1-(3-Chloro-2-pyridinyl)-4,5-dihydro-3-[(phenylsulphonyl)oxy]-1H-pyrazole-5-carboxylic acid ethyl ester

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:

October 2006 - December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

EC Number:

846-153-4

Cas Number:

653592-41-7

Molecular formula:

C17H16ClN3O5S

IUPAC Name:

ethyl 3-[(benzenesulfonyl)oxy]-1-(3-chloropyridin-2-yl)-4,5-dihydro-1H-pyrazole-5-carboxylate

Test material form:

solid

Method

Target gene:

histidine operon (for S. typhimurium) and tryptophan operon (for E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate, S9, prepared from male Sprague-Dawley rats induced with Aroclor 1254. 10% S9 mix prepared immediately prior to its use.

Test concentrations with justification for top dose:

First experiment (toxicity-mutation):
33.3, 66.7, 100, 333, 667, 1000, 3333, 5000 µg/plate with and without metabolic activation

Second experiment (mutagenicity):
333, 667, 1000, 3333, 5000 µg/plate with and without metabolic activation

Based on the results from the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate.

Vehicle / solvent:

- Vehicle/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest concentration that was tested in the study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthrazene: 2.5 μg/plate for TA100 (+S9), TA1535 (+ S9), TA1537 (+ S9), 25.0 μg/plate for WP2uvrA (+ S9); Acridine mutagen ICR-191: 2.0 μg/plate for TA1537 (- S9); 4-nitroquinoline-N-oxide: 1.0 μg/plate for WP2uvrA (- S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION: Exposure duration: 48 h

NUMBER OF REPLICATIONS:
All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate.
All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.

METHOD FOR MEASUREMENT OF CYTOTOXICITY: Inspection of the bacterial background lawn

OTHER EXAMINATIONS:
The presence of precipitation of the test compound on the plates was assessed by visual examination.
Aliquots of the vehicle control and each test substance concentration were taken to confirm dose concentrations, and stability. Data from the analysis of the samples during the study indicate that the test substance was at the targeted concentrations and stable under the conditions of the study. Test substance was not found in the 0 mg/mL sample.

Evaluation criteria:

Revertant colonies for a given tester strain and condition were counted by an automated colony counter. Plates that could not be counted automatically were counted by hand.
A test substance was classified as positive when the mean number of revertants in any strain except TA1535 and TA1537 and at any test substance concentration was at least 2 times greater than the mean number of revertants in the concurrent negative control and occurred in a positive dose-response relationship. For strains TA1535 and TA1537, a mean number of revertants of at least 3 times greater than negative control was needed to be considered a positive response.

Statistics:

Data for each tester strain were evaluated independently. For each tester strain, the mean number of revertants and the standard deviation at each concentration in the presence of and absence of exogenous metabolic activation system were calculated. No further statistical analyses were conducted.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Test substance precipitation was observed starting at 1000 μg/plate in the non activated test system and starting at 3333 μg/plate in the activated testing system.

In the initial toxicity-mutation test a >50% reduction in mean number of revertants was observed at 3333 μg/plate for TA1537 without S9 activation; however, this reduction occurred at an intermediate dose level with no dose related correlation. In the confirmatory mutagenicity test a >50% reduction in mean number of revertants was observed at 333 μg/plate for TA1537 with S9 activation; however, this reduction occurred at a low dose level with no dose related correlation.

Any other information on results incl. tables

Table 1: Summary of average revertants/plate without activation

Compound	Conc. μg/plate	TA98		TA100		TA1535		TA1537		WP2 uvrA
		Trial I	Trial II	Trial I	Trial II	Trial I	Trial II	Trial I	Trial II	Trial I	Trial II
Test item	0	16	18	105	95	11	13	10	7	25	36
	33,3	17	-	112	-	9	-	13	-	18	-
	66,7	19	-	109	-	9	-	9	-	31	-
	100	22	-	107	-	13	-	10	-	29	-
	333	26	17	101	113	12	13	9	10	34	34
	667	15	18	108	109	7	14	10	5	30	35
	1000	24	15	111	101	10	13	9	13	36	32
	3333	14	18	108	111	11	12	4	7	42	40
	5000	19	12	104	115	17	12	7	7	36	42
NAAZ	2,0	-	-	883	866	979	75	-	-	-	-
ICR-191	2,0	-	-	-	-	-	-	2285	1561	-	-
2NF	1,0	99	131	-	-	-	-	-	-	-	-
4NQ	1,0	-	-	-	-	-	-	-	-	366	669

Trial I – An average of 2 replicates per dose level
Trial II – An average of 3 replicates per dose level
- = Not evaluated
2NF = 2-nitrofluorene; NAAZ = sodium azide; ICR-191 = acridine mutagen ICR-191; 4NQ= 4-nitroquinoline N-oxide

 

Table 2: Summary of average revertants/plate with activation

Compound	Conc. μg/plate	TA98		TA100		TA1535		TA1537		WP2 uvrA
		Trial I	Trial II	Trial I	Trial II	Trial I	Trial II	Trial I	Trial II	Trial I	Trial II
Test item	0	19	22	130	119	9	12	8	12	40	36
	33,3	31	-	128	-	15	-	8	-	52	-
	66,7	32	-	134	-	8	-	7	-	41	-
	100	33	-	125	-	16	-	10	-	41	-
	333	28	25	109	135	9	13	9	5	39	63
	667	27	18	130	121	12	15	10	8	40	46
	1000	28	32	134	123	14	15	10	11	41	56
	3333	27	26	132	126	15	9	11	7	47	58
	5000	25	22	136	127	9	10	9	10	41	43
2AA	2,5	-	-	2678	2987	208	218	186	292	-	-
	25	-	-	-	-	-	-	-	-	330	366
B[a]P	2,5	464	287	-	-	-	-	-	-	-	-

Trial I – An average of 2 replicates per dose level
Trial II – An average of 3 replicates per dose level
b = No colonies on plates due to test substance toxicity
- = Not evaluated
2AA = 2-aminoantracene; B(a)P = benzo[a]pyrene

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test item was negative for mutagenic activity in non-activated and S9-activated test systems.

Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse MutationTest using the plate incorporation method according to OECD Guideline 471.

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and in Escherichia coli strain WP2 uvrA, with and without an exogenous metabolic activation system (Aroclor-induced rat liver S9). In the initial toxicity-mutation test, dose levels of 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate were evaluated using standard plate incorporation methods. In the confirmatory mutagenicity test, dose levels of 333, 667, 1000, 3333 and 5000 μg/plate were evaluated. The highest dose level was set based on the solubility of the test substance, and the limit dose for this test system (OECD 471). The test substance was administered to the test system as a solution in dimethyl sulfoxide (DMSO) at a maximum concentration of 50 mg/mL.
The number of revertants at all concentrations of the test substance was similar to concurrent controls in trials both with and without activation. No toxicity was observed at any dose level with any tester strain in either the absence or the presence of S9 activation. In the initial toxicity-mutation test a >50% reduction in mean number of revertants was observed at 3333 μg/plate for TA1537 without S9 activation; however, this reduction occurred at an intermediate dose level with no dose related correlation. In the confirmatory mutagenicity test a >50% reduction in mean number of revertants was observed at 333 μg/plate for TA1537 with S9 activation; however, this reduction occurred at a low dose level with no dose related correlation. Test substance precipitation was observed starting at 1000 μg/plate in the non-activated test system and starting at 3333 μg/plate in the activated testing system.
Under the conditions of this study, the test item was negative for mutagenic activity in non-activated and S9-activated test systems.