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EC number: 251-257-2 | CAS number: 32846-21-2
- 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:
- 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 997
- Report date:
- 1997
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (only four strains were tested)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate
- EC Number:
- 251-257-2
- EC Name:
- Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate
- Cas Number:
- 32846-21-2
- Molecular formula:
- C23H26N4O6S2.Na
- IUPAC Name:
- sodium 6-amino-5-({2-[cyclohexyl(methyl)sulfamoyl]phenyl}diazenyl)-4-hydroxynaphthalene-2-sulfonate
- Test material form:
- other: solid
Constituent 1
- Specific details on test material used for the study:
- Name: FAT 20'003/I
Batch No.: not indicated by the sponsor
Vers. No.: 9-5-97
Aggregate State at Room Temperature: Solid
Colour: red
Purity: approx 90%
Stability in Solvent: Stable for atleast 24 hours at room temperature and under test conditions at 37°C in water, PEG 400, saline, FCA/NaCl-solution and CMC solution.
Storage: room tempearture
Expiration Date: Feb 2001.
On the day of the experiment, the test article FAT 20003/I was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Method
- Target gene:
- Histidine gene
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- (concurrent untreated)
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- (for TA 1535 and TA100 without metabolic activation)
- Untreated negative controls:
- yes
- Remarks:
- (concurrent untreated)
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- (for TA 1537 and TA98 without metabolic activation)
- Untreated negative controls:
- yes
- Remarks:
- (concurrent untreated)
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- (for all strains with metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)
For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2,000 µL: Overlay agar
Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark. - Evaluation criteria:
- The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates.
A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test substance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the criteria described above or not. - Statistics:
- A statistical analysis of the data is not required.
Results and discussion
Test results
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (only in plate incorporation method)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. The frequency of spontaneous revertant colony formation in strain TA 1535 in the second experiment slightly exceeded the historical control range (36 colonies versus 10 - 29 colonies) in the absence of metabolic activation. This increase is most likely based upon a slight increase in the bacterial cell density during pre-incubation and does not represent statistical fluctuations since all test points are at or somewhat above the upper limit of the historical range of negative controls. A slight or moderate increase in the bacterial cell density is not uncommon during pre-incubation since small amounts of histidine containing medium is transferred together with the bacteria and the overall dilution during the preincubation step is considerably lower as compared to the direct plate incorporation method. Therefore, this slightly elevated frequency of spontaneous revertants was judged as biologically irrelevant within the scope of the assay.
No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Applicant's summary and conclusion
- Conclusions:
- The test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
- Executive summary:
An in vitro study was performed to investigate the potential of the test substance (at ca. 90 % purity) to induce gene mutations according to OECD Guideline 471 in compliance with GLP with deviation (i.e., only four strains were tested).
The assay was performed in two independent experiments both with and without liver microsomal activation using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Experiment I was performed as a plate incorporation assay and experiment II was performed as a pre-incubation assay. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate.
Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 with and without S9 mix at 5000 µg/plate in Experiment I. In Experiment II, no toxic effects were observed. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the four tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Taking into consideration the above findings, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
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