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Reaction mass of 3,3'-(1E,1'E)-(4,4'-(6-(bis(2-hydroxyethyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl)bis(3-methoxy-4,1-phenylene))bis(diazene-2,1-diyl)dibenzenesulfonic acid and disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]
EC number: 943-704-1 | CAS number: -
- 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:
- weight of evidence
- Study period:
- From May 17 to June 05, 1989
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Remarks:
- Source study has reliability 1. Details on the read across are attached in section 13.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 989
- Report date:
- 1989
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted on May 26, 1983
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Similar Substance 02
- IUPAC Name:
- Similar Substance 02
- Test material form:
- solid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELL CHECK
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory, according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
STORAGE
Strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Metabolic activation:
- with and without
- Metabolic activation system:
- hamster liver microsomal fraction
- Test concentrations with justification for top dose:
- 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
According to results of the pre-experiment, concentrations applied in main experiments were chosen. - Vehicle / solvent:
- - Vehicle: bidest. water.
- Justification for choice of vehicle: solubility properties and non-toxicity to bacteria.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- congo red
- other: 4-nitro-o-phenylene-diamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: pre-incubation.
The following materials were mixed in a test tube and gently shaken at 30 °C for 30 min.: 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).
After pre-incubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.
NUMBER OF REPLICATIONS: the assay was performed in two independent experiments, using identical procedures. Each concentration, including the controls, was tested in triplicate.
CONDITIONS
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium.
- Nutrient medium: the nutrient medium contains per litre 8 g Difco Nutrient Broth and 5 g NaCl. - Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used.
- Dished preparation: each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O and 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.
PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in the pre-experiment are the same as described for the main experiments.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial
DATA RECORDING
The colonies were counted using the BIOTRAN III counter. The counter was connected to an IBM XT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters. After cervical dislocation the livers of the animals were removed, washed in 0.1 M sodium phosphat buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidestilled water and homogenised. The homogenate, diluted 1:3 in sodium phosphat buffer was centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.
S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.
The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 33 mM KCl, 8 mM MgCl2, 20 mM glucose 6-phosphate, 2.8 units/ml glucose 6-phosphate dehydrogenase, 4 mM NADP, 2 NADH, 2 FMN in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The preparation was performed according to Ames et al. and and Mitchell. - Evaluation criteria:
- The generally accepted conditions for evaluation of results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related 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 test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of test article regardless whether the highest dose induced the above described enhancement factors or not.
Results and discussion
Test results
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weak toxic effects
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).
The plates incubated with test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.
Weak increases of revertant colony numbers in strain TA 1535 (exp. I, without S9 mix at 10.0 and 1000.0 µg/plate), in TA 98 (exp. I, with S9 mix at 10.0 µg/plate) and in TA 1537 (exp. II, with S9 mix at 333.3 µg/plate) are considered not to be relevant. The obtained effects were not reproduced in the independent experiment.
CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
PRE-EXPERIMENT FOR TOXICITY
The plates with test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.
Applicant's summary and conclusion
- Conclusions:
- Non-mutagenic in the Salmonella typhimurium reverse mutation assay.
- Executive summary:
The potential of test substance to induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 was assessed.
The assay was run as pre-icubation test in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including controls, was tested in triplicate. Test article was tested at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.
Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).
Plates incubated with test substance showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
Test substance induced a slight increase in number of revertants in strain TA 1535 at 10.0 and 1000.0 µg/plate without metabolic activation (exp. I), in TA 98 at 10.0 µg/plate (exp. I) and in TA 1537 at 333.3 µg/plate (exp. II) both with metabolic activation.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. Metabolic activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, test substance did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
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