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EC number: 212-977-2 | CAS number: 897-06-3
- 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:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- May to Aug 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- (1997)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Androst-4-ene-3,17-dione
- EC Number:
- 200-554-5
- EC Name:
- Androst-4-ene-3,17-dione
- Cas Number:
- 63-05-8
- Molecular formula:
- C19H26O2
- IUPAC Name:
- androst-4-ene-3,17-dione
- Details on test material:
- - Name of test material (as cited in study report): Androstendion
- Lot/batch No.: 72009028
- Purity: 99.86 %
- Stability under test conditions: The stability of the test substance in the vehicle was analytically verified for at least 4 hours.
Constituent 1
Method
- Target gene:
- HPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM with supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix from the liver of phenobarbital/ß-naphthoflavone induced rats.
- Test concentrations with justification for top dose:
- Experiment I: treatment time 4 hours, with and without S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, and 240.0 µg/mL
Experiment II: treatment time 4 hours, with S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, 180.0, and 240.0 µg/mL; treatment time 24 hours, without S9 mix 1.89, 3.75, 7.5, 15.0, 30.0, 60.0, 90.0, and 120.0 µg/mL. - Vehicle / solvent:
- 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:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulfonate (EMS; 1.2 mM), 7,12-dimethylbenzanthracene (DMBA; 4.3 µM)
- Remarks:
- EMS was used without and DMBA with metabolic activation
- Details on test system and experimental conditions:
- PRE-TEST: Test item concentrations ranged between 15.0 ¿g/mL and 1920 ¿g/mL in the pre-experiment. As result no relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hour treatment. A transient reduction of the cloning efficiency to 44.1 % at 480 ¿g/mL without metabolic activation was judged as precipitation artefact rather than true cytotoxicity as the cloning efficiency was back to normal at higher concentrations. Following 24 hours treatment without metabolic activation cytotoxic effects occurred at 120 ¿g/mL and above.
METHOD OF APPLICATION: in medium
Approximately 1.5×10exp6 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 ¿l/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
Three or four days after treatment 1.5×10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory¿s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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
- Additional information on results:
- No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Water solubility: The maximum concentration of the pre-experiments (1920 ¿g/mL) was based on the solubility properties of the test item in DMSO and aqueous medium.
- Precipitation: No precipitation of the test item was observed up to the maximum analyzable concentration in both main experiments. In the pre-experiments precipitation occurred at 120 ¿g/mL and above after 4 hours treatment with and without metabolic activation, and at 480 ¿g/mL and above following 24 hours treatment without metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by a relative cloning efficiency I or relative cell density below 50 % of the corresponding solvent control were noted at 120 ¿g/mL in the first main experiment without metabolic activation. In the presence of metabolic activation no cytotoxicity was observed at the maximum analyzable concentration of 120 ¿g/mL. At the next higher concentration of 240 ¿g/mL exceedingly severe cytotoxic effects precluded analysis of any data on mutagenicity. In the second experiment cytotoxicity as described above occurred at 180 ¿g/mL with and at 120 ¿g/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I or relative cell density was covered with and without metabolic activation. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Applicant's summary and conclusion
- Conclusions:
- negative
- Executive summary:
The test item did not induce substantial and reproducible dose dependent increase of the mutation frequency in a mammalian cell gene mutation assay (HPRT) according to OECD TG 476. In this assay two independent experiments were conducted with V79 cells being treated for either 4 hours with and without metabolic activation or 24 hours without metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Therefore, the substance was considered to be non-mutagenic in the specified test.
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