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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 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:
- experimental study
- Remarks:
- source of read across study record
- Adequacy of study:
- key study
- Study period:
- From April 25 to May 11, 1990
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 990
- Report date:
- 1990
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 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 01
- IUPAC Name:
- Similar Substance 01
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Regular checking: regular checking of the properties of the 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.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- 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. The nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl. The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal activation system
- Test concentrations with justification for top dose:
- 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
- Vehicle / solvent:
- - Vehicle: aqua dest, for test item and sodium azide. DMSO for 4-NOPD and 2-AA.
- Justification for choice of vehicle: the vehicle was chosen because of its solubility.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation, two independent experiments.
AGAR
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: the 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.
NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.
EXPERIMENTAL PERFORMANCE
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);
2000 µl overlay agar
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.
DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in this pre-experiment were the same as described for the experiment.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 preparation
The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU (weight ca. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in RC1 was centrifuged cold at 9,000 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: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 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. - Evaluation criteria:
- 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 significant response is described as follows: 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 the 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.
CYTOTOXICITY
Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).
The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Applicant's summary and conclusion
- Conclusions:
- The test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
- Executive summary:
The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.
The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.
Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 at 5000.0 µg/plate (exp. I; without S9 mix), in TA 1537 at 1000.0 and 5000.0 µg/plate (exp. I; without S9 mix), and in TA 98 at 1000.0 µg/plate (exp.II; with S9 mix), and 5000.0 µg/plate (exp.I, and II; with S9 mix).
The plates incubated with the 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, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Conclusion
It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
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