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EC number: - | 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
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- 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:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate
- Molecular formula:
- n.a.
- IUPAC Name:
- Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- his+/- and trp-
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat Liver S9 Homogenate
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 30.9 mg/mL (Lot. No.: 080721K) in experiment I.
Rat S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Hamster Liver S9 Homogenate
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters (not induced).
The S9 was prepared and stored according to the currently valid version of the SOP for hamster liver S9 preparation.
The protein concentration of the S9 preparation was 26.9 mg/mL (Lot. No.: 150621) in experiment II.
Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yielded the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al (2) and Prival and Mitchell (1).
S9 Mix Substitution Buffer
The S9 mix substitution buffer contained per litre:
700 mL 100 mM sodium-ortho-phosphate-buffer pH 7.4
300 mL KCl solution 0.15 M
During the experiment, the S9 mix substitution buffer was stored in an ice bath - Test concentrations with justification for top dose:
- 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate for the plate incorporation test (experiment I)
33; 100; 333; 1000; 2500; and 5000 µg/plate for the Prival modification (Experiment II)
The top dose was selected based on a preliminary toxicity test; In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. - Vehicle / solvent:
- On the day of the experiment, the test item was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- congo red
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium; in agar (plate incorporation) for experiment I and preincubation for experiment II
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition or reduction of spontaneous revertants
METHODS FOR MEASUREMENTS OF GENOTOXICIY: count of reversal bacteria coloniesa by a validated computer system , which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to the intense color of the test item the colonies were partly counted manually
- OTHER: - 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 negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- not mandatory according to the guidelines
Results and discussion
Test resultsopen allclose all
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- follogin Prival modification and pre-incubatio design
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- 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:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.
Any other information on results incl. tables
see attached file for individual tables of results
Applicant's summary and conclusion
- Conclusions:
- The substance was tested for in vitro gene mutation toxicity to bacteria following OECD 471. Under the experimental conditions the substance induced gene mutation based on the result for strain WP2 uvrA with non-induced Hamster liver S9 mix (Prival modification).
- Executive summary:
This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.
In experiment I a minor increase in revertant colony count, neither reaching nor exceeding the threshold of twofold the revertant colony count of the corresponding solvent control, was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence of S9 mix. The historical control data of the negative and solvent control were exceeded from 2500 µg/plate onwards.
In experiment II a substantial increase in revertant colony numbers was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence S9 mix. The threshold of twofold the colony count of the corresponding solvent was exceeded at concentrations ranging from 1000 to 5000 µg/plate.Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies
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