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EC number: 224-546-6 | CAS number: 4403-90-1
- 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 bacteria
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From October 03, 2002 to November 11, 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- The test was conducted by means of Read Across approach. The reliability of the source study report is 1.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Acid Green 025 - Similar Substance 01
- IUPAC Name:
- Acid Green 025 - Similar Substance 01
- Test material form:
- not specified
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- Source of S9: rat-liver post mitochondrial supernatant (S9 fraction) was prepared from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland.
- Method of preparation of S9 mix: the animals were treated with Aroclor 1254 (Analabs lnc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80°C for no longer than one year.
- Concentration or volume of S9 mix and S9 in the final culture medium: the amount of S9 supernatant was 10% v/v in the mixture.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. - Test concentrations with justification for top dose:
- 312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO; Merck, Germany,99.5%).
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- Remarks:
- without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: 2-aminoanthracene
- Remarks:
- with S9
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: two indipendent experiments.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not reported.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: plate incorporation method (first experiment); pre-incubation method (second experiment with S9).
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes.
- Exposure duration/duration of treatment: 48 hours.
- Harvest time after the end of treatment (sampling/recovery times): not specified.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: to evaluate the toxicity of the test item a range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test item. The concentrations applied were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate. One plate was prepared per test item concentration, negative and positive controls. The plates were inverted and incubated for about 48 hours at 37+2°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn.
METHODS FOR MEASUREMENTS OF GENOTOXICIY:
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have intedered with automating counting. The results were sent online to a computer. The operator checked them on a random basis. Observations indicating precipitates of the test item in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the results section. ln case of an increased number of revertant colonies, reaching the level for a positve response, is obtained at any test item concentration, ten colonies of each respecitive plate are tested on selective agar plates for their mutant properties, in order to avoid false positive or false negative results.
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of solvent controls such an increase is not considered biologically relevant. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of solvent controls such an increase is not considered biologically relevant. - Statistics:
- A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- The substance was tested following the OECD TG 471 (bacterial reverse mutation assay). ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S.Typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in DMSO and tested at five concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate.
ln order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.
Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control (reference item). Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S.Typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate.
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.
ln the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with the test substance at any concentrations.
ln the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations.
ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. Due to a growth inhibiting effect of the test item in the pre-incubation assay on strain TA 1537, the number of revertant colonies was reduced at the concentration of 5000 µg/plate. No similar effect was observed with the other strains. The test item did not precipitate on the surface of the agar plates.
ln the experiments negative (solvent) and positive control (reference items) treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The reference items induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
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