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EC number: 255-037-7 | CAS number: 40690-89-9
- 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
- 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 October 1998 to 23 October 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 999
- Report date:
- 1999
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:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- solid: particulate/powder
- Details on test material:
- Disperse Orange 288
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix from rat liver
- Test concentrations with justification for top dose:
- Concentration range in the main test (with metabolic activation): 50, 160, 500, 1600, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 160, 500, 1600, 5000 µg/plate - Vehicle / solvent:
- Solvent: DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without metabolic activation: sodium azide (TA 100, TA 1535), 9-aminoacridine hydrochloride hydrate (TA 1537), 2-nitrofluorene (TA98), 1-methyl-3-nitro-1-nitrosoguanidine (WP2uvrA) With metabolic activation: 2-aminoanthracene – all strains.
- Details on test system and experimental conditions:
- Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the department conducting the study according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.
Preparation of S9-mix
Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (batch no. 98/5 for the experiment, protein concentration 24.8 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCI2 33 mM KCI 5 mM glucose-6-phosphate 4 mM NADP 100 mM phosphate buffer pH 7.4
Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A.. The strain of £ coli was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine and/or tryptophan requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled as described (2, 3).
Assay procedure
The mutation test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 pg/plate were used.
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
If the total number of concentrations selected for evaluation in the plate incorporation test does not allow for a statement of genotoxicity of the test substance to be made, an additional plate incorporation test, based on the toxicity results of the first test, has to be performed. If negative or equivocal results obtained, a second mutation experiment was performed on the basis of toxicity results in the plate incorporation test as a preincubation test.
For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound suspension (suspended in DMSO)
After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 deg C in the dark, colonies (his* and trp* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present. - Evaluation criteria:
- Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- 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
Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system. - Statistics:
- As above.
Results and discussion
Test results
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Highest concentration 1600 µg/plate
- Vehicle controls validity:
- other: Historical data
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- other: Historical data
- Additional information on results:
- Sterility checks and control plates
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test materia! and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control.
Solubility and toxicity
The test compound was suspended in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in the mutation experiment as the plate incorporation method.
Visible precipitation of the test compound on the plates was observed at 500 ug/plate and above.
Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 ug/plate and lower doses.
The test compound proved to be not toxic to the bacterial strains in the mutagenicity experiment.
Mutagenicity
In the mutation test T-9601 was tested for mutagenicity with the same concentrations as described in section 5.3. The number of colonies per plate with each strain as well as mean values of 3 plates are given.
In the absence of the metabolic activation system the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 98, TA 100, TA 1537. In the presence of metabolic activation the test compound resulted in relevant increases in the number of revertant colonies with the Salmonella strains TA 98, TA 100, TA 1535, TA 1537. No increases of the revertants were induced at the Eschericia coli strain WP 2uvrA with and without S9-mix.
All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.
CONCLUSION
The results lead to the conclusion that T-9601 is mutagenic in these bacterial test systems with and without an exogenous metabolizing system. - Remarks on result:
- other: other: preliminary test
- Remarks:
- Migrated from field 'Test system'.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
positive with and without metabolic activation
The results lead to the conclusion that the substance is mutagenic in these bacterial test systems with and without an exogenous metabolizing system - Executive summary:
Study data conducted to EEC-Guideline B.13 & B14 of the Directive 92/68/EEC, OECD Guidleines for Testing of Chemicals 471 and US.EPA; OPPTS 870.5100 Heath Effects Test Guidelines in compliance with GLP.
The results lead to the conclusion that the susbstance is mutagenic in the bacterial systems with and without an exogenous metoblizing system. However it should be noted that the specific method according to Privall - Mitchell for azo dyes was not utilised in this study; hence the results may be due to redundancies within the method employed. This statement is further enforced by the negative mutagenicity results observed in the other in vitro studies conducted.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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