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EC number: 421-750-9 | CAS number: 57280-22-5 TRIOXABICYCLOOCTAN; TRIOXABICYCLOOCTANE
- 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
- Bioaccumulation
- 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 vivo
Administrative data
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2003-01-24 to 2003-06-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 003
- Report date:
- 2003
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Version / remarks:
- adopter 21 July 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- unscheduled DNA synthesis
Test material
- Reference substance name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
- EC Number:
- 421-750-9
- EC Name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
- Cas Number:
- 57280-22-5
- Molecular formula:
- C7H12O3
- IUPAC Name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Hanlbm: WIST (SPF)
- Details on species / strain selection:
- The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 6-10 weeks
- Weight at study initiation: Mean value 183.6 g (SD*± 15.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single, Makrolon Type II, with wire mesh top (Ehret, D-79312 Emmendingen) with granulated soft wood bedding (Altromin, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (Altromin, D-32791 Lage/Lippe) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiol. saline
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.9% NaCl solution. The vehicle was chosen to itsnon-toxicity for the animals. The animals received a single standard volume of 10 mL/kg body weight orally - Duration of treatment / exposure:
- treatment once and isolation of hepatocytes after 2 and 16 h
- Frequency of treatment:
- once
- Post exposure period:
- 2 and 16 h
Doses / concentrationsopen allclose all
- Dose / conc.:
- 2 000 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- Dose / conc.:
- 0 mg/kg bw/day
- No. of animals per sex per dose:
- 8
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- none; no data; 2-acetylaminofluorene; N, N`-dimethylhydrazinehydrochloride
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw and 100 mg/kg bw, respectively
Examinations
- Tissues and cell types examined:
- liver, hepatocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with two animals per group and sex under identical conditions as in the UDS study concerning: starvation period, animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally (gavage) and examined for acute toxic symptoms at intervals of 1 h, 2-4 h, 6 h, 24 h, and 48 h after administration of the test item.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment} before receiving the test item, the positive or the vehicle control substance. Water was available ad libitum. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weigiht of the animals. The animals received the test item once. Four animals (males) were treated per dose group. After anaesthetizing the rats with Na-thiopental (Trapanal, Byk Gulden, D-78467 Konstanz) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein} supplemented with collagenase (0.05 % (w/v), Boehringer Mannheim, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 37° C
DETAILS OF SLIDE PREPARATION: The washed hepatocytes were centrifuged and transferred into Williams medium E. At least three cultures were established from each animal. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO 2 humidified incubator at 37° C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 mL culture medium (WME, 1 % (v/v), FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried. The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara, D-35463 Fernwald) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developed with KODAK Dektol Developer (Tecnomara, D-35463 Fernwald) at room temperature, fixed in TETENAL (Tetenal, D-22844 Norderstedt) and stained with hematoxylin/eosin.
METHOD OF ANALYSIS: Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of the most heavily lableled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal; and 50 cells per slide were evaluated. Heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting. Three animals per group were evaluated as described above.
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system. - Evaluation criteria:
- see above
- Statistics:
- non-parametric Mann-Whitney test
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: not reported
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur.
- Evidence of cytotoxicity in tissue analysed: No
- Rationale for exposure: Based on preliminary acute toxicity testing
Any other information on results incl. tables
Treatment | Period | Animal No. | Viability* [%] | Number of isolated cells [x E+06] |
Vehicle control 0.9% NaCl solution | 2 h | 2 | 75 | 341 |
3 | 80 | 216 | ||
4 | 90 | 576 | ||
500 mg/kg bw | 2 h | 5 | 79 | 111 |
6 | 75 | 229 | ||
7 | 92 | 354 | ||
2000 mg/kg bw | 2 h | 9 | 72 | 310 |
10 | 70 | 294 | ||
11 | 81 | 126 | ||
40 mg/kg bw DMH | 2 h | 13 | 84 | 185 |
15 | 76 | 247 | ||
16 | 84 | 139 | ||
Vehicle control 0.9% NaCl solution | 16 h | 17 | 77 | 370 |
18 | 83 | 593 | ||
19 | 90 | 171 | ||
500 mg/kg bw | 16 h | 22 | 76 | 365 |
23 | 91 | 578 | ||
24 | 75 | 420 | ||
2000 mg/kg bw | 16 h | 25 | 71 | 540 |
26 | 78 | 270 | ||
27 | 86 | 357 | ||
100 mg/kg bw 2-AAF | 16 h | 29 | 72 | 266 |
30 | 81 | 150 | ||
31 | 80 | 164 |
Treatment | Period | Grains per nucleus | Grains per cytoplasmic area | Net grains per nucleus | |||
Mean * | ± SD** | Mean * | ± SD** | Mean * | ± SD** | ||
Vehicle control 0.9% NaCl solution | 2 h | 9.41 | 4.69 | 14.58 | 5.29 | -5.17 | 5.13 |
1000 mg/kg bw | 2 h | 10.68 | 5.00 | 17.65 | 7.24 | -6.97 | 5.80 |
2000 mg/kg bw | 2 h | 13.77 | 7.01 | 18.44 | 7.27 | -4.67 | 6.69 |
40 mg/kg bw DMH | 2 h | 17.91 | 11.19 | 12.02 | 6.83 | 5.89 | 7.82 |
Vehicle control 0.9% NaCl solution | 16 h | 8.86 | 4.30 | 11.40 | 4.49 | -2.54 | 4.69 |
1000 mg/kg bw | 16 h | 10.20 | 5.40 | 15.47 | 6.29 | -5.27 | 5.05 |
2000 mg/kg bw | 16 h | 10.23 | 4.14 | 17.25 | 5.42 | -7.02 | 5.57 |
100 mg/kg bw 2-AAF | 16 h | 33.98 | 13.96 | 16.90 | 7.41 | 17.08 | 11.80 |
* Mean of 3 animals ** Standard deviation |
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Trioxabicyclooctan did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
- Executive summary:
In a test conducted according to OECD test guideline 486 (1995) male Wistar rats (3 animals per group) were treated with Trioxabicyclooctan at doses of 0, 1000 and 2000 mg/kg bw once by oral gavage. Hepatocytes were isolated after 2h and after 16h after administration and incubated with 3HTdR for 4 h. Subsequently the hepatocytes were cultured over night with unlabelled thymidine. After the last incubation the DNA repair was measured as incorporation of 3HTdR into the DNA of the treated cells. The amount of incorporated radioactivity is determined by silver grain counting.
The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups.
No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals. as compared to the current vehicle controls.
Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.) were used as positive controls. In vivo treatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts as indication of induced DNA-repair.
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