3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

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 February 2020 to 11 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

JMAFF 2-1-19-1, November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

Molecular formula:

C9H7BrClN3

IUPAC Name:

3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

Test material form:

solid: particulate/powder

Details on test material:

Batch/Lot Number: 628-044-00
Analysed Concentration: 974.9 ± 3.3 g/kg
Physical State: Beige Solid
Date of Manufacture: 15 October 2019
Date of Expiry: 15 October 2021

Specific details on test material used for the study:

Storage Condition (at JRF):
Storage Temperature: Room temperature (15 to 30 °C).
Storage Condition: Cool and dry conditions.
Storage Container: In original container as supplied by the Sponsor.
Storage Location: Test Item Control Office, JRF.

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Aroclor 1254 induced rat liver S9 fraction, procured from Moltox (Lot N° 4149), was used in the study.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The S9 fractions procured from Molecular Toxicology Inc., U.S.A. (MOLTOX) (Lot N ° 4149). Species: Rat, Sex: Male, Tissue: Liver.
- Method of preparation of S9 mix : The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay.
- Concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of top agar, mixed thoroughly and this mixture was poured onto an MGA plate.

Test concentrations with justification for top dose:

Initial toxicity-mutation test, in the absence and presence of the metabolic activation (5% v/v S9 mix):
Eight test concentration levels: 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 µg/plate.

Confirmatory mutation test, in the absence and presence of the metabolic activation (10% v/v S9 mix):
Seven test concentration levels (three plates/concentration): 78.125, 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate.

Vehicle / solvent:

- Solvent used: DMSO.
- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water, (Stock A, 50000 µg/mL) while found to be soluble in dimethyl sulfoxide (Stock B, 50000 µg/mL). Hence, dimethyl sulfoxide (Stock B, 50000 µg/mL) was selected as the vehicle for treatment.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxid (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Number of replications:
Initial toxicity-mutation test:
Eight concentration levels (three plates/concentration) from 1.5 to 5000 µg/plate for the strains (TA1537, TA1535, TA98, TA100, and TA102), in the absence and presence of the metabolic activation (5% v/v S9 mix).

Confirmatory mutation test:
- Six concentration levels (three plates/concentration) from 156.25 to 5000 µg/plate for TA1535, TA98, TA100, TA102 and 781.25 to 2500 μg/plate for strain TA1537 in the absence and presence (10% v/v S9 mix) of the metabolic activation.
After 48 hours of incubation at 37 ± 1°C, revertant colonies were enumerated.

Solubility and Precipitation Test:
The test item was insoluble in distilled water, (Stock A, 50000 µg/mL) while found to be soluble in dimethyl sulfoxide (Stock B, 50000 µg/mL). Hence, dimethyl sulfoxide (Stock B, 50000 µg/mL) was selected as the vehicle for treatment. A volume of 100 µL from stock B was added to 2 mL of top agar and poured on a Minimal Glucose agar plate to assess the precipitation. Precipitation was not observed at the tested concentration of 5000 µg/plate. Hence, 5000 µg/plate was selected as the highest concentration to be tested for the initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Cell Viability Test:
The cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range.

Genotype Confirmation Test:
The genotype of the tester strain was confirmed for all the strains regularly (once a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Sterility Check for the Operating System
A sterility check for the operating system was performed along with a mutagenicity test for the following:
- Top Agar: Top agar prepared and used for the study (2 mL) was poured aseptically onto an MGA plate.
- Solvent: A volume of 0.1 mL of dimethyl sulfoxide (DMSO), used as a vehicle was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto an MGA plate.
- Test Item: A volume of 0.1 mL of the test item stock solution of the highest concentration was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto an MGA plate.
- ONB Solution: A volume of 0.1 mL of the ONB solution was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to an MGA plate.
- 0.2 M Sodium Phosphate Buffer: A volume of 0.1 mL of 0.2 M of sodium phosphate buffer was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to an MGA plate.

Tester Strain Integrity:
All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation.
All the tester strains demonstrated the requirement of the biotin except strain TA102 which is biotin independent. All the tester strains showed sensitivity to UV exposure except TA102 which is wild type.
All the tester strains demonstrated requirement of histidine for their growth. Tester strains TA98, TA100 and TA102 exhibited resistance to ampicillin, demonstrating presence of the pKM101 plasmid. Tester strains TA102 exhibited resistance to tetracycline, demonstrating the presence of pAQ1 plasmid.

Characteristic Number of Spontaneous Revertants:
All negative control cultures exhibited their characteristic number of spontaneous revertants per plate, demonstrating the requirement for histidine (Salmonella typhimurium).

Tester Strain Culture Density:
Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1–2 × 10 to the power of 9 bacterial cells/mL, demonstrating that appropriate numbers of bacteria were plated.

Positive Control Values in the Absence of Metabolic activation System (-S9):
The mean value of the positive control for each tester strain exhibited clear increase over the mean value of the negative control for that strain, demonstrating that tester strains were capable of identifying a mutagen.

Positive Control Values in the Presence of Metabolic activation System (+S9):
The mean value of the positive control for each tester strain exhibited clear increase over the mean value of the negative control for that strain, demonstrating that the S9 mix was capable of metabolising a pro-mutagen to its mutagenic form(s). An acceptable positive control in the presence of S9 for a specific strain was evaluated as having demonstrated integrity of the S9 mix and the ability of the tester strain to detect a mutagen.

Evaluation criteria:

Cytotoxicity Evaluation Criteria
Six analysable doses were available to evaluate the assay data. Cytotoxicity was detected by a decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).

Assay Evaluation Criteria
A result was considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Biological relevance of the result was considered first.
- Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
- Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

Statistics:

Statistical Analysis of the Results: Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of revertant colonies.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity : no.
- Individual plate counts : yes.
- Mean number of revertant colonies per plate and standard deviation : yes.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes.
- Negative (solvent/vehicle) historical control data: yes.

Any other information on results incl. tables

Negative Control

Results of this study indicate that values of the negative control in all tester strains were within the historical ranges of respective strains.

 

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Initial Toxicity-Mutation Test):

Concentration of 3-bromo-1-(3-chloro-2- pyridyl)-5-methyl-1H-pyrazole (µg/plate)	His^+Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	12.67	1.15	18.33	2.08	27.33	3.06	144.67	7.51	244.33	10.21
1.5	7.00	2.00	15.67	2.52	22.67	3.79	129.67	3.79	224.33	11.15
5	9.00	3.61	18.67	1.53	23.67	4.73	140.00	5.29	236.67	2.08
15	5.67	2.52	14.00	3.46	24.33	4.04	127.67	6.03	240.33	8.62
50	8.33	3.21	17.00	1.00	21.67	3.06	140.67	4.04	234.67	3.21
150	8.00	2.65	15.33	1.53	27.00	3.46	137.67	7.09	238.00	4.36
500	6.00	2.65	16.33	2.52	22.00	4.36	128.00	6.08	230.67	2.89
1500	9.67	2.08	15.33	1.53	24.67	3.51	139.67	4.93	238.67	9.71
5000	-	-	14.67	1.15	23.33	3.51	124.67	5.51	223.33	4.04
PC	209.33	69.01	350.33	95.47	530.33	88.79	669.00	54.81	986.33	50.02
2Aa	-	-	-	-	-	-	130.67	3.51	-	-

Key: SD = Standard Deviation, NC = Negative Control, DMSO =Dimethyl sulfoxide, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 μg/plate), TA1535 = Sodium Azide (0.5 μg/plate),

TA98 = 2-Nitrofluorene (7.5 μg/plate), TA100 = Sodium Azide (5 μg/plate), TA102 = Mitomycin-C (0.5 μg/plate)},

2-Aa = 2-Aminoanthracene (5 μg/plate for TA100), - = Not Applicable.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Initial Toxicity-Mutation Test):

Concentration of 3-bromo-1-(3-chloro-2- pyridyl)-5-methyl-1H-pyrazole (µg/plate)	His+ Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	12.67	3.79	18.00	1.00	26.33	5.51	143.33	4.51	248.67	2.31
1.5	10.00	3.61	17.33	2.52	23.33	2.52	130.67	11.02	233.67	3.51
5	8.33	3.21	15.67	1.53	25.00	4.36	134.67	0.58	235.00	7.94
15	8.00	1.00	17.67	1.53	26.67	2.08	137.33	5.51	238.00	3.61
50	9.00	1.73	17.00	2.65	26.00	2.00	126.67	2.08	234.67	0.58
150	9.67	3.06	14.67	2.52	27.67	2.31	140.33	9.61	238.67	6.81
500	12.00	2.65	18.00	2.65	24.33	2.52	131.00	4.36	236.00	2.65
1500	10.33	2.08	15.67	1.53	27.67	2.08	141.67	4.73	233.67	8.74
5000	8.00	2.00	14.33	1.53	23.00	1.00	123.00	3.61	224.33	5.13
PC – 2Aa	201.33	56.76	351.00	69.91	486.67	85.22	730.33	61.45	1023.67	39.80

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive control

{2Aa = 2-Aminoanthracene (10 μg/plate for TA1537, TA1535, TA102 and 5 μg/plate for TA98 and TA100)}.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Test):

Concentration of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole (µg/plate)		His^+Revertant Colonies/Plate [Absence of Metabolic Activation]
		TA1537		TA1535		TA98		TA100		TA102
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)		8.33	3.06	19.67	2.89	22.67	2.52	125.00	19.31	225.00	11.27
#78.125	*156.25	8.33	0.58	16.33	2.08	23.67	2.52	133.00	14.11	227.00	16.52
#156.25	*312.5	9.67	2.52	18.33	2.52	26.00	1.00	124.33	21.94	229.67	11.72
#312.5	*625	8.67	2.52	18.67	1.53	23.67	0.58	118.00	1.00	222.00	6.24
#625	*1250	9.00	4.36	17.67	0.58	25.33	2.89	134.33	17.04	230.33	19.09
#1250	*2500	10.33	0.58	18.33	0.58	22.67	2.52	130.00	22.61	229.67	5.86
#2500	*5000	4.00	1.00	18.67	2.08	23.33	4.73	133.33	17.04	228.00	14.73
PC		217.33	12.58	312.67	40.20	385.00	44.14	606.00	59.81	991.67	25.66
2Aa		-	-	-	-	-	-	126.33	10.02	-	-

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive control

{TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 μg/plate), TA1535 = Sodium Azide (0.5 μg/plate),

TA98 = 2-Nitrofluorene (7.5 μg/plate), TA100 = Sodium Azide (5 μg/plate), TA102 = Mitomycin-C (0.5 μg/plate)},

2-Aa = 2-Aminoanthracene (5 μg/plate for TA100), - = Not Applicable.

Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Test):

Concentration of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole (µg/plate)		His^+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
		TA1537		TA1535		TA98		TA100		TA102
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)		9.67	0.58	16.67	2.89	24.00	4.36	132.33	6.11	228.00	19.00
#78.125	*156.25	11.00	2.65	21.33	0.58	24.33	2.52	124.67	11.68	240.33	17.95
#156.25	*312.5	9.33	2.08	20.33	0.58	23.33	4.51	121.67	11.68	238.00	4.58
#312.5	*625	11.33	0.58	21.33	2.31	22.33	4.04	129.67	13.87	226.67	9.45
#625	*1250	9.33	2.08	19.00	3.46	22.33	4.51	134.00	7.00	229.00	6.08
#1250	*2500	10.00	1.00	18.00	2.65	26.67	2.52	137.67	11.72	233.00	2.00
#2500	*5000	3.67	1.15	20.33	1.53	22.67	2.52	135.33	18.01	230.67	1.15
PC-2Aa		233.67	18.77	311.00	34.00	394.33	63.14	768.00	135.28	960.67	96.15

Key: SD = Standard Deviation, NC = Negative Control, DMSO = Dimethyl sulfoxide, PC = Positive Control

{2Aa = 2-Aminoanthracene (10 μg/plate for TA1537, TA1535, TA102 and 5 μg/plate for TA98 and TA100)}.

Positive Controls

2-Aminoanthracene was used as the positive control, in the presence of the metabolic activation system for all tester strains during the initial toxicity and the confirmatory mutation test. The large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control, in the presence of the metabolic activation system. The batch of S9 used in this study was characterised with benzo(a)pyrene by the supplier which required the metabolic activation system with microsomal enzymes.

Positive controls exhibited a clear increase in the number of the revertant colonies when compared with that of the concurrent negative controls. This demonstrated the efficiency of the test system and the suitability of the procedures employed in the assay.

An increase in revertants was not observed in the tester strain TA100 (initial toxicity-mutation test and confirmatory mutation test), treated with 2-aminoanthracene in the absence of the metabolic activation but a clear increase was observed in the presence of the metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

 

Initial Toxicity-Mutation Test

Cytotoxicity was characterised by the inhibition of the bacterial background lawn and/or reduction in the number of the revertant colonies.

A normal bacterial background lawn with no significant reduction in the number of revertant colonies, was observed up to 5000 μg/plate in tester strains TA98, TA1535, TA100 and TA102, and up to 1500 μg/plate in tester strain TA1537, both in the absence and presence of the metabolic activation (5% v/v S9 mix).

 

A complete inhibition with the micro colonies was observed at 5000 μg/plate in the absence and presence of the metabolic activation (5 % v/v S9 mix) in tester strain TA1537.

No increase in the number of the revertant colonies was observed, either in the absence or presence of the metabolic activation (5% v/v S9 mix) at any tested concentration level, in any tester strain.

Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TA100, and TA102. Statistical analysis did not reveal any significant effect.

 

Based on the results of the initial toxicity mutation test, six concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole /plate for TA1535, TA98, TA100, TA102 and 78.125, 156.25, 312.5, 625, 1250 and 2500 μg 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole /plate for TA1537 were selected for the confirmatory mutation test with 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole/plate, in the absence and presence of the metabolic activation system (10% v/v S9 mix).

 

 

Confirmatory Mutation Test

A normal bacterial background lawn with no significant reduction in the number of the revertant colonies, was observed up to 5000 μg/plate in tester strains TA98, TA1535, TA100 and TA102, and up to 1250 μg/plate in tester strain TA1537, both in the absence and presence of metabolic activation (5% v/v S9 mix).

A partial inhibition with micro colonies was observed at 2500 μg/plate in the absence and presence of the metabolic activation (10 % v/v S9 mix) in the tester strain TA1537.

 

No increase in the number of the revertant colonies was observed, either in the absence or presence of the metabolic activation system (10% v/v S9 mix) in any tester strain.

Results revealed that there was no positive mutagenic effect in TA1537 at 78.125, 156.25, 312.5, 625, 1250 and 2500 μg/plate of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole and in TA1535, TA98, TA100 and TA102 at 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate of 3- bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole, either in the absence or presence of the metabolic activation system (10% v/v S9 mix), when compared with that of the concurrent negative control. Statistical analysis did not reveal any significant effect.

Applicant's summary and conclusion

Conclusions:

From results of this study, it is concluded that that 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is non-mutagenic to histidine requiring strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102) when tested under both the absence and presence of the metabolic activation system. According to EC regulation 1272/2008 the substance is not classified as mutagenic.

Executive summary:

This study was performed to evaluate the mutagenic potential of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole by the bacterial reverse mutation test, using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102).

3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole was tested in two independent experiments, in the absence and presence of the metabolic activation system. Bacterial cultures were exposed to the test item at eight concentration levels (three plates/concentration) from 1.5 to 5000 μg/plate in the initial toxicitymutation test. A normal bacterial background lawn was observed up to 5000 μg/plate in tester strains (TA1535, TA98, TA100, and TA102), in the absence and presence of the metabolic activation (5% v/v S9 mix).

A complete inhibition of the bacterial background lawn with microcolonies was observed at dose level 5000 μg/plate, in the absence and presence of the metabolic activation in the tester strain TA1537. No increase in the number of the revertant colonies (no mutagenic effect) was observed, either in the absence or presence of the metabolic activation system (5% v/v S9 mix) in tester strains (TA1537, TA1535, TA98, TA100 and TA102). To confirm the negative results obtained in the initial toxicity mutation test, a confirmatory mutation test was conducted with an increased S9 concentration, i.e., 10% v/v S9 mix and modified concentration spacing. Based on the results of the initial toxicity mutation test, bacterial cultures were exposed to 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole at six concentration levels (three plates/concentration) from 78.125 to 2500 μg/plate for TA1537 and 156.25 to 5000 μg/plate for TA1535, TA98, TA100, TA102, and in the absence and presence (10% v/v S9 mix) of the metabolic activation. After 48 hours of incubation at 37 ± 1°C, revertant colonies were enumerated.

 

3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole did not induce any significant increase in the number of the revertant colonies, in trials, with or without S9 mix, in any tester strain. All values for the negative control were within historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the sensitivity and efficiency of the test system.

 

All criteria for a valid study were met, as described in the study plan. From results of this study, under the specified experimental conditions, 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium in the absence and presence of the metabolic activation system.