2-(3-Chloro-2-pyridinyl)-5-oxo-3-pyrazolidinecarboxylic 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:

June 2006 - August 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)

Version / remarks:

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 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-carboxylate

EC Number:

610-490-3

Cas Number:

500011-88-1

Molecular formula:

C11H12ClN3O3

IUPAC Name:

ethyl 2-(3-chloropyridin-2-yl)-5-oxopyrazolidine-3-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 (S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA)

Second experiment (mutagenicity):
333, 667, 1000, 3333, 5000 µg/plate with metabolic activation (S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA)
333, 667, 1000, 3333, 5000 µg/plate without metabolic activation (TA98 and WP2uvrA)
100, 333, 667, 1000, and 3333 μg/plate without activation (TA100, TA1535, and TA1537)

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-aminoanthracene

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.

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:

cytotoxicity

Remarks:

starting at 3333 μg/plate (- S9) and at 5000 μg/plate (+ S9)

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

starting at 3333 μg/plate (- S9) and at 5000 μg/plate (+ S9)

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

starting at 3333 μg/plate (- S9) and at 5000 μg/plate (+ S9)

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

starting at 3333 μg/plate (- S9)

Vehicle controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

- Precipitation: no test substance precipitation was found

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	14	17	91	112	15	14	8	7	31	29
	33,3	23	-	96	-	12	-	7	-	27	-
	66,7	15	-	96	-	13	-	7	-	25	-
	100	20	-	102	116	13	10	7	10	32	-
	333	19	21	105	127	12	11	4	9	32	30
	667	17	16	101	118	11	12	5	7	34	29
	1000	14	15	102	137	11	10	3	7	29	25 (a)
	3333	7	8	2	12	2	1	2	0 (b)	41	20
	5000	1	2	0 (b)	-	1	-	1	-	18	15
NAAZ	2,0	-	-	788	1120	781	897	-	-	-	-
ICR-191	2,0	-	-	-	-	-	-	1352	1568	-	-
2NF	1,0	126	181	-	-	-	-	-	-	-	-
4NQ	1,0	-	-	-	-	-	-	-	-	558	564

Trial I – An average of 2 replicates per dose level
Trial II – An average of 3 replicates per dose level
a = An average of 2 replicates
b = No colonies on plates due to test substance toxicity
- = 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	21	27	116	144	14	13	7	10	21	28
	33,3	24	-	106	-	7	-	12	-	36	-
	66,7	24	-	128	-	19	-	5	-	30	-
	100	29	-	124	-	16	-	12	-	40	-
	333	20	28	111	141	14	13	6	9	32	38
	667	26	29	131	157	15	16	5	9	33	29
	1000	19	23	115	153	16	18	8	6	36	35
	3333	20	17	76	119	6	9	5	10	27	26
	5000	6	9	2	8	0	4	4	5	16	15
2AA	2,5	-	-	3346	3371	252	322	303	283	-	-
	25	-	-	-	-	-	-	-	-	519	346
B[a]P	2,5	443	479	-	-	-	-	-	-	-	-

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 substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or absence of Aroclor induced rat liver S9. The test substance was concluded to be negative in this study.

Executive summary:

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

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the presence and absence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

The test was performed in 2 phases. The first phase was the toxicity-mutation test which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test which evaluated and confirmed the mutagenic potential of the test substance.

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.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. No positive mutagenic responses were observed at any dose level in any tester strain in the presence or absence of S9 metabolic activation. In the non-activated testing system toxicity was observed starting at 1000 μg/plate with tester strain TA1537, starting at 3333 μg/plate with TA100 and TA1535, and at 5000 μg/plate with TA98. In the presence of S9 metabolic activation toxicity was observed at 5000 μg/plate with both tester strains TA98 and TA100 and starting at 3333 μg/plate with TA1535. No test substance precipitation was observed at any dose level with any tester strain in the presence or absence of S9 metabolic activation.

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 μg/plate for all tester strains in the activated testing system, as well as, TA98 and WP2uvrA in the absence of S9 metabolic activation. Tester strains TA100, TA1535, and TA1537 were evaluated at 100, 333, 667, 1000, and 3333 μg/plate in the absence of S9 metabolic activation. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the presence or the absence of S9 metabolic activation. Toxicity was observed starting at 3333 μg/plate in the absence of metabolic activation with TA98, TA100, TA1535, and TA1537. Toxicity was also observed at 5000 μg/plate for TA98, TA100, and TA1535 with S9 metabolic activation. No test substance precipitation was observed at any dose level with any tester strain in either the presence or the absence of metabolic S9.

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was concluded to be negative in this study.