Pyrazole

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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%

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.