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EC number: 277-539-5 | CAS number: 73570-52-2
- 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 gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Oct 2002
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 3,7-bis(diethylamino)phenoxazin-5-ium nitrate
- EC Number:
- 277-539-5
- EC Name:
- 3,7-bis(diethylamino)phenoxazin-5-ium nitrate
- Cas Number:
- 73570-52-2
- Molecular formula:
- C20H26N3O.NO3
- IUPAC Name:
- 3,7-bis(diethylamino)phenoxazin-5-ium nitrate
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- Hypoxanthine-guanine phosphoribosyltransferase (HPRT1)
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:MEM (minimal essential medium) with Hank's salts and 25 mM Hepes-buffer
- Properly maintained: ye
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (rat)
- Test concentrations with justification for top dose:
- Test groups with and without metabolic activation: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, 50* µg/mL
* because of high toxicity in the main experiment, no mutant selection was performed
Control groups
negative controls:
a: untreated control
b: cultures treated with the solvent
positive controls:
a: without metabolic activation:EMS (Ethyl methane sulfonate)
b: with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene) - Vehicle / solvent:
- No vehicle, substance was directly dissolved into test system (MEM)
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO for positive controls
- Positive controls:
- yes
- Positive control substance:
- 9,10-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- In preliminary toxicity experiments approximately 1000 cells were seeded in each well of a microtiter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. After 4 to 5 days, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 1000 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of 4 or 5 days.
Two independent mutation tests were performed.
Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25% (v/v) trypsin ready for use (mfr. Gibco). A single cell suspension was prepared.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency (see above).
The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 1000 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6E5 - E6 cells in 182 cm² flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.
Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.
Day 5 or 6: Subculturing of b) in 182 cm² flasks
Day 9: Subculturing of b) in five 75 cm² flasks with culture medium containing 6-thioguanine: Mutant selection (about 300 000 cells/flask);
subculturing of b) in two 25 cm² flasks for plating efficiency (about 400 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.
All incubations were carried out at approx. 37°C and 4% CO2 , Staining was performed with approx. 10% (v/v) methylene blue in approx. 0.01% (w/v) KOH solution. Only colonies with more than 50 cells were counted. - Rationale for test conditions:
- Based on results from preliminary study for solubility and toxicity
- Evaluation criteria:
- Criteria for a valid assay
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50%
Criteria for a positive response
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- survival of the responding dose group is at least 30%.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
Results and discussion
Test results
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 25 µg/mL onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No increase of mutant colony numbers in two independent experiments.
Positive controls: valid
Cytotoxicity: yes, from 25 µg/mL in both with and without S9 mix
Applicant's summary and conclusion
- Conclusions:
- The test item did not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described (two independent experiments). The test item is therefore considered to be non-mutagenic in the HPRT assay with mammalian cells. The test item was shown to cause cytotoxic effects from 25 µg/mL onwards.
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