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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test on S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (Sivak, 1983): inconclusive due to severe toxic effects

- QSAR prediction (DEREK, 2017): No structural alerts for mutagenicity

- Mutagenicity in mammalian cells (OECD 476, Rel.1, K): not mutagenic with and without metabolic activation.

- Micronucleus test (OECD 487, Rel.1, K): not clastogenic with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Jul 1982 - 15 Dec 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

Principles of method if other than guideline:

Not applicable.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material as cited in the report : exo-tetrahydrodi(cyclopentadiene); JP-10
- Storage conditions: stored at room temperature in a safety cabinet.

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver microsomal fraction, S9

Test concentrations with justification for top dose:

1, 5, 10, 20 and 50 µl/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

the highest volume of DMSO used

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

JP-10 which was immiscible with the top agar overlay was assayed using a preincubation modification of the Ames assay which alters the conditions of exposure of tester strains to test chemicals to enhance exposure. As for the standard assay, bacteria (0.05 ml), the metabolic activation mixture (0.5 ml) or saline (for assays in the absence of S9) and the test substance (0.05 ml or less) were combined in a test tube. Before the addition of 2.0 ml of top agar, however, the tubes containing bacteria, enzymes and test material were incubated at 37°C for 30 minutes with end-over-end rotation. At that time, the 2.0 ml of top agar was added and the top agar layer was plated in the usual way. The doses of positive control chemicals per plate were as follows: 2-aminoanthracene, 25 µg; 2-nitrofluorene, 5 µg; sodium azide, 1 µg; and 9-aminoacridine, 50 µg.

Rationale for test conditions:

A liquid pre-incubation modification (standard operating procedure #CB/M-818) was used to test JP-10 which was not compatible with the standard plate-incorporation assay since it formed oil-like droplets on the surface of the top agar layer.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

even at the lowest dose tested (1µl/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Even at the lowest dose tested (1 µl/plate) toxicity was severe enough to result in sparse background lawns. Many plates had no revertants rather than the number normally obtained as a result of spontaneous reversion.

Table of results: see attached document

Conclusions:

Under the test conditions, the test material is too cytotoxic to determine its mutagenic potential.

Executive summary:

JP-10 was assayed by a preincubation modification of the Ames assay because it was immiscible with the agar medium used in this assay. Varying volumes of the undiluted test material were added directly to the bacteria and S9 (or saline) mixes for preincubation because JP-10 was not sufficiently soluble in DMSO to obtain the necessary concentrates for the desired doses. Even at the lowest dose tested (1 µl/plate) toxicity was severe enough to result in sparse background lawns. Many plates had no revertants rather than the number normally obtained as a result of spontaneous reversion.

The results of this assay are inconclusive due to severe toxicity of JP-10 on the tester strains. Although a repeat assay with shorter exposure times is recommended, we consider that further modifications of the standard protocol for this particularly toxic test substance are outside the scope of this present work assignment.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

20 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

DEREK is a validated (Q)SAR software, widely used to predict genotoxicity properties

Justification for type of information:

1. SOFTWARE
Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.

2. MODEL (incl. version number)
Derek KB 2015 v2.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C3CC2C1CCC(C1)C2C3

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached report

6. ADEQUACY OF THE RESULT
See attached report

Qualifier:

no guideline followed

Principles of method if other than guideline:

Software used: Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.

GLP compliance:

no

Type of assay:

other: Derek Nexus evaluation for mutagenicity

Target gene:

Not applicable

Species / strain / cell type:

bacteria, other: Salmonella typhimurium and Escherichia coli

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Not applicable

Vehicle / solvent:

Not applicable

Details on test system and experimental conditions:

Not applicable

Rationale for test conditions:

Not applicable

Evaluation criteria:

Not applicable

Statistics:

Not applicable

Key result

Species / strain:

bacteria, other: Salmonella typhimurium and Escherichia coli

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Mutagenicity in vitro in bacterium is INACTIVE: No misclassified or unclassified features
Mutagenicity in vitro in Escherichia coli is INACTIVE: No misclassified or unclassified features
Mutagenicity in vitro in Salmonella typhimurium is INACTIVE: No misclassified or unclassified features

Details
The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

None

Conclusions:

DEREK Nexus evaluation showed no alerts for mutagenicity.

Executive summary:

DEREK software (Derek Nexus: 5.0.2, Nexus: 2.1.1, Lhasa Ltd.) was used to predict the mutagenicity of tetrahydrodicyclopentadiene.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

DEREK Nexus evaluation showed no alerts for mutagenicity.

Reference 3

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.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

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

Target gene:

HPRT locus

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.

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

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.

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.

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

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.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 June to 09 October, 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 487 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Commission Regulation No. 2017/735. Method B.49: Mutagenicity – In Vitro Mammalian Cell Micronucleus Test. OJ L 112/109.

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:

in vitro mammalian cell micronucleus test

Target gene:

not applicable (in vitro micronucleus test of: clastogenic and aneugenic potential)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor)
- Whether whole blood or separated lymphocytes were used:
Human blood was collected aseptically from two healthy adult and diluted with HML media. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34 to 39°C, and the cells were re-suspended (twice daily) by gentle inversion.
- Whether blood from different donors were pooled or not: yes pooled (in equal volumes from each donor).
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

MEDIA USED:
The following media, obtained from a suitable supplier, was used:
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L glutamine.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Covance
- method of preparation of S9 mix: S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at -90 to -70°C.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% v/v
- Other: S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilized before use.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test (3 hours with and without S9 mix) and (20 hours without S9 mix): 2.66, 5.31, 10.63, 21.25, 42.5, 85, 170, 340, 680 and 1360 µg/mL
- Main tests:
* Without S9 mix (3 hours): 10, 20, 25, 30, 35, 40 and 45 µg/mL
* With S9 mix (3 hours): 20, 40, 45, 50, 55, 60, 65, 70, 75 and 80 µg/mL
* Without S9 mix (20 hours): 1, 10, 15, 20, 25, 30, 35 and 40 µg/mL

Additional main tests:
* Without S9 mix (3 hours): 5, 12.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL
* With S9 mix (3 hours): 40, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 and 160 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- 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 soluble at 272 mg/mL in ethanol. Ethanol was therefore used as the vehicle for this study. The final volume of ethanol added to the cultures was 0.5% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

Remarks:

Without metabolic activation.

Details on test system and experimental conditions:

CULTURE OF LYMPHOCYTES: Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34-39 °C, and the cells were resuspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HML Media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

DURATION
- Exposure duration: 3 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 20 h continuous exposure (-S9) in main tests
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at a final concentration of 6 μg/mL.

STAIN (for cytogenetic assays): Slides were stained with Acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Main test: Duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls; two slides were prepared from each culture.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34-39 °C, cultures were then incubated for 3 minutes at 34-39 °C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4 °C until slide preparation.
Slide Preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The remaining cell cultures were stored at approximately 4 °C until slide analysis was complete.
Slide Staining: Slides were stained with Acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
Microscopic Examination: The prepared slides were examined by fluorescence microscopy. The incidences of mononucleate, binucleate and polynucleate cells were assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted.

NUMBER OF CELLS EVALUATED:
- Scoring of Micronuclei: Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis for micronucleated cells was based on the following criteria (Fenech and Morley 1985 and Fenech, 1993):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

OTHER:
At least three concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5% cytotoxicity (approximately) when compared with the concurrent vehicle control or, where no cytotoxicity was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) All results are inside the distribution of the historical vehicle control data (below the 95% confidence limit).

The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Tetrahydrodicyclopentadiene treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using two-tailed t tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2017).

Key result

Species / strain:

lymphocytes: Human lymphocytes

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 item in medium was tested at 1360 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: In all exposure conditions the highest concentration tested was 1360 μg/mL, a precipitate of the test item was observed at the end of treatment at 680 μg/mL and above following 3-hour exposure and 1360 μg/mL following a 20-hour exposure, as assessed in concurrently treated HML media-only cultures.
- Definition of acceptable cells for analysis: Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

RANGE-FINDING/SCREENING STUDIES: In all exposure conditions the highest concentration tested was 1360 μg/mL, a precipitate of the test item was observed at the end of treatment at 680 μg/mL and above following 3-hour exposure and 1360 μg/mL following a 20-hour exposure, as assessed in concurrently treated HML media-only cultures.
After 3-hour treatment in the absence of S9-mix, a reduction in the CBPI compared with vehicle control values, equivalent to 46.5% cytostasis, was obtained with
Tetrahydrodicyclopentadiene at 42.5 μg/mL. After 3-hour treatment in the presence of S9-mix, a reduction in the CBPI compared with vehicle control values, equivalent to 83.9% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 85 μg/mL.
After 20-hour treatment in the absence of S9-mix, a reduction in the CBPI compared with vehicle control values, equivalent to 95.5% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 42.5 μg/mL.
These data were used to select concentrations for the main test.

MAIN STUDY RESULTS
- Concurrent vehicle negative and positive control data: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

- Results from cytotoxicity measurements:
* 3-Hour Treatment in the Absence of S9 Mix: The highest concentration tested was 45 μg/mL. A reduction in the CBPI compared to vehicle control values equivalent to 55.9% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 30 μg/mL. Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 10, 25 and 30 μg/mL.
* Additional 3-Hour Treatment in the Absence of S9 Mix: The highest concentration tested was 40 μg/mL. A reduction in the CBPI compared to vehicle control values equivalent to 50.1% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 40 μg/mL. Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 30, 35 and 40 μg/mL.
* 3-Hour Treatment in the Presence of S9 Mix: The highest concentration tested was 80 μg/mL. A reduction in the CBPI compared to vehicle control values equivalent to 17.8% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 80 μg/mL. As the required increase in cytotoxicity was not achieved an additional test using a modified concentration range was performed.
* Additional 3-Hour Treatment in the Presence of S9 Mix: The highest concentration tested was 160 μg/mL. A reduction in the CBPI compared to vehicle control values equivalent to 52.9% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 100 μg/mL. Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 60, 70 and 100 μg/mL.
* 20-Hour Treatment in the Absence of S9 Mix: The highest concentration tested was 40 μg/mL. A reduction in the CBPI compared to vehicle control values equivalent to 49.7% cytostasis, was obtained with Tetrahydrodicyclopentadiene at 20 μg/mL. Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 1, 10 and 20 μg/mL.

- Genotoxicity results:
* 3-Hour Treatment in the Absence of S9 Mix: Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear doseconcentration relationship. The mean micronucleus frequencies for the vehicle treated cultures were within the laboratory historical 95% confidence limits. The mean micronucleus frequencies for the test item treated cultures at 30 μg/mL was above the laboratory historical 95% confidence limits. It was noted that there was a huge inter-culture difference in MN frequency at this dose level. These results were considered to be equivocal, therefore an
additional test was performed to confirm the result.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei,compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system.

* Additional 3-Hour Treatment in the Absence of S9 Mix:
Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear doseconcentration relationship. The mean micronucleus frequencies for the vehicle and for the test item treated cultures at 35 and 40 μg/mL were within the laboratory historical 95% confidence limits. The mean micronucleus frequencies for the test item treated cultures at 30 μg/mL was above the laboratory historical 95% confidence limits, but within the maximum value. The equivocal result seen in the previous test was not repeated and therefore was considered to be natural variation within the test system with no biological relevance.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system. therefore was considered to be natural variation within the test system with no biological relevance.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system
* Additional 3-Hour Treatment in the Presence of S9 Mix:
Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear doseconcentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.
The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
* 20-Hour Treatment in the Absence of S9 Mix:
Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear doseconcentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system.

Conclusions:

Under the conditions of this study,Tetrahydrodicyclopentadiene did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls.

The study consisted of a preliminary toxicity test, a main micronucleus test and two additional main micronucleus tests. Human lymphocytes in whole blood culture, were exposed to Tetrahydrodicyclopentadiene for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 1360 μg/mL, dosed at 0.5% v/v, in order to test up to 10 mM, the maximum concentration as recommended in the current OECD Guideline 487 (2016). Vehicle (ethanol) and positive control cultures were included in all appropriate test conditions.

Three Tetrahydrodicyclopentadiene concentrations were assessed for determination of induction of micronuclei. The highest concentration selected was that which caused a reduction in the cytokinesis-block proliferative index (CBPI) equivalent to approximately 55±5% cytostasis.

Following 3-hour treatment in the absence of S9 mix, a reduction in the CBPI equivalent to 55.9% cytostasis was obtained with Tetrahydrodicyclopentadiene at 30 μg/mL.

Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 10, 25 and 30 μg/mL. Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a

linear dose-concentration relationship. The mean micronucleus frequency for the vehicle treated cultures were within the laboratory historical 95% confidence limits. The mean micronucleus frequency for the test item treated cultures at 30 μg/mL was above the laboratory historical 95% confidence limits. These results were considered to be equivocal, therefore an additional test was performed to confirm the result.

Following an additional 3-hour treatment in the absence of S9 mix, a reduction in the CBPI equivalent to 50.1% cytostasis was obtained with Tetrahydrodicyclopentadiene at 40 μg/mL.

Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 30, 35 and 40 μg/mL. Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship. The mean micronucleus frequencies for the vehicle and for the test item treated cultures at 35 and 40 μg/mL were within the laboratory historical 95% confidence limits. The mean micronucleus frequency for the test item treated cultures at 30 μg/mL was above the laboratory historical 95% confidence limits, but within the maximum value. The equivocal result seen in the previous test was not repeated and therefore was considered to be natural variation within the test system with no biological relevance.

Following an additional (the original test did not provide an acceptable range of toxicity) 3-hour treatment in the presence of S9 mix, a reduction in the CBPI equivalent to 52.9% cytostasis was obtained with Tetrahydrodicyclopentadiene at 100 μg/mL. Concentrations of

Tetrahydrodicyclopentadiene selected for micronucleus analysis were 60, 70 and 100 μg/mL.

Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear doseconcentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.

Following a 20-hour exposure in the absence of S9 mix, a reduction in the CBPI equivalent to 49.7% cytostasis was obtained with Tetrahydrodicyclopentadiene at 20 μg/mL.

Concentrations of Tetrahydrodicyclopentadiene selected for micronucleus analysis were 1, 10 and 20 μg/mL. Tetrahydrodicyclopentadiene caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear

dose-concentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

It was observed that Tetrahydrodicyclopentadiene did not induce an increase in the number of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. Therefore, it was concluded that Tetrahydrodicyclopentadiene is not clastogenic or aneugenic and has no genotoxic potential in this test system.

This study is considered as acceptable and satisfies the requirement for the mammalian cell chromosomal aberration endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation:

Gene mutation in bacteria: two studies are available and considered as a WoE. In an old study performed in 1983, tetrahydrodicyclopetadiene, designed as "JP-10", was assayed by a preincubation modification of the Ames assay because it was immiscible with the agar medium used in this assay. Varying volumes of the undiluted test material were added directly to the bacteria and S9 (or saline) mixes for preincubation because JP-10 was not sufficiently soluble in DMSO to obtain the necessary concentrates for the desired doses (from 1 to 50 µl/plate).

Even at the lowest dose tested (1 µl/plate) toxicity was severe enough to result in sparse background lawns. Many plates had no revertants rather than the number normally obtained as a result of spontaneous reversion. The results of this assay are then inconclusive due to severe toxicity of JP-10 on the tester strains.

However, the mutagenic potential of tetrahydrodicyclopentadiene was evaluated with Derek software. The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.

Gene mutation in mammalian cells:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to the test substance efor 3 h, with and without metabolic activation.Test item did not induce any statistically significant increases in mutant frequency at concentrations up to 40 μg/mL in absence of metabolic activation system, where RS was reduced to 20%.Test item did not induce any statistically significant increases in mutant frequency at concentrations up to 70 μg/mL in presence of S9, where RS was reduced to 19%.The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.It was concluded thatthe test itemdid not demonstrate mutagenic potential in thisin vitro HPRT cell mutation assay, under the experimental conditions.

Chromosomal aberration

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test substance in the presence and absence of a metabolic activation system. In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.It was concluded that test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Justification for classification or non-classification

Harmonised classification

The test material has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification

No additional classification is proposed regarding in vitro genetic toxicity according to the criteria of Annex I to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.