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EC number: 601-147-9 | CAS number: 111988-49-9
- 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
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- Nanomaterial pour density
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- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 6 Dec 1994 - 7 Apr 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 996
- Report date:
- 1996
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- adopted 2016
- Deviations:
- yes
- Remarks:
- less than 2 million cells were seeded, data was not pooled for data analysis
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 1984
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- 3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
- EC Number:
- 601-147-9
- Cas Number:
- 111988-49-9
- Molecular formula:
- C10H9ClN4S
- IUPAC Name:
- 3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
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):
- CELLS USED
- Type and source of cells: V79 cells, provided by University of Ulm, Germany
- Suitability of cells: standard cell for this assay
- Normal cell cycle time (negative control): 10-14 h
For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: subculturing twice a week
- Cell cycle length, doubling time or proliferation index: 10-14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: cells were cultured in hypoxanthine-free Eagle's Minimal Essential Medium (MEM), supplemented with nonessential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHC03 , penicillin (100 units/mL), streptomycin (100 μg/mL) and heat-inactivated fetal calf serum (FCS, 10%). This medium is the culture medium. For treatment, the FCS was reduced to 2%. Cells were kept at 37 °C, 5% CO2.
- Cytokinesis block (if used):
- None
- Metabolic activation:
- with and without
- Metabolic activation system:
- The metabolic activation system was cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with Aroclor 1254. S9 fraction was purchased commercially.
The protein concentration of the S9 preparation was 37.4 mg/mL.
S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 40% v/v in the S9 mix. The cofactors in the final S9 mix were concentrated as follows: 8 mM MgCI2 x 6 H2O, 33 mM KCI, 5 mM Glucose-6-phosphate, 1 mM NADP in buffer.
The final concentration of S9 in the culture was 5% (S9 mix) and 1.6% (S9 preparation).
The S9 was tested for its capability to activate the positive control 7,12-dimethylbenzanthracene (DMBA) during the test. - Test concentrations with justification for top dose:
- The concentrations in the main experiment were based on a pre-test. Nine concentrations were tested ranging from 9 to 1000 µg/mL for 5 h +/- S9 mix. Without S9 mix, cytotoxicity was not seen up to the highest concentration. Precipitation occurred at 500 μg/mL and above.
Therefore, the following concentrations were chosen for the main test: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL - Vehicle / solvent:
- - Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
- Percentage of solvent in the final culture medium: 1 % (v/v)
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2 (- S9 mix), 3 (+ S9 mix)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4x10^6 cells per 250 mL flask (exposure), 1.5 x 10^6 cells (expression), 3x10^5 (selection)
- Test substance added in medium (2% FCS)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment: 13 - 14 days
FOR GENE MUTATION:
- Expression time: 7 days (subcultured once)
- Selection time: 6-7 days
- Fixation time: 13-14 days
- Selective agent: 6-TG, final concentration 10 μg/mL, 8 days
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment (relative cloning efficiency), relative population growth, the ability of cells to form colonies at the time of mutant selection (absolute cloning efficiency)
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Mutation frequency = (number of mutation colonies * 100/number of evaluated dishes x 3 x 10^5 x CE)
with CE = cloning efficiency - Rationale for test conditions:
- The present study was conducted according to the recommendations of the OECD test guideline 476 version dated 1984, which was the standard at the time the study was conducted.
- Evaluation criteria:
- Acceptability
The assay is considered acceptable if it meets the following criteria
- the assay is performed twice
- at least 5 dishes per conditions are used to determine mutant frequency (better: 8)
- the numbers of mutant colonies per million cells found in the negative and/or solvent controls fall within the laboratory historical control data range
- the positive control substances produce a significant increase in mutant colonies (three times of
positive control)
- the average cloning efficiency of the negative and/or solvent controls must exceed 50%
- spontanous mutant frequency should be lower than 25x10^-6 cells
- absolute CE should be 10% or greater
An assay is considered positive if an increase in mutant frequencies is
- concentration-dependent (desirable over 3 concentrations, but at higher concentrations, reproducibiliy is sufficient to classify as mutagenic)
- significant (at least two to three times the mutant frequency of the solvent control)
- reproducible (seen in parallel cultures)
The results should be reproduced in the second experiment.
A test substance is considered equivocal if there is no concentration-dependency but one or more concentrations induced a reproducible, significant mutant frequency in all assays.
The assay is considered negative if none of the criteria described above are met (for a range of applied concentrations which extends to toxicity causing about 30% survival or less). - Statistics:
- Mutation frequencies were submitted to weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. The weighted analysis of variance is followed by a Dunnett's test (nominal significance level of a = 0.05).
Mutation frequencies of 5 plates are too few to be considered for statistics. If the relative population growth was below 10%, the concentration is also discarded for evaluation.
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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated at 500 μg/mL and above. At 500 μg/mL (the highest soluble concentration), osmolality was not affected.
RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was conducted with a series of 9 concentrations ranging from 9 μg/mL to 1000 μg/mL, with and without S9 mix. Cytotoxicity was not affected up to the highest concentration tested but precipitation occurred at 500 µg/mL and above. Based on these results, 6 concentrations were retained for the main experiments ranging from 15.6 μg/mL to 500 μg/mL both, with and without S9 mix.
CYTOTOXICITY:
No toxicity occurred up to the highest concentration tested, both with and without S9 mix.
STUDY RESULTS
Without metabolic activation:
No relevant increase in mutation frequencies was observed. At 62.5 μg/mL, an increased mutant frequency compared to the vehicle controls was observed but this was not reproducible, not concentration-related and did not exceed the range, which is typical of vehicle control variation between trials. Therefore, the finding was considered incidental.
With metabolic activation:
No relevant increase in mutation frequencies was observed. A slight increase occurred at 125, 250 and 500 μg/mL in one culture compared to the vehicle controls but this was not reproduced in the parallel culture or the other trials. The joint statistical assessment of the 3 trials resulted in no statistically significant increase of the mutation frequency over the concurrent vehicle controls at any of the tested concentrations
Solvent, negative and positive controls gave the expected outcome.
HISTORICAL CONTROL DATA
Historical control data was provided by the testing facility. The data came from 17 and 16 experiments with and without metabolic activation, respectively. The experiments were conducted from February 1994 to April 1995.
Mutation frequency of negative controls
- S9-activation 6.1 ± 3.8 (range 0.5 to 15.0)
+ S9-activation 5.7 ± 5.9 (range 0.5 to 28.8)
Mutation frequency of vehicle controls
- S9-activation 5.6 ± 4.4 (range 1.0 to 22.6)
+ S9-activation 5.0 ± 5.5 (range 0.5 to 26.7)
Mutation frequency of positive controls
- S9-activation (EMS) 887.3 ± 193.9 (range 531.1 to 1291.5)
+ S9-activation (DMBA) 93.4 + 42.2 (range 21.8 to 240.2)
Applicant's summary and conclusion
- Conclusions:
- The present study was conducted according to the OECD guideline 476 dated 1984, under GLP conditions. Under the conditions of the assay, the test item did not induce any gene mutation at the HPRT locus in V79 cells, neither in the presence nor absence of metabolic activation.
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