(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-06-24 to 2019-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EC Commission Regulation No. 440/2008. OJ L 142/262.

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2019-04-02 / Signed on 2019-08-01.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene

EC Number:

220-585-8

EC Name:

(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene

Cas Number:

2825-82-3

Molecular formula:

C10H16

IUPAC Name:

(1R,2S,6R,7S)-tricyclo[5.2.1.0^{2,6}]decane

Test material form:

liquid

Details on test material:

- Physical appearance: clear liquid
- Storage conditions: at room temperature (15 - 25°C), under nitrogen.

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cells: CHO-K1 cells were obtained from the European Collection of Cell Cultures. Cells are stored at -196 to -150°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).
- Type and identity of media:
Ham’s Nutrient Mixture F12, supplemented with with 1 mM L glutamine and 50 ng/mL amphotericin B / 20 IU/mL penicillin / 20 μg/mL streptomycin. The resulting medium is referred to as H0.
H0 medium supplemented with 10% HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 µg/mL.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes, 4 days prior to exposure, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine for three days.
All cell cultures are maintained between 34 and 39°C in a atmosphere of 5% CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was obtained from Envigo - Shardlow and stored at -90 to -70°C.
S9 mix contained: S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 10.63, 21.25, 42.5, 85, 170, 340, 680 and 1360 µg/mL (10 mM).
Main test 1 (-S9 mix): 0, 10, 20, 25, 30, 35, 40 and 45 µg/mL (3 hours)
Additional test 1 (- S9 mix): 0, 25, 30, 31, 32, 33, 34, 35, 40 µg/mL (3 hours)
Main test 1 (+S9 mix): 0, 20, 40, 45, 50, 55, 60, 65, 70, 75, 80 µg/mL (3 hours)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol. The final volume of ethanol added to the cultures was 1% v/v in the preliminary toxicity test and 0.5% v/v in the main tests.
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test item in vehicles compatible with this test system was assessed. Tetrahydrodicyclopentadiene was found to be insoluble at 136 mg/mL in dimethyl sulphoxide and found to be soluble at 136 mg/mL in ethanol. Ethanol was therefore used as the vehicle for this study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

250 µg/mL in ethanol

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/mL in ethanol

Positive control substance:

3-methylcholanthrene

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
The cells were incubated for approximately 20 hours at 37°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test substance on Day 1.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound and positive controls, four individual cultures for solvent controls.

NUMBER OF CELLS EVALUATED: 10E06 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)

Rationale for test conditions:

Tested up to cytotoxic concentrations.

Evaluation criteria:

The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
Demonstration of reproducibility in any increase in mutant frequency;
The mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10E6 survivors with a corresponding survival rate of 20% or greater.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

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: No fluctuations in pH of the medium were observed at 1360 µg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test substance in medium was tested at concentrations of 1360 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation:
* Precipitate was observed by eye at the end of treatment in the absence of S9 mix at concentrations of 42.5 μg/mL and above and in the presence of S9 mix at 85 μg/mL
* Main test and additional main test (3-hour treatment in the absence of S9 Mix): No precipitate was seen by eye at the end of treatment.
* Main test (3-hour treatment in the presence of S9 Mix): No precipitate was seen by eye at the end of treatment.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Tetrahydrodicyclopentadiene was dosed at concentrations up to 1360 μg/mL (10mM). Precipitate was observed by eye at the end of treatment in the absence of S9 mix at concentrations of 42.5 μg/mL and above and in the presence of S9 mix at 85 μg/mL and these were, therefore, the highest concentrations plated for determination of relative survival (RS) in the absence and presence of S9 mix. Exposure to Tetrahydrodicyclopentadiene for 3 hours at concentrations from 10.63 to 42.5 μg/mL in the absence of S9 mix resulted in RS values from 117 to 5%. Exposure to Tetrahydrodicyclopentadiene for 3 hours at concentrations from 10.63 to 85 μg/mL in the presence of S9 mix resulted in RS values from 124 to 3%. Concentrations for the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Additional main test (- S9 mix): None of the Tetrahydrodicyclopentadiene treated cultures had mutant frequencies above the laboratory historical 95% confidence limit.
- Main test (+ S9 mix): None of the Tetrahydrodicyclopentadiene treated cultures had mutant frequencies above the laboratory historical 95% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Main test (3-hour treatment in the absence of S9 Mix):Exposure to Tetrahydrodicyclopentadiene resulted in mean RS values from 136 to 8%. As the required range of toxicity was not achieved this test was abandoned and an additional test was performed with modified dose concentrations.
- Additional Main Test (3-hour treatment in the absence of S9 Mix): At 25 to 35 μg/mL the mean RS values ranged from 92 to 20% relative to the vehicle control.
- Main test (3-hour treatment in the presence of S9 Mix): At 20 to 70 μg/mL the mean RS values ranged from 101 to 19% relative to the vehicle control.

Any other information on results incl. tables

Table 7.6.1/2: Summary table

Treatment	Concentration (µg/mL)	Additional 3-hour Treatment -S9 mix		3-hour Treatment +S9 mix
		Mean RS (%)	Mean MFa	Mean RS (%)	Mean MFa
Ethanol	0	100	11.23	100	4.56
Tetrahydrodicyclopentadiene	20	NT	NT	101	4.74
Tetrahydrodicyclopentadiene	25	92	9.51	NT	NT
Tetrahydrodicyclopentadiene	30	81	7.87	NT	NT
Tetrahydrodicyclopentadiene	32	61	14.14	NT	NT
Tetrahydrodicyclopentadiene	33	61	14.10	NT	NT
Tetrahydrodicyclopentadiene	34	23	13.46	NT	NT
Tetrahydrodicyclopentadiene	35	20	12.55	NT	NT
Tetrahydrodicyclopentadiene	40	NT	NT	84	3.22
Tetrahydrodicyclopentadiene	45	NT	NT	77	3.58
Tetrahydrodicyclopentadiene	50	NT	NT	97	3.70
Tetrahydrodicyclopentadiene	55	NT	NT	92	2.24
Tetrahydrodicyclopentadiene	60	NT	NT	70	3.72
Tetrahydrodicyclopentadiene	65	NT	NT	53	3.70
Tetrahydrodicyclopentadiene	70	NT	NT	19	1.84
Ethyl methanesulphonate	250	100	115.27***	NT	NT
3-methylcholanthrene	5	NT	NT	96	67.63***

 

a. Mutant frequencies expressed per 106 viable cells

RS: Relative Survival

MF: Mutant Frequency

NT: Not tested

*** p<0.001; all other cultures p≥0.05. Treated groups were compared to the vehicle control using

one-tailed Dunnett’s tests for an increase and the positive control was compared to

the vehicle control using a one-tailed t-test for an increase

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary cells (CHO-K1) were exposed to the test item diluted in ethanol, in duplicate in the presence and absence of metabolic activation (S9-mix).Three independent tests are performed, two in the absence of exogenous metabolic activation (S9 mix) and one in the presence of S9 mix.Three-hour exposures were used both with and without activation (S9) in all tests.

 

The highest final concentration used in the preliminary toxicity test was 1360 μg/mL (10 mM). This is the standard limit concentration within this test system as recommended in the regulatory guidelines. Precipitate was observed by eye at the end of treatment at 42.5 μg/mL and above in the absence of S9 mix and 85 μg/mL and above in the presence of S9 mix, and these were therefore the highest concentrations plated for determination of toxicity. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to Tetrahydrodicyclopentadiene in the absence of S9 mix at concentrations from 10.63 to 42.5 μg/mL the RS values ranged from 117 to 5% and in the presence of S9 mix at concentrations from 10.63 to 85 μg/mL the RS values ranged from 124 to 3%.

 

In the main mutation test in the absence of S9 mix (Test 1) the required range of toxicity was not achieved, therefore this test was abandoned, and an additional test (Test 2) was performed with modified dose concentrations (from 25 to 40μg/mL). No precipitate was observed by eye at the end of treatment. At 25 to 35μg/mL the mean RS values ranged from 92 to 20% relative to the vehicle control. Tetrahydrodicyclopentadiene did not induce a statistically significant increase in mutant frequency. None of the Tetrahydrodicyclopentadiene treated groups had mutant frequencies above the laboratory historical 95% confidence limit and a test for linear trend was applied across all treatment

groups, which was not statistically significant. A test for non-linearity was applied across all treatment groups which was statistically significant (p= 0.036).

 

In the main mutation test in the presence of S9 mix (Test 3), cells were exposed to Tetrahydrodicyclopentadiene at concentrations from 20 to 80μg/mL. No precipitate was observed by eye at the end of treatment. At 20 to 70μg/mL the mean RS values ranged from 101 to 19% relative to the vehicle control. Tetrahydrodicyclopentadiene did not induce a statistically significant increase in mutant frequency. None of the Tetrahydrodicyclopentadiene treated groups had mutant frequencies above the laboratory historical 95% confidence limit. A test for linear trend and non-linearity were applied across all treatment groups, both were statistically significant (p<0.001 andp=0.017, respectively).

The linear trend was produced by a non-toxicologically relevant inverse response.

 

The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test item did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation,under the experimental conditions described.

 

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