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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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From 2010-02-25 to 2010-03-04
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Referenceopen allclose all
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Criteria for Mutagenicity Test in Bacterial Systems (Ministry of Labour, Japan, Notification No. 77, September 1, 1988; Notification No. 67, June 2, 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Health, Labor and Welfare; Ministry of Economy, Trade and Industry; and Ministry of the Environment; Heisei 15.11.21 Yakushokuhatsu Notification No. 1121002, Heisei 15.11.13 Seikyoku Notific. No. 2, Kanpokihatsu Notific. No. 031121002
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- - Purity: 100%
- Lot No.: 10SC8156928-2-2
Method
- Target gene:
- his operon (S. typhimurium strains)
trp operon (E. coli strain)
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):
- - Selection reason: because they have high sensitivity to detect mutagens and are widely used in the reverse mutation test
-Source and: The Salmonella strains were obtained from Dr. B. N. Ames (University of California, Berkeley, U.S.A.). The Escherichia strain was obtained from Dr. T. Matsushima (Institute of Medical Science, the University of Tokyo, Tokyo, Japan).
- Preparation: A culture of the test strain was mixed with DMSO using a ratio of 8 parts cell culture stock and 0.7 parts DMSO by volume and frozen in an acetone-dry ice bath. These frozen stocks were kept in a deep freezer at -80℃ until use.
The stock cultures of each strain were checked for their phenotypic characteristics [the amino acid requirement, UV sensitivity and presence of rfa and R factors]. The results confirmed that all strains retained their own phenotypic characteristics. These strains also yielded solvent and positive control substances-induced revertants within the frequency ranges expected from the historical control data. Each bacterial frozen stock culture was thawed and 0.1 mL of bacterial suspension was added to 60 mL of nutrient broth medium in a flask of 300 mL volume and cultured for 10 hours at 37℃ with shaking.
- Composition of nutrient broth medium (per 1000 mL of purified water):
Nutrient broth (Bacto Nutrient Broth): 8g; NaCl: 5g
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)
- method of preparation of S9 mix: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 used in this study was characterised with mutagen requiring metabolic activation, based on the assay data certificate of S9 batch used. - Test concentrations with justification for top dose:
- Range-finding experiment (all strains): 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate with and without metabolic activation
Main experiment (all strains): 156, 313, 625, 1250, 5000 μg/plate with and without metabolic activation
Since neither precipitation of the test substance nor cytotoxicity to bacteria was observed in the dose-finding assay, the dose of 5000 μg/plate was selected as the highest dose in the main assay as specified in the guidelines. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance has a water solubility of ≥ 50 mg/mL and is stable at this concentration for 24 h in room temperature
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see 'Remarks' field
- Remarks:
- 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), Sodium azide (NaN3), 9-aminoacridine (9-AA), 2-Aminoanthracene (2-AA) (see 'Details on test system and conditions' for concentrations)
- Details on test system and experimental conditions:
- DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiments
METHOD OF TREATMENT/ EXPOSURE:
Test substance solution (0.1 mL), either 100 mM Na-phosphate buffer (0.5 mL, pH 7.4) or S9 mix (0.5 mL), and bacterial suspension (0.1 mL) were mixed in a small test tube and incubated for 20 minutes at 37℃ with shaking. After addition of 2.0 mL of the top agar solution, the mixture was poured onto a minimal glucose agar plate, spread evenly and solidified at room temperature. All plates were incubated for 48 hours at 37℃.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of revertant colonies on a plate was counted using an automatic colony counter.
DETERMINATION OF CYTOTOXICITY
- Method: examination of bacterial backgroud lawn under stereomicroscope.
OTHER:
Positive control concentrations: AF-2 (0.01 μg/plate in DMSO, -S9, TA100 and WP2 uvrA; 0.1 μg/plate in DMSO, -S9, TA98); NaN3 (0.5 μg/plate in water, -S9, TA1535); 9-AA (80 μg/plate in DMSO, -S9, TA 1537); 2-AA (1.0 μg/plate in DMSO, +S9, TA100; 2.0 μg/plate in DMSO, +S9, TA1535 and TA1537; 0.5 μg/plate in DMSO, +S9, TA98; 10.0 μg/plate in DMSO, +S9, WP2 uvrA) - Evaluation criteria:
- When the test substance shows a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control, the response is judged to be positive. The study director judges the result with scientific consideration of biological relevance. When the positive response is reproducible, the test substance is judged to have mutagenic potential.
- Statistics:
- Mean values were calculated at each concentration for the two experiments. For the historical control data, mean values and standard deviation were calculated for each test strain. Any statistical analysis was not applied to evaluate the test results.
Results and discussion
Test resultsopen allclose all
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed.
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES:
Neither precipitation of the test substance nor cytotoxicity to bacteria was observed. The results of the range-finding study were evaluated together with the main study.
COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, see Table 1 under 'Any other information on materials and methods incl tables'.
Any other information on results incl. tables
Table 1. Results of dose range-finding assay (preincubation method)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 2 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
Sterilised purified water |
104 |
15 |
24 |
37 |
11 |
– |
1.22 |
87 |
9 |
22 |
28 |
11 |
- |
4.88 |
92 |
12 |
32 |
32 |
10 |
– |
19.5 |
92 |
11 |
21 |
39 |
10 |
- |
78.1 |
97 |
16 |
22 |
39 |
10 |
– |
312 |
88 |
11 |
20 |
44 |
11 |
- |
1250 |
84 |
12 |
23 |
39 |
10 |
– |
5000 |
94 |
14 |
23 |
42 |
12 |
Positive controls, –S9 |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
9-AA |
Concentrations (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
Mean No. of colonies/plate (average of 2 plates) |
594 |
356 |
102 |
335 |
540 |
|
+ |
Sterilised purified water |
93 |
9 |
20 |
38 |
15 |
+ |
1.22 |
93 |
8 |
30 |
40 |
15 |
+ |
4.88 |
105 |
8 |
28 |
38 |
12 |
+ |
19.5 |
80 |
13 |
20 |
42 |
15 |
+ |
78.1 |
85 |
12 |
30 |
39 |
18 |
+ |
313 |
94 |
7 |
22 |
36 |
17 |
+ |
1250 |
88 |
9 |
23 |
43 |
9 |
+ |
5000 |
86 |
7 |
23 |
33 |
14 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Mean No. of colonies/plate (average of 2 plates) |
760 |
206 |
570 |
214 |
133 |
AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: sodium azide
9-AA: 9-aminoacridine
2-AA: 2 -aminoanthracene
Table 2. Results of main assay (preincubation method)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 2 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
Sterilised purified water |
96 |
10 |
25 |
28 |
13 |
– |
156 |
90 |
7 |
21 |
19 |
11 |
- |
313 |
98 |
9 |
23 |
26 |
12 |
– |
625 |
94 |
7 |
18 |
27 |
13 |
- |
1250 |
90 |
12 |
23 |
26 |
13 |
– |
2500 |
95 |
9 |
17 |
30 |
9 |
- |
5000 |
98 |
11 |
26 |
31 |
10 |
Positive controls, –S9 |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
9-AA |
Concentrations (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
Mean No. of colonies/plate (average of 2 plates) |
696 |
311 |
110 |
317 |
535 |
|
+ |
Sterilised purified water |
82 |
10 |
25 |
35 |
19 |
+ |
156 |
100 |
10 |
29 |
30 |
21 |
+ |
313 |
83 |
10 |
23 |
28 |
11 |
+ |
625 |
86 |
5 |
18 |
27 |
15 |
+ |
1250 |
85 |
12 |
29 |
30 |
19 |
+ |
2500 |
86 |
5 |
31 |
37 |
18 |
+ |
5000 |
103 |
11 |
29 |
35 |
11 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Mean No. of colonies/plate (average of 2 plates) |
729 |
193 |
480 |
216 |
120 |
AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: sodium azide
9-AA: 9-aminoacridine
2-AA: 2 -aminoanthracene
Applicant's summary and conclusion
- Conclusions:
- The test item is not mutagenic under the test conditions.
- Executive summary:
The test item was evaluated for its mutagenic potential by a reverse mutation test with four strains of Salmonella typhimurium (TA100, TA98, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) based on OECD 471. The test was conducted in the presence and the absence of a rat liver drug metabolizing enzyme system (S9 mix).
In a dose-finding assay, the highest dose of test item was set at 5000 μg/plate, and the test was conducted at dose levels ranging from 5000 to 1.22 μg/plate in duplicate plating. Neither precipitation of the test substance nor cytotoxicity to bacteria was observed.
In the main assay, the test item was tested at doses ranging from 5000 to 156 μg/plate for all five test strains with and without S9 mix in duplicate plating based on the results of the dose-finding assay.
Throughout the tests, the test item did not show any statistically significant dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control with or without S9 mix in any of the strains. Positive control substances showed marked increases in the number of revertant colonies.
Based on these results, it is concluded that test item is not mutagenic under the test conditions.
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