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EC number: 458-880-0 | CAS number: -
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 004
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Version / remarks:
- 21. July 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
- Version / remarks:
- 19. May 2000
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- unscheduled DNA synthesis
Test material
- Reference substance name:
- -
- EC Number:
- 458-880-0
- EC Name:
- -
- Cas Number:
- 85209-93-4
- Molecular formula:
- Hill formula: C29 H42 O4 P Li
- IUPAC Name:
- 2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo- [d,g][1,3,2]dioxaphosphocin 6-oxide, lithium salt
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The rat 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.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS: Wistar HanIbm: WIST (SPF)
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Füllinsdorf
- Weight at study initiation: 183.7 ± 15.5 (SD) g (males)
- Number of animals: 32 (males)
- Assigned to test groups randomly: yes
- Housing: single housing
- Diet (e.g. ad libitum): pelleted standard diet (Altromin) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12; artificial light 6.00 a.m. - 6.00 p.m.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: 0.5% w/v sodium carboxymethylcellulose (CMC)
- Concentration of test material in vehicle: 0, 1000, 2000 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw (10 mL/kg bw for positive control substances). - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% CMC (Fluka, Cat. no: 21902). The vehicle was chosen to its non-toxicity for the animals. The animals received a single standard volume of 20 ml/kg body weight orally (the positive control groups were treated with 10 mL/kg body weight). - Duration of treatment / exposure:
- The test item was administered once for 2 h or or 16 h.
- Frequency of treatment:
- single treatment
- Post exposure period:
- 2 h and 16 h after dosing.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 4 males per dose group (main experiment).
2 female and 2 male per dose (pre-test). - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - 4 male rats administered N,N'-dimethylhydrazinedihydrochloride (sym.) (DMH) (40 mg/kg bw) in 0.9 % NaCl solution (for 2 hours preparation interval).
- 4 male rats administered 2-acetylaminofluorene (2-AAF) (100 mg/kg bw) in dimethylsulfoxide/polyethylene glycol 400 (1 + 9) (for 16 hours preparation interval).
Examinations
- Tissues and cell types examined:
- hepatocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Preliminary study at max. dose of 2000 mg/kg body weight in 2 male and female rats
TREATMENT
The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test item, the positive or vehicle control substance. Water was available ad libitum. Animals were weighed and the volume to be administered was adjusted to the body weight of each animal. The animals received the test item once. Four animals (males) were treated per group.
ISOLATION OF PRIMARY HEPATOCYTES
After anesthesizing the rats with Na-thiopental (Trapanal, Byk Gulden) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL) supplemented with 0.05 % w/v collagenase (Boehringer Mannheim) adjusted to pH 7.4 and maintained at 37 °C.
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension. Cell viability was determined using trypan blue dye exclusion. In addition, the number of cells was determined.
CULTURE CONDITIONS
The washed cells were centrifuged and transferred into Williams medium E (WME, Gibco/BRL) supplemented with Hepes (2.38 mg/mL), Penicillin (100 units/mL), Streptomycin (0.10 mg/mL), L-Glutamine (0.29 mg/mL), Insulin (0.5 µg/mL) and Fetal calf serum (FCS, 100 µl/mL). This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0 x 10^5 viable cells/mL) were added to 35 mm six-well dishes (Greiner) containing one 25 mm round plastic coverslip (Thermanox) per well coated with gelatine.
After 1.5 h in a 95 % air / 5 % CO2 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) in 2.0 mL culture medium (WME, 1 % v/v FCS) was added to the cultures. After a labelling for 4 h, cells were washed twice with WME, 1 % v/v FCS and 0.25 mM unlabelled thymidine and incubated overnight. 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 min each, rinsed with 96 % (v/v) ethanol, and air-dried.
AUTORADIOGRAPHIC PROCESSING
Coverslips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara) 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 developped with Ilford Phenisol (Ilford Imaging) at room temperature, fixed with Rapid Fixer (Ilford Imaging) and stained with hematoxylin/eosin.
QUANTIFICATION OF UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. 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 Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabelled cells undergoing replicative DNA synthesis were excluded from counting.
Three animals per group were evaluated as described above. The remaining animal per test group would have been evaluated if an animal died spontaneously or in case of technical problems concerning the isolation of hepatocytes. This was not the case in the present study.
METHOD OF ANALYSIS: light microscopy - Evaluation criteria:
- 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. - Statistics:
- A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- The treated animals expressed toxic reactions such as a reduction of spontaneous activity (2/4), ruffled fur (4/4) and eyelid closure (0/1) 2 h after treatment with 1000 / 2000 mg/kg b.w. of T-71.
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- see attached "Tables 10.4 Mean Nucleus, Cytoplasmic Area, and Net Grains" for details of results.
Applicant's summary and conclusion
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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