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EC number: 220-585-8 | CAS number: 2825-82-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
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- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- 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.
Constituent 1
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.
Controlsopen allclose all
- 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.
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.
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