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EC number: 275-148-4 | CAS number: 71033-19-7
- 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:
- From the 12nd of May to the 27th of May, 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Test conducted according to an internationally accepted test guideline.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
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)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)
- EC Number:
- 275-148-4
- EC Name:
- Disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)
- Cas Number:
- 71033-19-7
- Molecular formula:
- C32H26N10Na2O8S3
- IUPAC Name:
- disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected.
The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth.
The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured.
The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus.
Since for azo-dyes and diazocompounds, a reductive metabolic activation system could be more appropriate than the standard liver metabolizing system, in Main Assay II, the pre-incubation method was used and the metabolising system was prepared with S9 liver fraction from uninduced hamsters, supplemented with flavin mononucleotide cofactor (Prival et al. 1984).
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):
- TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens.
In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to
some mutagenic compounds.
The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface
of the bacterial cell wall and increases permeability to certain classes of chemicals.
All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens.
TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens.
In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- Toxicity test: 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
Main assay I: 5000, 2500, 1250, 625 and 313 µg/plate.
Main assay II: 5000, 2500, 1250, 625 and 313 µg/plate. - Vehicle / solvent:
- Sterile water
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- sterile water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- Remarks:
- Absence S9
- Untreated negative controls:
- yes
- Remarks:
- sterile water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- Presence of S9
- Untreated negative controls:
- yes
- Remarks:
- sterile water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive control substance:
- other: 2-aminoanthracene, Congo Red, Trypan Blue
- Remarks:
- Presence of S9
- Details on test system and experimental conditions:
- Permanent stocks of these strains are kept at -80°C in the laboratory.
Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement
No Growth onMinimal plates+Biotin. Growth on Minimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates. Growth onMinimal plates+Tryptophan.
uvrA, uvrB
Sensitivity to UV irradiation.
rfa
Sensitivity to Crystal Violet.
pKM101
Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.
MEDIA
Nutrient Broth
Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar
Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar
Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar
"Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).
S9 tissue homogenate
Standard S9 mix
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics: Rat, Strain Sprague Dawley, Liver, Inducing Agents Phenobarbital – 5,6-Benzoflavone, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3488
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.
S9 mix Privall modification (Main Assay II)
Species Syrian hamster, Strain GSH, Tissue Liver, Inducing Agents None, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3342.
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.
PRELIMINARY TOXICITY TEST
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assay. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.
Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
MAIN ASSAY
A Main Assay was performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates wereused at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrientagar plates to establish the number of bacteria in the cultures.
Main Assay I was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.
Main Assay II was performed using a pre-incubation method. The components were added in turn to an empty test-tube.
The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours, in the preliminary toxicity test, and immediately scored by counting the number of revertant colonies on each plate, in the Main Assay I and II. - Evaluation criteria:
- Acceptance criteria
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.
Criteria for outcome of the assays
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. - Statistics:
- The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a +bx
Results and discussion
Test results
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Solubility
Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0mg/mL (expressed in terms of active ingredient). This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.
Toxicity test
The test item Acid Yellow 166 was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
No precipitation of the test item was observed, at the end of the incubation period, at any concentration tested, with any tester strain, in the absence or presence of S9 metabolism.
A yellow dose-related colouring, which did not interfere with scoring neither with the evaluation of the background lawn, was observed, at the end of the incubation period, with all tester strains, in the absence and presence of S9 metabolism.
No toxic effect, as indicated by thinning of the background lawn and/or reduction in revertant numbers, neither increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.
Main assays
Two Main Assays were performed.
On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
No toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, neither increases in revertant numbers were observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step usinga reductive metabolic activation system, was included for all treatments ofMain Assay II. The test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
No toxicity, as indicated by thinning of the background lawn and/or reduction in revertantn numbers, neither increases in revertant numbers were observed with any tester strain, at any concentration tested, in the absence or presence of S9 Prival metabolic system. Both in Main Assay I and Main Assay II, a yellow dose-related colouring, which did not interfere with scoring neither with the evaluation of the background lawn, was observed, at the end of the incubation period, with all tester strains, in the absence and presence of S9 metabolism.
No relevant increases in the number of revertant colonies were observed in the plate incorporation
or pre-incubation assay, at any dose level, with any tester strain, in the absence or
presence of S9 metabolism.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence ofccolonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Applicant's summary and conclusion
- Conclusions:
- The test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under
the reported experimental conditions - Executive summary:
Method
The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, according to the OECD471, in GLP.
The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone (Standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification), in Main Assay II.
The test item was used as a solution in sterile water for injection and, as requested by the Sponsor, concentrations were expressed in terms of active ingredient.
Toxicity test: the test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
Main Assay I: on the basis of toxicity test results, inMain Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
Main Assay II: as no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was performed. Based on the chemical structure of the test item (azo-dyes), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactory). The test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
Observation
Toxicity test: at the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
No toxicity neither increases in revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.
Main test I: no precipitation of the test item was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
No toxicity neither increases in revertant colonies were observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
Main test II:
No precipitation of the test item was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
No toxicity neither mutagenicity was noticed with any tester strain, at any dose level, in the absence or presence of S9 Prival metabolizing system.
The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.
Conclusion
It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
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