absence and presence of the metabolic activation (2% v/v S9 mix) up to the concentration of 50 µg/mL, in absence and 100 µg/mL in the presence of metabolic activation.

Phase II (Absence of metabolic activation and exposure for 24 h and 30 minutes):
The observed reduction in the % mitotic index was 51.65%. Hence, based on results of reduction in mitotic index, concentrations 12.5, 25, and 50 µg/mL were scored for chromosomal aberrations. Statistically significant reduction in mitotic index was observed at 50 µg/mL and 100 µg/mL, in absence and presence of the metabolic activation, respectively. Sclareolide did not induce a statistically significant or biologically relevant increase in the percent aberrant cells, up to the concentration 50 µg/mL
Sclareolide did not induce an increase in the number of polyploid cells in the absence of the metabolic activation up to the concentration of 50 µg/mL.

Number of cells with chromosomal aberrations found in the negative control cultures was within the laboratory historical control data range. The positive controls Mitomycin-C and Cyclophosphamide produced significant increase in the frequency of aberrant cells during phase I and II of the main study experiment. Therefore, it is concluded that test conditions were adequate and that the metabolic activation (S9 mix) functioned properly.

Conclusions:

Sclareolide did not show any potential to induce chromosomal aberration, in the absence and presence of the metabolic activation under the present experimental conditions and is considered to be negative for clastogenicity.

Executive summary:

An OECD 473 compliant in vitro mammalian chromosomal aberration test was conducted on Sclareolide using human peripheral blood lymphocytes under GLP conditions.

Based on results of preliminary solubility and cytotoxicity tests,50 µg/mL and 100 µg/mLwere selected as the highest concentration level, without metabolic activation and with metabolic activation system, respectively, in the main study.

Sclareolidewas tested in two phases, without metabolic activation and with metabolic activation (2% v/v S9 mix) for 4 hours (Phase-I) and without metabolic activation for 24 hours and 30 minutes (Phase-II). Cultures were exposed tosclareolideat five different concentration levels between3.13 and 50 µg/mL of culture (phase-I and phase-II), in the absence and 12.5 and 100 µg/mL of culture (phase-I), in the presence of the metabolic activation (2% v/v S9 mix), in duplicate.  

Sclareolidedid not induce statistically significant or biologically relevant increase in the number of percent aberrant cells, in the absence and presence of the metabolic activation system for the short term (phase-I) and in the absence of S9, long term (phase-II) exposure period. No effect of sclareolideon the number of polypoid cells was observed, in the absence (phase-I and phase-II) and presence of S9-mix (phase-I). All negative controls were comparable to the laboratory historical control limits. Positive controls showed an increase in the incidence of cells with chromosomal aberrations.

All criteria for a valid study were met.From results of this study, it is concluded thatsclareolide didnot show any potential to induce chromosomal aberrations, in the absence and presence of the metabolic activation system, under the described experimental conditions and is considered to be negative for clastogenicity.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-12 to 2018-12-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted by the Council on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cell line (free from mycoplasma contamination), a sub-clone of Chinese hamster ovary cell line was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 26 and 24 was used in cytotoxicity and mutagenicity tests, respectively.

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

A metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used.
The S9 fraction was buffered and supplemented with the essential co-factors β-NADP, KCl and Glucose-6- phosphate to form the “S9 mix”. The S9 fraction was used at a concentration of 2% in the final culture medium for Main Study.

Test concentrations with justification for top dose:

The following concentrations were tested in the Cytotoxicity test: 15.6, 31.3, 62.5, 125, and 250 µg/mL.
Based on the results of the cytoxicity test, the following concentrations were selected for the mutagenicity experiment: 15.6, 31.3, 62.5, 125, and 250 µg/mL

Vehicle / solvent:

Considering molecular weight and purity of the test item, the 10 mM concentration is higher than the guideline limit concentration (2000 µg/mL). Hence, test item solubility was tested for the required concentration at 2000 µg/mL. The test item was found insoluble in distilled water and found to form suspension (stock B, 200040.5 µg/mL) with DMSO even after sonication. The above suspension dissolves to its next lower concentration (stock C, 100020.3 µg/mL) after sonication. A dilution series was prepared to check pH and osmolality.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Cytotoxicity Test:
Based on the results of solubility, precipitation and pH tests, 250 µg/mL of culture medium was selected as the highest concentration to be tested for cytotoxicity test. The cytotoxicity test was performed both in the absence and presence of metabolic activation (2% v/v S9 mix) at the concentrations of 15.6, 31.3, 62.5, 125, and 250 µg/mL. Dimethyl sulfoxide was used as concurrent negative control. The pH and osmolality were also checked.
Based on the results of cytotoxicity test, the concentration of 250 µg Sclareolide/mL was selected as the highest concentration for main study experiment.

Mutagenicity Experiment:
Treatment was performed both in the absence and presence of metabolic activation (2% v/v S9 mix). Two replicate flasks were maintained for each test concentration, negative and positive controls.
Cultures were treated at the concentrations of 15.6, 31.3, 62.5, 125, and 250 µg/mL both in the absence and presence of metabolic activation (2% v/v S9 mix) for a period of 4 hours.
For the treatment, the first stock solution (stock A) of sclareolide was prepared by solubilising 125.008 mg of sclareolide in DMSO and volume was made up to 5 mL (250 µg/mL).

Treatment:
The day of treatment was recorded as day 1 in this experiment.
Twenty Eight (Fourteen each for treatment in absence and presence of metabolic activation) culture flasks prepared on ‘day 0’ were observed under an inverted microscope for their growth and culture conditions. Cultures free from contamination were used during main study.
The media in culture flasks was removed prior to treatment. Plain culture media (α-MEM) without serum was used for treatment. For treatment in presence of metabolic activation, media containing S9 was used at a final concentration 2% v/v S9 mix.
The untreated cultures maintained in the absence and presence of the metabolic activation system served as the negative control.
Two culture flasks were maintained for each test concentration including negative and positive controls. The cultures were incubated at 37 ± 1 °C and 5% CO2 in humidified air for 4 hours.
At the end of the exposure period, the treatment medium was removed from the flasks and the cell surface was rinsed using Dulbecco’s Phosphate Buffered Saline (DPBS). The washed cells were then trypsinized and suspended in complete medium (media containing serum) to obtain single cells. The cell concentration was determined by using a haemocytometer and adjusted accordingly with complete medium. A sample taken from the cell suspension was serially diluted with complete medium to approximately 1000 - 2000 cells/mL.
An aliquot of 100 µL was then dispensed on the center of 60 mm tissue culture dishes and 5 mL of complete medium was added. The plates were used determine relative survival (RS) as a maker of cytotoxic effects of the selected test concentrations.
Also, a convenient volume was transferred to fresh tissue culture flasks in such a way that the flasks received approximately 2 x 106 cells for each of treatment, negative and positive controls. These flasks were kept as expression flasks for mutation. All the plates and flasks were incubated in a CO2 incubator at 37 ± 1 °C and 5% CO2 for 8 days.

Subculture for Mutation Expression:
The cultures for mutation expression were subcultured on days 3 and 5, following the culture processing on day 1.
During the expression period (7 - 9 days) the cell concentration in the flasks was adjusted by subculturing on days 3 (one flask per replicate) and 5 (two flasks per replicate). The cells in each flask were trypsinized and the cell concentration was adjusted to approximately 2 x 106 cells/culture, provided with fresh complete medium and incubated under standard conditions.

Selection of Mutants and Cytotoxicity observation:
On day 8, the cells from the expression flasks were trypsinised for each test concentration, negative control and positive controls and were counted and plated for determination of survival and mutant frequency.
The cell concentration was adjusted to around 1 - 2 x 10e5 cells/mL for survival plating and 2 x 10e5 cells/mL for selection of mutants. A sample taken from the cell suspension was diluted to approximately 1000 - 2000 cells/mL by serial dilution. An aliquot of 100 µL was then dispensed on the center of 60 mm culture plate and 5 mL of complete medium was added. Duplicate plates were maintained for each replicate of treatment, negative and positive controls. The plates were then incubated for 8 days for the determination of survival. One mL of the suspension (2 x 10e5 cells/mL) was added into 100 mm culture plate with 10 mL of selective medium (α-MEM without nucleoside - complete medium containing 5 µg/mL of 6-thioguanine). For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5% CO2 in humidified air using a CO2 incubator for 8 days (i.e. up to Day 16). At the end of the 8 days incubation period on day 9, the cytotoxicity plates were removed and the medium was decanted. The colonies were fixed, stained and washed. The colonies formed on each plate were counted and recorded for calculation of relative survival/cloning efficiency.

Enumeration of Colonies:
On day 16 the plates were removed from the incubator and the medium was decanted off and colonies were fixed using 3.7% formaldehyde for 10 minutes. The fixative was removed and the colonies were stained using 0.4% methylene blue for 10 minutes. The number of colonies formed on each replicate plate was counted on day 17 and recorded. The number of colonies was used to calculate the cloning efficiency at the time of selection, number of clonable cells and the mutant frequency per 1 x 10e6 clonable cells.

Rationale for test conditions:

OECD guidleine protocol

Evaluation criteria:

A test item was considered positive in the mutation assay if:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is concentration-related when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit).
When all of these criteria are met, the test item was then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item will be considered clearly negative if, in all experimental conditions examined:
d. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
e. There is no concentration-related increase when evaluated with an appropriate trend test.
f. All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).
The test item will be, then considered unable to induce gene mutations in cultured mammalian cells in this test system.

In cases when the response is neither clearly negative nor clearly positive, as described above, or in order to assist in establishing the biological relevance of a result, the data will be evaluated by senior study director for further investigations. Performing a repeat experiment possibly using modified experimental conditions could be useful.

In rare cases, the data set will preclude making a conclusion. Therefore the test item response will be concluded to be equivocal (interpreted as equally likely to be positive or negative).
Historical control data for this laboratory will be taken into account in evaluating the results.

Negative and equivocal results will be confirmed by a repeat test (short duration), using a modification in test concentration and / or metabolic activation conditions.

Statistics:

Weighted regression analysis was performed to evaluate the concentration response relationship on treatment groups against the negative control group (excluding positive controls).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation, pH and Osmolality Tests:
A significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was not observed at 0 and 4 h in any of the tested concentration (62.5, 125, 250, 500, 1000, and 2000 µg/mL culture medium).
Precipitation was observed from 250 to 2000 µg/mL and at 125 to 2000 µg/mL after incubation of 4 h, respectively.

Cytotoxicity Test:
Cytotoxicity was assessed by calculating percent relative cloning efficiency following treatment.
No change in the pH or osmolality was found in any treated concentration, during the cytotoxicity experiment. Precipitation was also absent in any treated concentration, except 250 µg/mL.
The relative cloning efficiency (RCE) is also known as relative survival (RS). The percent relative cloning efficiency (%RCE) was comparable to the concurrent control, both in the absence of the metabolic activation (62-100 %) and in the presence of the metabolic activation system (2% v/v S9 mix, 62-97%), at the tested concentration up to 250 µg/mL of culture medium, respectively.
Based on results of cytotoxicity test, proposed concentrations for mutagenicity experiment are: 15.6, 31.3, 62.5, 125, and 250 µg/mL of culture medium both in the absence and presence of the metabolic activation.

Mutagenicity Test:
No relevant influence of the sclareolide on osmolality and pH value was observed in the absence and presence of the metabolic activation in the main study.
Relative Cloning Efficiency/percent relative survival (RCE) following the treatment with 0, 15.6, 31.3, 62.5, 125, and 250 µg/mL in the absence of metabolic activation was 100, 101.05, 86.13, 93.01, 76.64, 66.37 and in the presence of metabolic activation was 100, 98.64, 100.09, 96.08, 79.05 and 70.48, respectively.
Mean mutation frequency per 1 x 10e6 cells (MF) following treatment with 0, 15.6, 31.3, 62.5, 125, and 250 µg/mL in the absence of metabolic activation was 15.25, 17.47, 18.74, 18.09, 18.92 and 18.19 and in the presence of metabolic activation was 17.18, 16.57, 17.16, 16.92, 18.23 and 17.38, respectively.
Mean mutation frequency per 1 x 10e6 cells (MF) for positive control in the absence of metabolic activation was 301.20 and in the presence of metabolic activation was 335.68.
A weighted regression analysis was performed to evaluate the significant concentration-related effect in the mutation frequency of cultures treated with the concurrent negative control group.
The mean mutant frequency of the positive control exhibited a clear increase over the mean value of the negative control demonstrating that positive control had potential to induce gene mutations at the hprt locus of CHO-K1 both in the absence and presence of the metabolic activation. This also demonstrates that the S9 mix was capable of metabolising a pro-mutagen to its mutagenic form(s), thus, demonstrating integrity of the S9 mix.
The cloning efficiency in the negative control was above 60% during main study. The mutant frequency, in the negative control group was less than 20 per 106 clonable cells during main study, validating the acceptability of the test system .
A significant concentration-related increase in the mutation frequency was not observed in any treated concentration and the mutation frequency was comparable to that from the negative control group.

Conclusions:

Sclareolide does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of the metabolic activation (2% v/v S9 mix) under the present experimental conditions.

Executive summary:

An OECD 476 compliant mammalian cell gene mutation assay [Hprt locus] was conducted in CHO-K1 cells cultured in vitrounder GLP conditions. CHO-K1 cells were exposed to Sclareolideat different concentrations, both in the absence and presence of the metabolic activation (2% v/v S9 mix) for a period of 4 hours.

 

Cultures were exposed to sclareolideat 15.6, 31.3, 62.5, 125, and 250 µg/mL of the culture medium, in duplicate, both in the absence and presence of the metabolic activation (2% v/v S9 mix) for a period of 4 hours.

 

Mutation frequency was comparable to the control and any significant concentration-related increase in the mutation frequency was not observed at any tested concentration (15.6, 31.3, 62.5, 125, and 250 µg/mL of culture medium), both in the absence and presence of the metabolic activation system (2% v/v S9 mix).

 

All negative controls were showing normal mutation frequency while positive controls showed an increase in the mutation frequency. Relevant influences of sclareolide on pH or osmolality too were not observed both in the absence and presence of the metabolic activation.

 

All criteria for a valid study as described in the study plan were met. Based on results from this study, it is concluded that sclareolidedoes not have potential to induce gene mutations at thehprtlocus of CHO-K1 cells both in the absence and presence of metabolic activation system (2% v/v S9 mix) under the present experimental conditions.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available