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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
- 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:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experiment Starting Date: July 22, 2015 Experiment Completion Date: July 30, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
Reference
- Name:
- Unnamed
- Type:
- Constituent
- Type:
- impurity
- Test material form:
- solid: particulate/powder
Method
- Target gene:
- S. typhimurium - histidine locus
E. coli - tryptophan locus
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:
- S9 mix (prepared from from induced rat liver)
- Test concentrations with justification for top dose:
- Main test 1:
+/- S9 mix
All strains: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Main test 2:
- S9 mix
TA100: 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/plate
TA1535: 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/plate
WP2uvrA: 156, 313, 625, 1250, 2500 and 5000 µg/plate
TA98 and TA1537: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
+ S9 mix
All strains: 313, 625, 1250, 2500 and 5000 µg/plate
Dose selection:
Main test 1:
5000 µg/plate was the highest dose and five lower doses were diluted with a geometric progression of 4.
Main test 2:
The result of the main test 1 showed that the number of revertant colonies in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/plate or more for TA100, TA1535, TA98 and TA1537 and at 5000 µg/plate for WP2uvrA without S9 mix. The precipitation of the test substance was not observed in either the presence or absence of S9 mix.
Therefore, in consideration of the increase of the number of revertant colonies and the bacterial growth inhibition, the dose levels described above for main test 2 were selected. - Vehicle / solvent:
- Vehicle- DMSO
The test substance was insoluble in distilled water at 50.0 mg/mL and soluble in DMSO at 50.0 mg/ml. The test substance solution of 50,0 mg/mL prepared with DMSO was considercd to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 hours after preparation. Therefore, DMSO was selected as a vehicle.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.01 µg/plate for strain TA100 and WP2uvrA, 0.1 µg/plate for strain TA98
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
- Remarks:
- - S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.5 µg/plate for strain TA1535
- Positive control substance:
- sodium azide
- Remarks:
- - S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.5 µg/plate for strain TA1537
- Positive control substance:
- other: ICR-191
- Remarks:
- - S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.5 µg/plate for strain TA98, 1 µg/plate for strain TA100, 2 µg/plate for strains TA1535 and TA1537, 10 µg/plate for strain WP2uvrA
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- + S9 mix
- Details on test system and experimental conditions:
- Medium and S9 Mix:
Medium:
- Minimal glucose agar plate
- Soft agar: A solution containing 0.5 mM histidine and 0.5 mM biotin for S.typhimurium strains or 0.5 mM tryptophan for E.coli strain was added to a soft agar solution containing 0.6 w/v% agar and 0.5 w/v% NaCl at a volume ratio of 1 to 10.
S9 mix:
Rat liver S9: S9 was prepared from the liver of 7-week old rats administered with phenobarbital and 5,6-benzoflavone.
Composition of S9 mix: S9 mix was prepared just before use. 1 ml of S9 mix consisted of 8 µmol MgCl2, 33 µmol KCl, 5 µmol Glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.
Pre-cultures of the tester strains:
Frozen stock culture of bacterial strains, 20 µl for TA100, TA98, TA1535 and TA1537, 5 µl for WP2uvrA, were respectively inoculated to 11 ml of Nutrient broth No. 2 in L- tube (volume: 27 ml). The culture was incubated at 37 ± 0.5 °C for 9 hours with shaking at about 50 times/minute by a seesaw type of shaker.
The number of viable cells was calculated from O.D value at 660 nm measured by a photometer (miniphoto 518R, TAITEC) at the end of incubation. It was confirmed that the numbers of viable cells were more than 1.0 x 10^9 cells/ml.
The final numbers of viable cells are shown below:
TA100: 2.1 x 10^9 cells/ml
TA1535: 2.0 x 10^9 cells/ml
WP2uvrA: 4.7 - 4.8 x 10^9 cells/ml
TA98: 2.2 x 10^9 cells/ml
TA1537: 1.8 - 1.9 x 10^9 cells/ml
Preparation of test substance solution and positive control substance solutions:
a) Preparation of test substance solution
1) Preparation method
The test substance was weighed, added into DMSO and mixed by laboratory mixer to make a 50.0 mg/ml as original solution. The test substance solutions of each required concentration were prepared by diluting with the same vehicle.
2) Timing of preparation
The test substance solutions were prepared just before use, kept under yellow light at room temperature and used within 2 hours.
b) Preparation of positive control substance solutions
1) Preparation method and storage conditions
NaN3 was dissolved in distilled water, AF-2, ICR-191 and 2AA were dissolved in DMSO. Positive control solutions were stored below -80°C. The positive control substances were thawed just before use.
Methods:
The pre-incubation method was used and the test was carried out in triplicate (for negative control, positive control and test substance treatment groups).
Procedures:
After 0.1 ml of the test substance solution, vehicle or positive control solutions, 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 ml of the bacterial culture were added to a test tube, the mixture was shaken at 37 ± 0.5 ºC for 20 minutes. Two millilitres of the soft agar were then added to each tube and the mixture was then poured onto a minimal agar plate. The number of revertant colonies was counted after incubation at 37 ± 0.5 ºC for 48 hours.
Confirmation of Sterility:
The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto each minimal glucose agar plate in order to examine bacterial contamination. Bacterial contamination was judged with those plates after incubation at 37 ± 0.5 ºC for 48 hours.
Observation:
The precipitation of the test substance was observed macroscopically and the bacterial growth inhibition was observed by using a stereomicroscope at the end of the incubation
Colony counting:
For the plates in which the bacterial growth inhibition was observed, the number of colonies was counted manually, and for the other plates with a colony analyser. Square correction and miscounting correction were conducted when counting with the colony analyser. - Rationale for test conditions:
- The ability of the test substance to induce mutations was investigated by using S.typhimurium and E.coli.
- Evaluation criteria:
- The test substance was judged to be positive when the number of revertant colonies increased twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative. No statistical methods were used.
In the case of a positive result, the specific activity (number of revertant colonies/mg) was calculated by the following formula for the dose levels at which the number of revertant colonies was shown twice or more than that in the negative control, and the highest value in the specific activity wa designated as the specific activity of the test substance.
Specific activity = (N – N0) / D
N: mean number of revertant colonies at the corresponding dose
N0: mean number of revertant colonies in the negative control
D: the corresponding dose (mg/plate)
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- (with and without S9-mix)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (without S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9 mix only
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9 mix only
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Main Test 1:
The number of revertant colonecs in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/Plate or more for TA100, TA1535, TA98 and TA1537, and at 5000 µg/Plate for WP2uvrA without S9 mix.
The precipitation of the test substance was not observed in either the presence or absence of S9 mix.
Main Test 2:
The number of revertant colonies in the test substance treatment groups was increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix. The bacterial growth inhibition was observed at 1250 µg/plate or more for TA100, at 625µg/plate or more for TA1535, at 5000 µg/plate for WP2uvrA, and at 625 µg/plate or more for TA98 and TA1537 without S9 mix. The precipitation of the test substance was not observed in either the presence or absence of S9 mix.
Any other information on results incl. tables
The results of tests 1 and 2 are shown in attached background material (result tables and dose-response curves).
Applicant's summary and conclusion
- Conclusions:
- It was concluded that CIM-43 had ability to induce mutations under the test conditions
- Executive summary:
The ability of CIM-43 to induce mutations was investigated using Salmonella typhurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).
The mutagenicity of the test substance was judged to be positive because the number of revertant colonies in the test substance treatment groups increased to twice or more than that in the negative control for TA100 and TA1535 without S9 mix and for TA100 with S9 mix, and the reproducibility was confirmed. In the other test conditions, the number of revertant colonies in the test substance treatment groups was less than twice that in each negative control.
Consequently, it was concluded that the CIM-43 had ability to induce mutations under the present test conditions.
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