1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 24 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-1,2,3,4-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

EC Number:

810-161-6

Cas Number:

1229654-66-3

Molecular formula:

C22 H16 Cl F3 N10 O2

IUPAC Name:

1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-1,2,3,4-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was chosen as maximal concentration since minor toxic effects were observed in the pre-test/experiment I.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent DMSO was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Remarks:

+ S9: 2-AA (2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100; 10 µg/plate in TA 102) - S9: sodium azide (10 µg/plate in TA 1535, TA 100), 4-NOPD (10 µg/plate in TA 98; 50 µg/plate in TA 1537), methylmethanesulfonate (2.0 µL/plate in TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertants

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-experiment/experiment 1 the test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 μg/plate. In experiment II precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 2500 to 5000 μg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: A pre-test (experiment I, plate incorporation method) was performed with all strains used to evaluate the toxicity of the test item and to choose a concentration range for experiment II. The test substance was tested with and without metabolic activation at eight concentrations ranging from 3 - 5000 µg/plate. A reduction in the number was observed in one strain only (TA 1537) at 2500 and 5000 µg/plate (precipitating concentrations) in the absence of metabolic activation. Thus, the same concentrations with the exception of 3 µg/plate were used in experiment II.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1).
- Negative (solvent/vehicle) historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1).

Any other information on results incl. tables

Table 1 Historical control data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	14	2.37	9	23	20	3.75	11	35
	Untreated control	14	2.87	7	25	20	3.82	10	32
	Positive control	1751	226.44	710	2385	367	93.12	126	703
TA 1537	Solvent control	12	3.31	5	26	16	4.34	7	30
	Untreated control	12	4.03	5	29	18	4.92	6	33
	Positive control	88	38.92	61	448	342	144.31	77	809
TA 98	Solvent control	30	5.60	17	47	40	6.08	21	58
	Untreated control	31	6.36	17	55	42	6.83	24	68
	Positive control	372	78.05	158	595	2167	717.60	249	4089
TA 100	Solvent control	142	29.42	86	243	156	29.4	99	249
	Untreated control	450	28.19	86	248	163	31.26	94	281
	Positive control	1741	488.75	569	3082	2642	796.59	825	4503
TA 102	Solvent control	380	43.64	305	510	502	89.88	321	677
	Untreated control	372	41.71	300	479	511	87.27	315	659
	Positive control	2499	898.06	1080	4678	2329	598.00	1091	3972

Mean = mean value of revertants/plate; SD = standard deviation; Min = minimal value; Max = maximal value

Table 2. Test results of the pre-experiment/experiment I (plate incorporation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
-	0 (DMSO)	13 ± 3	10 ± 4	33 ± 3	88 ± 4	355 ± 22
-	0	14 ± 1	11 ± 2	37 ± 6	98 ± 6	329 ± 24
-	3	15 ± 2	10 ± 5	34 ± 6	83 ± 2	325 ± 49
-	10	11 ± 2	7 ± 2	30 ± 6	85 ± 4	366 ± 29
-	33	12 ± 4	9 ± 1	24 ± 3	90 ± 6	366 ± 19
-	100	16 ± 5	8 ± 0	35 ± 9	89 ± 23	311 ± 29
-	333	13 ± 3	7 ± 2	30 ± 1	87 ± 12	317 ± 9
-	1000	13 ± 8^P	8 ± 2^P	27 ± 6^P	90 ± 23^P	316 ± 2^P
-	2500	11 ± 2^P,M	4 ± 2^P,M	28 ± 4^P,M	94 ± 9^P,M	224 ± 27^P,M
-	5000	9 ± 2^P,M	3 ± 1^P,M	21 ± 1^P,M	92 ± 12^P,M	172 ± 9^P,M
Positive controls, - S9	Na-azide, 10 µg/plate	1913 ± 54			1949 ± 122
	4-NOPD, 10 µg/plate			254 ± 69
	4-NOPD, 50 µg/plate		72 ± 6
	MMS, 2.0 µL/plate					3681 ± 508
+	0 (DMSO)	20 ± 3	18 ± 4	29 ± 6^B,M	137 ± 10	484 ± 36
+	0	16 ± 5	16 ± 1	36 ± 6^B,M	147 ± 10	533 ± 17
+	3	19 ± 6	18 ± 3	27 ± 5^B,M	131 ± 10	460 ± 34
+	10	16 ± 4	16 ± 4	26 ± 6^B,M	137 ± 11	494 ± 26
+	33	21 ± 4	17 ± 5	23 ± 3^B,M	126 ± 8	512 ± 65
+	100	18 ± 7	15 ± 2	28 ± 4^B,M	140 ± 3	459 ± 47
+	333	17 ± 4	21 ± 2	29 ± 2^B,M	132 ± 11	439 ± 9
+	1000	18 ± 4	12 ± 3	31 ± 7^B,M	111 ± 8	485 ± 39
+	2500	16 ± 3^P	10 ± 1^P,M	31 ± 4^B,P,M	115 ± 11^P	514 ± 11^P
+	5000	12 ± 2^P,M	10 ±3^P,M	33 ± 8^B,P,M	119 ± 6^P,M	410 ± 1^P,M
Positive controls, + S9	2-AA, 2.5 µg/plate	414 ± 23	373 ± 35	1905 ± 100^B,M	3106 ± 300
	2-AA, 10 µg/plate					3158 ± 70

Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

P = precipitate; M = manual count, B = extensive bacterial growth

Table 3. Test results of the experiment II (preincubation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
-	0 (DMSO)	14 ± 5	9 ± 0	25 ± 9	90 ± 3	363 ± 4
-	0	15 ± 4	8 ± 4	32 ± 12	98 ± 5	384 ± 19
-	10	12 ± 6	9 ± 2	25 ± 7	101 ± 15	391 ± 18
-	33	13 ± 3	8 ± 0	25 ± 4	81 ± 3	381 ± 20
-	100	11 ± 3	9 ± 1	25 ± 3	98 ± 12	359 ± 27
-	333	13 ± 5	9 ± 3	28 ± 2	85 ± 7	368 ± 34
-	1000	12 ± 2	8 ± 1	22 ± 7	98 ± 19	398 ± 22
-	2500	14 ± 1^P	8 ± 2^P,M	24 ± 6^P	87 ± 11^P	399 ± 10^P
-	5000	11 ± 3^P,M	5 ± 3^P,M	20 ± 2^P,M	89 ± 11^P,M	330 ± 42^P,M
Positive controls, - S9	Na-azide, 10 µg/plate	1939 ± 64			2144 ± 26
	4-NOPD, 10 µg/plate			342 ± 28
	4-NOPD, 50 µg/plate		68 ± 7
	MMS, 2.0 µL/plate					3761 ± 15
+	0 (DMSO)	20 ± 3	18 ± 4	44 ± 8	129 ± 13	522 ± 27
+	0	18 ± 3	20 ± 5	45 ± 3	152 ± 10	493 ± 6
+	10	20 ± 4	17 ± 3	42 ± 5	133 ± 17	522 ± 25
+	33	22 ± 9	15 ± 7	37 ± 3	137 ± 21	527 ± 37
+	100	18 ± 4	19 ± 6	47 ± 15	137 ± 11	546 ± 63
+	333	19 ± 6	21 ± 2	41 ± 6	142 ± 14	476 ± 29
+	1000	23 ± 3	17 ± 2	50 ± 2	127 ± 9	509 ± 19
+	2500	20 ± 3^P	15 ± 5^P	61 ± 4^P	124 ± 4^P	557 ± 22^P
+	5000	13 ± 5^P,M	12 ± 4^P,M	46 ± 8^P,M	127 ± 9^P,M	534 ± 23^P,M
Positive controls, + S9	2-AA, 2.5 µg/plate	415 ± 40	269 ± 8	2314 ± 458	3638 ± 20
	2-AA, 10 µg/plate					2217 ± 259

Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

P = precipitate; M = manual count