(1-[2-[5-METHYL-3-(TRIFLUOROMETHYL)-1H-PYRAZOL-1-YL]ACETYL]-4-HETEROMONOCYCLECARBOTHIOAMIDE)

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:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

yes

Remarks:

The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Japan 59-Nousan-4200, Testing Guidelines for Toxicity Studies

Deviations:

yes

Remarks:

The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible

GLP compliance:

yes

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT Assay)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Purity: 93.6%

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: Nine concentrations of the test substance ranging from 0.5 to 3340 μg/mL (approximately 10 mM) in the presence and absence of S9 reaction mixture.
The test substance formed clear solutions in DMSO from 0.05 to 334 mg/mL. Visible precipitate was observed in the treatment medium at concentrations ≥ 1500 μg/mL at the beginning and end of treatment. Cloning efficiency relative to the solvent control (relative cloning efficiency) at 3340 μg/mL was 98% in the absence of S9 activation and 101% in the presence of S9 activation. Selection of concentrations for the mutagenesis assay was based on the precipitate profile observed in the preliminary toxicity assay.
Mutagenesis Assays: Based on the preliminary toxicity assay, the concentrations chosen for the mutagenesis assay ranged from 250 to 1500 μg/mL for both the non-activated and S9-activated cultures.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on solubility information provided by the Sponsor and compatibility with the target cells. The Sponsor indicated that the test substance is soluble in DMSO at a concentration of 334 mg/mL.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Remarks:

BaP for S9-activated test system. EMS for non-activated test system.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The mutagenesis assay was performed according to a protocol developed from published methodologies (Hsie et al., 1981 and O'Neill et al., 1977). Treatment flasks were identified with the test substance number and a code to designate the treatment condition and test phase. Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5+Hx at a density of 1x1000000 cells/25 cm2 flask and were incubated at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air for 18-24 hours. F12FBS5+Hx is Ham's F12 medium with hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 μg streptomycin/mL and 2 mM L-glutamine/mL
- Cell density at seeding (if applicable): 1x1000000 cells/25 cm²

DURATION
- Preincubation period: 18-24 hours
- Exposure duration: 5±0.5 hours at 37±1ºC
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 7 to 10 days

SELECTION AGENT (mutation assays): For selection of the 6-thioguanine (TG, 2-amino-6-mercaptopurine)-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x100000 cells/100 mm dish in F12FBS5 without Hypoxanthine (F12FBS5-Hx) containing 10 μM TG.

NUMBER OF REPLICATIONS: Mutagenesis assay: Duplicates, Cytotoxicity: Triplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were fixed with methanol, stained with 0.05% to 0.06% Crystal Violet, counted and cloning efficiency determined

NUMBER OF CELLS EVALUATED: 2X100000 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: Relative cloning efficiency
- Any supplementary information relevant to cytotoxicity: The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2x1000000 cells: 10 plates at 2x100000 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 1000000 clonable cells.

References:
Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O'Neill, and B.L.Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. A report of the Gene-Tox Program, Mutation Research 86:193-214.
O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Research 45:91-101.

Evaluation criteria:

All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data:
The test material was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of >40 mutants per 1000000 clonable cells.
If a single point above 40 mutants per 1000000 clonable cells was observed at the highest dose, the assay was considered equivocal.
If no culture exhibited a mutant frequency of >40 mutants per 1000000 clonable cells, the test substance was considered negative.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No positive responses

Any other information on results incl. tables

Table 1: Preliminary Toxicity Assay using IN-QEU76
Treatment (µg/mL)	Non-Activated					S9-Activated
	Plate counts			Cloning Efficiency (%)	Relative Cloning Efficiency (%)	Plate counts			Cloning Efficiency (%)	Relative Cloning Efficiency (%)
	1	2	3			1	2	3
Solvent	126	118	117	60	100	149	139	140	71	100
0.5	132	164	144	73	122	149	132	153	72	101
1.5	94	91	83	45	74	129	118	90	56	79
5	124	110	123	60	99	137	134	145	69	97
15	118	103	113	56	93	139	149	165	76	106
50	125	124	134	64	106	184	179	147	85	119
150	114	140	132	64	107	153	178	167	83	116
500	98	112	112	54	89	147	152	138	73	102
1500 P	173	144	154	79	130	150	167	152	78	110
3340 P	121	116	117	59	98	139	156	137	72	101
Solvent = DMSO, P – Precipitating concentration
Cloning efficiency = average colonies divided by 200 cells per dish X 100 Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 2: Concurrent Cytotoxicity Test using IN-QEU76
Treatment (µg/mL)	Plate counts			Cloning Efficiency (%)	Relative Cloning Efficiency (%)
	1	2	3
Non-Activated Cultures
Solvent A	160	143	166	78	100
Solvent B	*	156	156
250 A	151	157	160	73	93
250 B	131	131	144
500 A	124	157	132	72	93
500 B	144	156	154
750 A	151	156	156	74	95
750 B	140	141	147
1000 A	123	143	129	67	86
1000 B	146	126	138
1500 A (P)	143	149	139	74	94
1500 B (P)	154	152	146
EMS (0.2μL/mL) A	106	72	88	45	58
EMS (0.2μL/mL) B	100	86	87
Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration, * - Plate lost due to contamination
Cloning efficiency = average colonies divided by 200 cells per dish X 100 Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 2 Cont.: Concurrent Cytotoxicity Test using IN-QEU76
Treatment (µg/mL)	Plate counts			Cloning Efficiency (%)	Relative Cloning Efficiency (%)
	1	2	3
S9-Activated Cultures
Solvent A	137	150	138	82	100
Solvent B	170	198	193
250 A	172	184	147	83	102
250 B	169	177	152
500 A	156	158	160	84	102
500 B	184	183	163
750 A	169	185	186	86	104
750 B	157	164	166
1000 A	162	167	183	82	100
1000 B	160	167	145
1500 A (P)	179	196	172	86	104
1500 B (P)	146	171	165
B(a)P (4μL/mL) A	94	98	107	45	55
B(a)P (4μL/mL) B	81	88	74
Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration
Cloning efficiency = average colonies divided by 200 cells per dish X 100 Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 3: Non-activated (-S9) Study using IN-QEU76
Treatment (μg/mL)	Cloning Efficiency Plates					Cloning Efficiency	Selection (Mutation) Plates					Average Colonies	Mutants/106 Clonable Cells	Relative Cloning Efficiency (%)
	Plate Counts				Average Colonies		Plate Counts
	1		2	3			1	2	3	4	5
Solvent A	131	154		129	139.8	0.7	0	0	0	0	0	0.1	0.7	100
Solvent B	149	150		126			0	1	0	0	0
250 A	165	165		153	152.5	0.76	0	0	0	0	0	0.1	0.7	93
250 B	136	145		151			0	0	1	0	0
500 A	143	116		157	142.7	0.71	0	0	1	0	0	0.5	3.5	93
500 B	146	149		145			0	2	0	0	2
750 A	162	139		171	150.8	0.75	0	0	0	0	0	0	0	95
750 B	154	136		143			0	0	0	0	0
1000 A	156	159		130	157.7	0.79	2	0	0	0	0	0.3	1.9	86
1000 B	170	165		166			0	0	0	0	1
1500 A (P)	124	150		132	136.2	0.68	0	0	0	0	0	0.1	0.7	94
1500 B (P)	136	132		143			1	0	0	0	0
EMS (0.2μL/mL) A	105	99		108	108.5	0.54	50	39	52	62	44	45.5	419.4	58
EMS (0.2μL/mL) B	121	113		105			31	44	39	37	57
Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration
Cloning efficiency = average colonies divided by 200 cells per dish X 100 Mutants/106 clonable cells = average mutant colonies divided by cloning efficiency x 2 x 105 cells X 106

Table 4: Activated (+S9) Study using IN-QEU76
Treatment (μg/mL)	Cloning Efficiency Plates				Cloning Efficiency	Selection (Mutation) Plates					Average Colonies	Mutants/106 Clonable Cells	Relative Cloning Efficiency (%)
	Plate Counts			Average Colonies		Plate Counts
	1	2	3			1	2	3	4	5
Solvent A	132	162	191	156.2	0.78	0	1	1	0	0	1.1	7.0	100
Solvent B	139	153	160			5	3	0	1	0
250 A	180	186	187	186.8	0.93	0	0	0	0	0	0.5	2.7	102
250 B	204	210	154			2	0	0	2	1
500 A	176	163	175	174.8	0.87	2	0	1	2	0	0.6	3.4	102
500 B	166	171	198			0	0	0	1	0
750 A	175	192	160	172.5	0.86	0	2	2	5	5	2.0	11.6	104
750 B	166	171	171			2	1	3	0	0
1000 A	146	167	178	156.3	0.78	1	3	2	2	1	0.9	5.8	100
1000 B	144	141	162			0	0	0	0	0
1500 A (P)	177	156	191	169.2	0.85	1	1	0	0	1	0.4	2.4	104
1500 B (P)	149	167	175			0	0	1	0	0
B(a)P (4μg/mL) A	136	149	123	123.7	0.62	40	33	54	29	37	35.7	288.7	55
B(a)P (4μg/mL) B	90	121	125			29	36	28	40	31
Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration
Cloning efficiency = average colonies divided by 200 cells per dish X 100 Mutants/106 clonable cells = average mutant colonies divided by cloning efficiency x 2 x 105 cells X 106

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. Under the conditions of this study, the test material did not cause a dose-responsive increase in mutant frequency in the non-activated or S9-activated test systems in the CHO/HGPRT Mutation Assay. No positive responses were observed in either the presence or absence of S9 activation. Therefore, the test substance was concluded to be negative in this assay.

Executive summary:

The test material was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test substance. Dosing formulations were adjusted to compensate for the purity of the test substance (93.6%) using a correction factor of 1.07. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice.

The maximum concentration of the test substance in treatment medium was 3340 μg/mL (approximately 10 mM) in the preliminary toxicity assay. The test substance formed clear solutions in DMSO from 0.05 to 334 mg/mL. Visible precipitate was observed in the treatment medium at concentrations ≥ 1500 μg/mL at the beginning and end of treatment. No substantial toxicity (i.e., relative cloning efficiency) was observed at any concentration in the presence or absence of S9 activation.

The concentrations chosen for the mutagenesis assay ranged from 250 to 1500 μg/mL for both the non-activated and S9-activated cultures. Visible precipitate was observed in the treatment medium at a concentration of 1500 μg/mL at the beginning and end of treatment. No positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 10⁶ clonable cells, were observed in either the presence or absence of S9 activation. No substantial toxicity (i.e., relative cloning efficiency) was observed at any concentration in the presence or absence of S9 activation.

All criteria for a valid study were met. Under the conditions of this study, the test material did not cause a dose-responsive increase in mutant frequency in the non-activated or S9-activated test systems in the CHO/HGPRT Mutation Assay. No positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 10⁶ clonable cells, were observed in either the presence or absence of S9 activation. Therefore, the test substance was concluded to be negative in this assay.