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EC number: 277-633-6 | CAS number: 73912-21-7
- 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 / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-06-17 - 2015-07-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Well-documented GLP OECD 487 guideline study without deviations on the registered substance itself.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin
- EC Number:
- 277-633-6
- EC Name:
- 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin
- Cas Number:
- 73912-21-7
- Molecular formula:
- C27H35O3P
- IUPAC Name:
- 7,13-dicyclohexyl-5,15-dimethyl-9,11-dioxa-10-phosphatricyclo[10.4.0.0³,⁸]hexadeca-1(12),3(8),4,6,13,15-hexaen-10-ol
- Test material form:
- solid: pellets
- Details on test material:
- - Substance type: pure substance
- Storage condition of test material: In the refrigerator at 2-8°C, moisture protected
Constituent 1
Method
- Target gene:
- not applicable
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- Human lymphocytes
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (31 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Pre-Test: 1.2 to 1200.0 μg/mL (with regard to the solubility of the test item)
Experiment I, ±S9, 4h exposure: 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml
Experiment II, -S9, 20h exposure: 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
- Untreated negative controls:
- yes
- Remarks:
- solvent control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% DMSO (99.99% purity)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: demecolcin
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h (pulse exposure) or 20h (continuous exposure)
- Expression time (cells in growth medium): 16h or 0h in medium + 20h with CytB
- Fixation time (start of exposure up to fixation or harvest of cells): 40h
SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 parallel cultures
NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index as indication of cytostasis - Evaluation criteria:
- Evaluation of cytotoxicity and cytogenetic damage:
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI (Cytokinesis-block proliferation index) was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis. - Statistics:
- Statistical analysis
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation: Precipitation of the test item in the culture medium was observed in Experiment I at 37.5 μg/mL and above in the absence of S9 mix and at 75.0 μg/mL and above in the presence of S9 mix and in Experiment II in the absence of S9 mix at 86.8 μg/mL and above at the end of treatment.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity was observed. - Remarks on result:
- other: all strains/cell types tested
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results: negative
The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin to induce micronuclei in mammalian cells.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or the highest evaluable concentrations. - Executive summary:
In a mammalian cell cytogenetics assay, Micronucleus test (OECD 487), primary human lymphocyte cultures were exposed to 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin in DMSO at concentrations of 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml for 4h with and without metabolic activation and 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml for 20h without metabolic activation (Phenobarbital/β-naphthoflavone induced rat liver S9).
4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin was tested up to precipitating concentrations. Positive controls induced the appropriate response. There was no evidence or a concentration related positive response of Micronuclei induced over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.
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