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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 vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- 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 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 1997
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- 1998
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
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
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: human, cultured in vitro in whole blood culture
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Normal cell cycle time (negative control): cells do not divide unless stimulated with phytohaemagglutinin. In this laboratory the cell cycle time is approximately 13 - 14 hours.
For lymphocytes:
- Sex, age and number of blood donors: two male, non-smoking donors
- Whether whole blood or separated lymphocytes were used: whole blood was used
- Whether blood from different donors were pooled or not: pooled blood was used
- Mitogen used for lymphocytes: phytohaemagglutinin
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 supplemented with 10% foetal calf serum, 1 I.U./ml sodium heparin, 20 I.U./ml penicillin / 20 µg/ml streptomycin and 2.0 mM glutamine. Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin) of cell suspension were placed in sterile universal containers and incubated at 37°C for approximately 48 h. The cultures were gently shaken daily to resuspend the cells. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Colcemid
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Sprague-Dawley rats (Aroclor 1254 induced rat liver fraction)
- Test concentrations with justification for top dose:
- EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (S9): 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.
Due to a steep toxic response observed in both the absence and presence of S9 mix at concentrations above 78.13 μg/mL, a repeat test was performed using the following concentrations of T-71: 0, 6.25, 12.5, 25, 50, 75, 100 and 125 μg/mL.
EXPERIMENT 2:
Concentrations of T-71 were as follows:
Without S9 mix: 0, 3.13, 6.25, 12.5, 25, 50 and 75 μg/mL.
With S9 mix: 0, 6.25, 12.5, 25, 50 and 75 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle:
Information provided by the sponsor indicated that dimethyl sulphoxide, purified water and acetone were not suitable solvents. T-71 was also found to be insoluble in ethanol, however a dosable suspension was formed in culture medium at 10 mg/mL. On dosing at 50% v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, heavy precipitate was observed. In this case, the highest concentration used for subsequent testing was 5000 µg/mL.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Cyclophosphamide tested at 10 μg/mL, vehicle sterile purified water: with metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (continuous treatment), vehicle sterile purified water: without metabolic activation
- Details on test system and experimental conditions:
- CELL DIVISION STIMULANT:
Phytohaemagglutinin
METHOD OF APPLICATION:
in cell culture medium
DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
20 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 17 h].
- Final concentration of S9 mix:
Experiment 1: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
Experiment 2: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
- Fixation time (start of exposure up to fixation or harvest of cells):
20 hours in each of both experiments
SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 18 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.
STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.
NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.
NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced In cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;
Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes
Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.
- Evaluation criteria:
- An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
-Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
-The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level. - Statistics:
- One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the solvent control value.
FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- lymphocytes: human
- 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
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- in both experiments following 3 h treatment
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- With metabolic acitviation and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- See table on results 'Table CA NA-70.pdf' attached as background material.
Applicant's summary and conclusion
- Conclusions:
- It is concluded that the test substance T-71 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.
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