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EC number: 939-638-8 | CAS number: 90268-37-4
- 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:
- key study
- Study period:
- 2012-2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Reference substance 001
- Cas Number:
- 90268-36-3
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
Constituent 1
Method
- Target gene:
- hprt locus at the X-chromosome
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
*V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2.
*For subculturing, a trypsin (0.05%)-EDTA (ethylenediamine¬tetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.
*Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes.
- Periodically checked for Mycoplasma contamination: yes, by using the HOECHST stain 33258.
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes. The spontaneous mutation rate was continuously monitored.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test: 19.53, 39.06, 78.13, 156.3, 312.5, 625, and 1250 µg test item/mL
Main test without S9-mix: 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL
Main test with S9-mix: 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- aqua ad iniectabilia
- Positive controls:
- yes
- Positive control substance:
- other: Ethylmethanesulfonate in DMSO
- Remarks:
- 600 and 700 µg/mL, without S9-mix
- Positive controls:
- yes
- Positive control substance:
- other: 9,10-dimethyl-1,2-benzanthracene in DMSO
- Remarks:
- 20 and 30 µg/mL with S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.
DURATION
- Preincubation period: 24 hours
- Exposure duration: Without S9-mix:4 hours (1st experiment) or 24 hours (2nd experiment); With S9-mix: 4 hours
- Expression time (cells in growth medium): until day 8 with one subcultivation on day 5
- Selection time (if incubation with a selection agent): about 8 days (plating efficiency plates: cytotoxicity test) or 12 days (6-thioguanine plates: mutagenicity test).
SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL);
STAIN (cytogenetic assays): After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted.
NUMBER OF REPLICATIONS:
cytotoxicity: triplicate
mutagenicity: for selection of mutants 5 replicate plates; for the estimation of plating efficiencies (PE) 3 replicate plates.
DETERMINATION OF CYTOTOXICITY
- Method: other: relative plating efficiency is determined for each dose to obtain an accurate measure of the toxic effect of the chemical. - Evaluation criteria:
- So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion). Following pre¬determined descriptive criteria are used for interpretation of the results:
-lf in both independent experiments solvent and positive controls show results within the norm and if the test compound does not increase the mutation, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1 000 000 cells per condition have been evaluated, the compound is considered as negative in the test.
-In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the compound is considered as positive in the test.
Equivocal results, if applicable are clarified by further testing, in agreement with Sponsor and Study Monitor.
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:
- In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.
RANGE-FINDING/SCREENING STUDIES:
- In the preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
- The next higher concentrations resulted in complete cytotoxicity in the preliminary experiment. In addition, the number of mutants was also already considerable decreased in the main experiments at the highest employed concentrations pointing to general pronounced cytotoxicity. Finding a higher concentration with viable and evaluable cells/mutants was therefore not considered realistic.
- In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.
COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 10 6 survivors in S9 activation solvent controls.
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium without metabolic activation ranged from 3.78 to 15.74 x 10 -6 clonable cells. These results are within the normal range of the vehicle controls.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium with metabolic activation ranged from 4.44 to 13.04 x 10 -6 clonable cells. These results are within the normal range of the vehicle controls.
The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative with metabolic activation
negative without metabolic activation
Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects. - Executive summary:
The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study test item concentrations of 19.53, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL medium were employed . Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
Main study
Five concentrations 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.
Cytotoxicity
In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.
Experiments without metabolic activation
The mutation frequency of the vehicle control aqua ad iniectabilia was 14.35 and 16.67 x 10-6clonable cells. Hence, the vehicle controls were well within the expected range.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium ranged from 4.44 to 13.04 x 10‑6clonable cells. These results are within the normal range of the vehicle controls.
Experiments with metabolic activation
The mutation frequency of the vehicle control aqua ad iniectabilia was 17.87 and 16.05 x 10-6 clonable cells. Hence, the vehicle controls were well within the expected range (see below).
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium ranged from 3.78 to 15.74 x 10‑6 clonable cells. These results are within the normal range of the vehicle controls.
The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.
Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
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