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EC number: 200-657-5 | CAS number: 67-51-6
- 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:
- 15 March 2002 - 29 March 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA 712-C-98-247
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: CPMP/ICH/141/95
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: CPMP/ICH/174/95
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 3,5-dimethylpyrazole
- EC Number:
- 200-657-5
- EC Name:
- 3,5-dimethylpyrazole
- Cas Number:
- 67-51-6
- Molecular formula:
- C5H8N2
- IUPAC Name:
- 3,5-dimethyl-1H-pyrazole
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- - Physical state: White powder
- Storage condition of test material: In the dark at ambient temperature
Constituent 1
Method
- Target gene:
- his G 46 in TA 1535 and TA 100,
his C 3076 in TA 1537,
his D 3052 in TA 1538 and TA 98,
trpE locus in E. coli.
his G 46 is a mis-sense mutation which is reverted to prototrophy by a variety of mutagens that cause base-pair substitutions
his C 3076 contains a frameshift mutation which appears to have added a GC base pair. This mutation is reverted by 9-aminoacridine, ICR-191 and epoxides of polycyclic hydrocarbons.
his D 3052 also contains a frameshift mutation which is reverted by the deletion of 2 base-pairs, CG GC. It is readily reverted by aromatic amines and derivatives.
E.col WP2uvrA contains an ochre mutation at the trpE locus and can be mutated to tryptophan independence either by a base-pair reversion of an A-T base-pair at the tprE locus, or, more likely, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromome.
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):
- - Type and identity of media: Oxoid Nutrient Broth No. 2 (25 g per litre).
- Properly maintained: yes
- Samples of each strain were grown by culturing for 16 hrs at 37 ºC in nutrient broth. Cultures were kept for up to 4 days at +4 ºC to allow relevant checks to be performed but fresh cultures were used for the experiment. - Additional strain / cell type characteristics:
- other: rfa, uvrB, pKM101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9 mix
- Test concentrations with justification for top dose:
- - 17, 50, 167, 500, 1667 and 5000 µg per plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Two mutation assays were carried out; one with direct plate incorporation and the second with preincubation.
DURATION
- Direct plate method: Plates were prepared by mixing 0.5 mL of S9 mix or 0.05 M phosphate buffer and 0.1 mL of bacteria to 2 mL of soft agar, 0.1 mL aliquots of the solvent (DMSO) or test material were added last. Once mixed, the cooling agar was then poured onto minimal medium plates. These plates contained 25 mL of 1.5% purified agar, in Vogel-Bonner Medium E with 2% glucose. Once set the agar plates were inverted and incubated at 37º C.
- Preincubation period: Preparation began with adding 0.5 mL volumes of either S9 mix or 0.05 M phosphate buffer to sterile tubes, followed by 0.1 mL of bacteria and finally 0.1 mL of either the test solution or solvent (DMSO). Tubes were then sealed and placed in a shaking incubator at 37 ºC for 20 minutes. After which 2 mL of soft agar was added. The cooling contents were then mixed and poured into agar plates, as above. Once set the agar plates were inverted and incubated at 37 °C.
- Exposure duration: Plates were incubated for 2 or 3 days.
NUMBER OF REPLICATIONS: All concentrations were performed with and without S9 mix in triplicate for both tests.
COLONY EVALUATION: Colonies of ≥ 0.1 mm in diameter were counted.
TOXICITY TEST
- Strain: TA 100
- Concentrations: 17, 50, 167, 500, 1667 and 5000 µg per plate.
- Method: One plate was prepared for each concentration in the presence and absence of the S9 mix.
OTHER EXAMINATIONS
Quality Control: Each strain was tested for its resistance to amplicillin (indicating pKM101) and its sensitivity to ultraviolet light and crystal violet (indicating persistence of the uvrB and rfa mutations). - Evaluation criteria:
- EVALUATION CRITERIA
A positive response was recorded if the following criteria were met:
1) For S. typhimurium strains TA 1353, TA 1537, and TA 98 and for E. coli at least doubling of the mean concurrent vehicle control values at some concentration of the test material. For S. typhimurium stain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
2) A dose related response, although at high dose levels this relationship could be inverted because of, for example (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
3) A reproducible effect in independent tests.
ACCEPTABILITY
The test was considered acceptable if the following criteria were met:
1) The bacteria demonstrated their typical response to crystal violet, ampicillin and U.V. light.
2) At least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.
3) On at least 2 of the positive control plates, there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5.
4) No toxicity or contamination was observed in at least 4 dose levels.
5) In cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.
Results and discussion
Test results
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- other: All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not thought to have affected the integrity of the study results.
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precippitation was observed at any of the concentrations tested.
QUALITY CONTROL:
All strains were sensitive to crystal violet, whereas only the plasmid-containing stains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to U.V light emitted over a period of 5-10 s from a lamp set at 254 nm. Increased sensitivity to U.V. light was demonstrated. These results are consistent with the known properties of these bacteria.
TOXICITY TEST:
No toxicity to bacteria was observed and no precipitation of the test material occurred in either the presence or absence of S9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 1. Direct Plate Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix
Substance |
Concentrations ( µg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
Mean ± SD |
Mean ± SD |
Mean ± SD |
Mean ± SD |
Mean ± SD |
||
In the Presence of S9 |
||||||
DMSO |
100 µL |
22 ± 5 |
17 ± 6 |
25 ± 8 |
117 ± 8 |
9 ± 0 |
Test Material |
17 |
21 ± 1 |
22 ± 1 |
22± 6 |
115 ± 15 |
11 ± 5 |
50 |
22 ± 1 |
15 ± 3 |
20 ± 2 |
116 ± 11 |
9 ± 3 |
|
167 |
18 ± 3 |
16 ± 4 |
24 ± 3 |
111 ± 6 |
14 ± 2 |
|
500 |
18 ± 5 |
19 ± 4 |
21 ± 7 |
118 ± 3 |
10 ± 2 |
|
1667 |
20 ± 2 |
19 ± 3 |
26 ± 6 |
115 ± 9 |
16 ± 3 |
|
5000 |
18 ± 6 |
15 ± 8 |
25 ± 2 |
117 ± 8 |
13 ± 2 |
|
In the Absence of S9 |
||||||
DMSO |
100 µL |
24 ± 8 |
14 ± 3 |
11 ± 4 |
110 ± 6 |
12 ± 6 |
Test Material |
17 |
24 ± 11 |
15 ± 5 |
19 ± 4 |
121 ± 14 |
11 ± 3 |
50 |
20 ± 7 |
13 ± 3 |
11 ± 2 |
114 ± 12 |
9 ± 3 |
|
167 |
22 ± 7 |
13 ± 5 |
15 ± 4 |
112 ± 9 |
10 ± 3 |
|
500 |
23 ± 10 |
15 ± 3 |
16 ± 3 |
109 ± 6 |
12 ± 1 |
|
1667 |
23 ± 8 |
13 ± 2 |
14 ± 3 |
117 ± 5 |
13 ± 2 |
|
5000 |
22 ± 10 |
11 ± 1 |
13 ± 2 |
99 ± 13 |
14 ± 2 |
Table 2. Preincubation Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix
Substance |
Concentrations ( µg/plate) |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
Mean ± SD |
Mean ± SD |
Mean ± SD |
Mean ± SD |
Mean ± SD |
||
In the Presence of S9 |
||||||
DMSO |
100 µL |
20 ± 3 |
9 ± 1 |
40 ± 1 |
139 ± 14 |
10 ± 1 |
Test Material |
17 |
17 ± 5 |
16 ± 4 |
39 ± 8 |
146 ± 17 |
12 ± 3 |
50 |
21 ± 5 |
12 ± 2 |
41 ± 5 |
144 ± 16 |
15 ± 5 |
|
167 |
20 ± 3 |
9 ± 4 |
33 ± 6 |
149 ± 10 |
13 ± 1 |
|
500 |
19 ± 7 |
10 ± 1 |
38 ± 3 |
164 ± 7 |
12 ± 3 |
|
1667 |
16 ± 3 |
10 ± 1 |
37 ± 6 |
134 ± 8 |
12 ± 3 |
|
5000 |
20 ± 3 |
7 ± 2 |
41 ± 6 |
125 ± 12 |
13 ± 1 |
|
In the Absence of S9 |
||||||
DMSO |
100 µL |
20 ± 5 |
9 ± 1 |
26 ± 2 |
142 ± 11 |
11 ± 3 |
Test Material |
17 |
15 ± 3 |
8 ± 1 |
29 ± 5 |
125 ± 11 |
11 ± 6 |
50 |
18 ± 5 |
13 ± 3 |
25 ± 7 |
124 ± 13 |
12 ± 3 |
|
167 |
17 ± 2 |
8 ± 3 |
23 ± 3 |
130 ± 3 |
9 ± 3 |
|
500 |
19 ± 2 |
10 ± 3 |
27 ± 5 |
121 ± 9 |
10 ± 4 |
|
1667 |
18 ± 6 |
9 ± 4 |
31 ± 3 |
122 ± 9 |
8 ± 4 |
|
5000 |
19 ± 2 |
6 ± 2 |
20 ± 2 |
136 ± 5 |
10 ± 4 |
Table 3. Positive Control Results
Test Method |
Presence/Absence of S9 mix |
Compound |
Strain |
Concentration (µg/plate) |
Colony Count (mean ± SD) |
Direct Plate Method |
+ |
2AAN |
TA 1535 |
2 |
608 ± 40 |
TA 1537 |
2 |
705 ± 55 |
|||
TA 98 |
0.5 |
235 ± 29 |
|||
TA 100 |
0.5 |
1136 ± 71 |
|||
WP2uvrA |
20 |
701 ± 31 |
|||
- |
NaN₃ |
TA 1535 |
1 |
335 ± 80 |
|
9AA |
TA 1537 |
80 |
2945 ± 276 |
||
2NF |
TA 98 |
1 |
468 ± 30 |
||
NaN₃ |
TA 100 |
1 |
938 ± 61 |
||
ENNG |
WP2uvrA |
2 |
864 ± 26 |
||
Preincubation Method |
+ |
2AAN |
TA 1535 |
2 |
465 ± 32 |
TA 1537 |
2 |
441 ± 6 |
|||
TA 98 |
0.5 |
955 ± 34 |
|||
TA 100 |
0.5 |
1994 ± 23 |
|||
WP2uvrA |
20 |
673 ± 52 |
|||
- |
NaN₃ |
TA 1535 |
1 |
490 ± 26 |
|
9AA |
TA 1537 |
80 |
4048 ± 546 |
||
2NF |
TA 98 |
1 |
723 ± 73 |
||
NaN₃ |
TA 100 |
1 |
1160 ± 24 |
||
ENNG |
WP2uvrA |
2 |
902 ± 60 |
2-AAN = 2-Aminoanthracene
NaN₃ = Sodium azide
9AA = 9-Aminoacridine
2NF = 2-Nitrofluorene
ENNG = N-Ethyl-N-nitro-N-nitrosoguanidine
Table 4. Toxicity Test
Strain |
Concentration (µg/plate) |
S-9 mix |
Revertant Colony Count |
TA 100 |
DMSO |
- |
114 |
17 |
- |
112 |
|
50 |
- |
122 |
|
167 |
- |
114 |
|
500 |
- |
119 |
|
1667 |
- |
115 |
|
5000 |
- |
96 |
|
DMSO |
+ |
121 |
|
17 |
+ |
121 |
|
50 |
+ |
142 |
|
167 |
+ |
122 |
|
500 |
+ |
132 |
|
1667 |
+ |
112 |
|
5000 |
+ |
102 |
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
Under the conditions of the test, no mutagenic response was induced in any of the five strains tested in either the presence or absence of metabolic activation. - Executive summary:
In a GLP compliant study performed to standardised guidelines, the mutagenic potential of the test material was assessed in an Ames test. Both S. typhimurium and E. coli were exposed to the test material by both direct plate application and preincubation methods. The following strains were tested, with and without the presence of metabolic activation by S9 -mix: TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. Positive and solvent controls were run concurrently.
Under the conditions of the test, no mutagenic response was observed in any of the five strains tested in either the presence or absence of metabolic activation up to a maximum concentration of 5000 µg per plate. The results obtained in both mutation assays were similar. There was no toxicity to the bacteria and no precipitation of the test material at any of the concentrations tested. Positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not considered to have affected the integrity of the study results.
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