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EC number: 206-017-1 | CAS number: 288-13-1
- 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
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- Water solubility
- Solubility in organic solvents / fat solubility
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- Flash point
- Auto flammability
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- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- 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
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Carcinogenicity
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell transformation assay
Test material
- Reference substance name:
- Pyrazole
- EC Number:
- 206-017-1
- EC Name:
- Pyrazole
- Cas Number:
- 288-13-1
- Molecular formula:
- C3H4N2
- IUPAC Name:
- 1H-pyrazole
- Test material form:
- solid
- Details on test material:
- - Purity: 98-100%
Constituent 1
Method
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Cell cycle length, doubling time or proliferation index: doubling time of 12-14 hours
-Cloning efficiency: greater than 80%
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- Preliminary test concentrations: 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL
Main test concentrations: 42.6, 85.1, 170, 341 and 681 μg/mL
-The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: No precipitate was seen before or after tests
Controls
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
NUMBER OF REPLICATIONS: dulpicate at each concentration level
- OTHER:METHOD OF APPLICATION: in medium, Complete Ham’s F12 medium
- Cell density at seeding (if applicable): ~5E6 in 10 mL
DURATION
- Preincubation period: overnight at standard conditions
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): incubated under standard conditions for 7 days
SELECTION AGENT (mutation assays): Hypoxanthine-free Complete Ham’s F12 medium (Complete Ham’s F12 medium -Hx)
STAIN (for cytogenetic assays): crystal violet
NUMBER OF REPLICATIONS: tests done in duplicate
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Treatment
Cells were plated (on Day -1) in 75-cm2 cultures at a density of ~5 E6 in 10 mL Complete Ham’s F12 medium. Following an overnight incubation (on Day 0) at standard conditions, the cultures were washed twice with HBSS and re-fed with 5 mL treatment medium, or 4 mL treatment medium plus 1 mL S9 mix (adjusted for the test substance dose volume if >1%, v/v), as appropriate. Following addition of the test or control substance formulations (2.0 mL for vehicle control and test substance; 200 μL for positive control) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours (positive control substances were prepared in DMSO and added to the flasks using a 1% dose volume).
Subculture for Phenotypic Expression and Initial Survival
After the 5-hour treatment, the treatment media were removed, the cultures were washed twice with CMF-HBSS and then were trypsinized and counted. Cells were subcultured at ~2.4 E6 cells/225-cm2 flask in 30 mL Complete Ham’s F12 medium in duplicate (or all available into 1 or 2 flasks) for phenotypic expression and incubated under standard conditions (larger numbers of cells may be subcultured for phenotypic expression where decreases in cloning efficiency are observed in the Preliminary Toxicity Test; i.e., there should be ~2.4E6 viable cells for phenotypic expression). An additional aliquot of cells was plated at 200 cells/60-mm plate in 5 mL Complete Ham’s F12 medium in triplicate for initial survival. The 60-mm plates were incubated under standard conditions for 7 days and the resulting colonies were fixed in methanol, stained with crystal violet, and counted.
The cultures were subcultured for 7 days, at 2- to 3-day intervals, to maintain logarithmic growth and permit expression of the mutant phenotype. At each subculture, the flasks were washed once (CMF-HBSS), trypsinized, counted and subcultured at ~2.4E6 cells/225-cm2 flask in 30 mL Complete Ham’s F12 in duplicate (or all available into 1 or 2 plates). Subculture was as follows based upon visual observation of the monolayer:
• ≥ 50% of the vehicle control, only one flask was subcultured (into duplicate flasks) and the back-up flask was discarded
• between 25% to 50% of the vehicle control, both flasks were subcultured
• ≤ 25% of the vehicle control), the culture(s) were re-fed with fresh medium and re-incubated for an additional 2 to 3 days
Mutant Selection
Hypoxanthine-free Complete Ham’s F12 medium (Complete Ham’s F12 medium -Hx) was used for mutant selection and to determine cloning efficiency at the time of selection. At the end of the phenotypic expression period, 2.4E6 cells from each culture were plated at a density of 6E5 cells/150-mm plate (4 plates total) in 30 mL Complete Ham’s F12 -Hx containing 10 μM TG. Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Complete Ham’s F12 -Hx in triplicate, to determine the cloning efficiency at the time of selection. The plates were incubated under standard conditions for 7 days.
After the 7-day incubation period, the colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/E6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The average absolute cloning efficiency of the vehicle controls must be >60% (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle controls should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fell outside the 95% control limits, they may be acceptable as long as these data were not extreme outliers (indicative of experimental or human error). Spontaneous mutant frequencies were calculated separately for cultures with and without S9.
The positive controls must induce a significant increase in mutant frequency as compared to the concurrent vehicle controls (p≤ 0.01). A significant increase in the absence of S9 indicated the test system could identify a mutagen, while a significant increase in the presence of S9 was considered to have demonstrated the integrity of the S9 mix as well as the ability of the test system to detect a mutagen.
The highest concentration evaluated was the limit dose for this assay (2000 μg/mL or 10 mM), or must have induced 10 to 20% adjusted relative survival, or must be the highest concentration able to be prepared in the vehicle and administered (whichever is lowest). If increasing cytotoxicity was observed at precipitating concentrations, cytotoxicity was the determining factor. This latter requirement was waived if the highest concentration with acceptable cytotoxicity (>10% adjusted relative survival) was at least 75% of an excessively toxic concentration (cultures with adjusted relative survivals <10% were excluded from evaluation as excessively cytotoxic). There was no maximum concentration or toxicity requirement for test substances which clearly showed mutagenic activity.
A minimum of four acceptable concentrations was required for a valid assay. Fewer concentrations may be justified for test substances which clearly show mutagenic activity. - Statistics:
- Statistical analyses were performed using the method of Snee and Irr, with significance established at the 0.05 level.
Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows.
The test substance was considered to have produced a positive response if it induced a dose-dependent increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no adverse impact on pH
- Effects of osmolality: no adverse impact on osmolality of cultures [280 mmol/kg for the vehicle control and 268 mmol/kg for the highest concentration (681 μg/mL)]
- Evaporation from medium: not specified
- Precipitation: No visible precipitate was observed at the beginning or end of treatment
RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the concentrations tested were 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL. The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476. The test substance formed clear solutions in water from 0.0133 to 6.81 mg/mL. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures [280 mmol/kg for the vehicle control and 268 mmol/kg for the highest concentration (681 μg/mL). Adjusted relative survival was 64.89 and 105.47% at a concentration of 681 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 42.6, 85.1, 170, 341 and 681 μg/mL with and without S9.
HISTORICAL CONTROL DATA
- Positive historical control data:
0.2 μL/mL EMS (-S9 treatment) - mean: 259.7, st. dev: 135.1, 95% Control Limit: 0.0-529.9, Obs Range: 79.2-764.2
4.0 μL/mL B(a)P (+S9 treatment) - mean: 154.4, st. dev: 73.3, 95% Control Limit: 7.8-301.0, Obs Range: 34.9-314.5
- Negative (solvent/vehicle) historical control data: includes water and DMSO
-S9 treatment - mean: 4, st. dev: 3.3, 95% Control Limit: 0.0-10.6, Obs Range: 0.0-12.3
+S9 treatment - mean: 4.2, st. dev: 3.4, 95% Control Limit: 0.0-11.0, Obs Range: 0.0-14.0
The average adjusted relative survival was 100.47 and 94.52% at a concentration of 681 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. A statistically significant increase in mutant frequency, as compared to the concurrent vehicle control, was observed at a concentration of 85.1 μg/mL without S9 (p < 0.01). However, this increase was not dose-dependent, the mutant frequency for the vehicle control was on the lower end of the 95% control limit and the average mutant frequency at this test substance concentration was within the 95% control limit. Therefore, this increase was not biologically significant and considered spurious. No statistically significant or biologically relevant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at the remaining concentrations evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01).
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
Any other information on results incl. tables
Table 1: Summary of Results for Mutagenicity Assay -S9
Treatment | Dose (μg/mL) | S9 | Cells (x 10^6) | Cloning Efficiency | Relative Survival (adj., %) | TGr Mutants/Plate | Total Mutant Colonies | Cloning Efficiency | Mutant Frequency (x 10^6) | ||||||||||
(Colonies/Plate) | % | (Colonies/Plate) | % | Individual | Average | ||||||||||||||
Water | 0 | - | 0.577 | 139 | 139 | 137 | 69.17 | 94.86 | 1 | 1 | 0 | 0 | 2 | 191 | 203 | 217 | 101.83 | 0.82 | 0.65 |
Water | 0 | - | 0.609 | 158 | 153 | 149 | 76.67 | 105.14 | 0 | 0 | 0 | 1 | 1 | 166 | 190 | 167 | 87.17 | 0.48 | |
Test Substance | 42.6 | - | 0.715 | 131 | 138 | 132 | 66.83 | 91.66 | 3 | 2 | 4 | 1 | 10 | 140 | 163 | 163 | 77.67 | 5.36 | 6.94 |
42.6 | - | 0.688 | 171 | 160 | 146 | 79.50 | 109.03 | 7 | 3 | 5 | 2 | 17 | 182 | 162 | 155 | 83.17 | 8.52 | ||
85.1 | - | 0.716 | 169 | 192 | 153 | 85.67 | 117.49 | 3 | 6 | 4 | 6 | 19 | 154 | 162 | 137 | 75.50 | 10.49 | 9.99** | |
85.1 | - | 0.655 | 191 | 179 | 196 | 94.33 | 129.37 | 3 | 2 | 1 | 7 | 13 | 110 | 102 | 131 | 57.17 | 9.48 | ||
170 | - | 0.649 | 184 | 191 | 177 | 92.00 | 126.17 | 0 | 1 | 1 | 1 | 3 | 152 | 154 | 166 | 78.67 | 1.59 | 3.37 | |
170 | - | 0.707 | 141 | 140 | 132 | 68.83 | 94.40 | 4 | 2 | 2 | 1 | 9 | 141 | 145 | 152 | 73.00 | 5.14 | ||
341 | - | 0.653 | 166 | 179 | 177 | 87.00 | 119.31 | 2 | 7 | 7 | 7 | 23 | 141 | 120 | 147 | 68.00 | 14.09 | 8.9 | |
341 | - | 0.639 | 168 | 150 | 177 | 82.50 | 113.14 | 2 | 0 | 3 | 1 | 6 | 144 | 128 | 132 | 67.33 | 3.71 | ||
681 | - | 0.636 | 132 | 118 | 134 | 64.00 | 87.77 | 2 | 5 | 2 | 5 | 14 | 160 | 158 | 159 | 79.50 | 7.34 | 5.24 | |
681 | - | 0.65 | 133 | 152 | 158 | 73.83 | 101.26 | 1 | 1 | 2 | 1 | 5 | 130 | 133 | 135 | 66.33 | 3.14 | ||
EMS | 0.2a | - | 0.709 | 109 | 116 | 122 | 57.83 | 79.31 | 101 | 100 | 88 | 112 | 401 | C | 141 | 134 | 68.75 | 243.03 | 260.2** |
EMS | 0.2a | - | 0.679 | 103 | 103 | 97 | 50.50 | 69.26 | 134 | 102 | 104 | 116 | 456 | C | 134 | 140 | 68.50 | 277.37 | |
a: μL/mL | |||||||||||||||||||
Water aliquot volume: 100 μL/mL | |||||||||||||||||||
C = Contaminated | |||||||||||||||||||
** p<0.01 compared to the vehicle control (T-Test) |
Table 2: Summary of Results for Mutagenicity Assay +S9
Treatment | Dose (μg/mL) | S9 | Cells (x 10^6) | Cloning Efficiency | Relative Survival (adj., %) | TGr Mutants/Plate | Total Mutant Colonies | Cloning Efficiency | Mutant Frequency (x 10^6) | ||||||||||
(Colonies/Plate) | % | (Colonies/Plate) | % | Individual | Average | ||||||||||||||
Water | 0 | + | 0.514 | 169 | 182 | 187 | 89.67 | 101.13 | 0 | 0 | 0 | 0 | 0 | 179 | 196 | 176 | 91.83 | 0.00 | 3.66 |
Water | 0 | + | 0.626 | 169 | 192 | 165 | 87.67 | 98.87 | 3 | 3 | 4 | 5 | 15 | 186 | 148 | 179 | 85.50 | 7.31 | |
Test Substance | 42.6 | + | 0.726 | 187 | 145 | 179 | 85.17 | 96.05 | 3 | 10 | 4 | 3 | 20 | 197 | 168 | 205 | 95.00 | 8.77 | 9.15 |
42.6 | + | 0.886 | 90 | 115 | 99 | 50.67 | 57.14 | 8 | 2 | 5 | 1 | 16 | 132 | 148 | 140 | 70.00 | 9.52 | ||
85.1 | + | 0.647 | 164 | 176 | 170 | 85.00 | 95.86 | 2 | 4 | 2 | 4 | 12 | 203 | 207 | 202 | 102.00 | 4.90 | 5.39 | |
85.1 | + | 0.637 | 170 | 188 | 162 | 86.67 | 97.74 | 3 | 2 | 4 | 3 | 12 | 179 | 172 | 159 | 85.00 | 5.88 | ||
170 | + | 0.543 | 203 | 210 | 221 | 105.67 | 119.17 | 1 | 0 | 1 | 0 | 2 | 147 | 109 | 142 | 66.33 | 1.26 | 3.90 | |
170 | + | 0.668 | 195 | 170 | 175 | 90.00 | 101.50 | 4 | 3 | 3 | 3 | 13 | 172 | 152 | 174 | 83.00 | 6.53 | ||
341 | + | 0.646 | 185 | 201 | 221 | 101.17 | 114.10 | 1 | 1 | 2 | 3 | 7 | 172 | 180 | 174 | 87.67 | 3.33 | 8.25 | |
341 | + | 0.505 | 166 | 166 | 178 | 85.00 | 95.86 | 8 | 9 | 3 | 8 | 28 | 181 | 177 | 174 | 88.67 | 13.16 | ||
681 | + | 0.528 | 179 | 176 | 189 | 90.67 | 102.26 | 0 | 3 | 2 | 2 | 7 | 214 | 212 | 195 | 103.50 | 2.82 | 6.81 | |
681 | + | 0.539 | 192 | 165 | 168 | 87.50 | 98.68 | 7 | 5 | 5 | 6 | 23 | 190 | 187 | 156 | 88.83 | 10.79 | ||
EMS | 0.2a | + | 0.604 | 72 | 95 | 74 | 40.17 | 45.30 | 70 | 71 | 52 | 79 | 272 | 137 | 135 | 151 | 70.50 | 160.76 | 170.54** |
EMS | 0.2a | + | 0.579 | 77 | 77 | 90 | 40.67 | 45.86 | 38 | 72 | 92 | 93 | 295 | 146 | 135 | 128 | 68.17 | 180.32 | |
a: μL/mL | |||||||||||||||||||
Water aliquot volume: 100 μL/mL | |||||||||||||||||||
** p<0.01 compared to the vehicle control (T-Test) |
Applicant's summary and conclusion
- Conclusions:
- The test substance was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay.
- Executive summary:
The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) in accordance with OECD Guideline 476. The test substance was formulated in water and formed a clear solution at 6.81 mg/mL, the highest stock concentration used in the study.
In the preliminary toxicity assay, the concentrations tested were 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL. The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures. Adjusted relative survival was 64.89 and 105.47% at a concentration of 681 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 42.6, 85.1, 170, 341 and 681 μg/mL with and without S9.
In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 100.47 and 94.52% at a concentration of 681 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. A statistically significant increase in mutant frequency, as compared to the concurrent vehicle control, was observed at a concentration of 85.1 μg/mL without S9 (p < 0.01). However, this increase was not dose-dependent, the mutant frequency for the vehicle control was on the lower end of the 95% control limit and the average mutant frequency at this test substance concentration was within the 95% control limit. Therefore, this increase was not biologically significant and considered spurious. No statistically significant or biologically relevant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at the remaining concentrations evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
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