pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

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:

2 Jul 1991 to 12 Aug 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted May 1983

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1987

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: THE MINISTRY OF HEALTH AND WELFARE, Japan , Guidelines of Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Part 1), Notification No. 118 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare.

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

EC Number:

602-927-1

Cas Number:

123312-89-0

Molecular formula:

C10H11N5O

IUPAC Name:

6-methyl-4-[(E)-[(pyridin-3-yl)methylidene]amino]-2,3,4,5-tetrahydro-1,2,4-triazin-3-one

Test material form:

solid: particulate/powder

Method

Target gene:

his- (S. typhimurium), trp- (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:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

Range in the (preliminary) cytotoxicity test (with and without microsomal activation): 20.6, 61.7, 185, 555, 1666 and 5000 μg/plate
Range in the mutagenicity test (with and without microsomal activation): 312, 625, 1250, 2500 and 5000 μg/plate
Justification for top dose: 5000 μg/plate is the maximum recommended dose according to the test guideline

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-Aminoanthracene (2-AA); 4-Nitroquinoline (4-NQ)

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY ASSAY: A preliminary toxicity test was carried out with strains s. typhimurium TA 100 and E.coli WP2uvrA with and without microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was the highest applicable concentration (due to the solubility limit of the test substance in the solvent) or 5000 μg/plate. The five lower concentrations decreased by a factor of 3 (The number of concentrations tested and the factor of dilution were in deviation to the SOP effective at the time of this study). The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

METHOD OF APPLICATION: in agar (plate incorporation)

PROTOCOL
- Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
- 0.1 mL of the overnight cultures were mixed with 2 ml of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 mL of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
- The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and with the Escherichia coli strain WP2uvrA without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance (312 to 5000 μg/plate), a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was determined in the preliminary toxicity test and the four lower concentrations were each decreased by a factor of 2. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
- Colonies were counted electronically with an Artek counter. The results were sent online to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director

CRITERIA FOR A POSITIVE RESPONSE:
The test substance is considered to be mutagenic in this test system if one or both of the following conditions are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: s. typhimurium TA 98, TA 1535, TA 1537 and E. Coli WPuvrA.
-A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains. typhimurium TA 100.
- Generally a concentration-related effect should be demonstrable

Statistics:

None.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA100, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 50 and 25 mg/mL precipitates were visible. The highest concentration soluble in dimethylsulfoxide was found to be 12.5 mg/mL. The highest concentration tested was 5 mg/mL.

RANGE-FINDING/SCREENING STUDIES:
Six concentrations of the test substance ranging from 20.6 to 5000 μg/plate were tested with strains S. typhimurium TA 100 and E.coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 μg/plate without activation and 5000 μg/plate with activation.

NUMBER OF CELLS WITH REVERSE MUTATION
In the original experiment performed without and with microsomal activation, none of the tested concentrations led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control. In the confirmatory experiment performed without and with microsomal activation, again, the tested concentrations did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

HISTORICAL CONTROL DATA
- Positive and negative historical control data can be found in Table 1 and 2 in ‘any other information on results incl. tables’. The data is presented as Arithmetic Mean (m) and Standard Deviation (s) of colony counts obtained in 91 separate experiments over the period of January 15, 1990 to January 02, 1991. Acceptable range for mean colony counts (=mean of values) of spontaneous revertants.

Any other information on results incl. tables

Table 1. Historical data positive and negative control in absence of metabolic activation.

Strain	Substance	µg/plate	Mean	StDev	Acceptable range
TA 100	Negative control		146.9	28.2	8-220
	sodium azide	2.0	762.9	257.2
TA 1535	Negative control		11.4	3.3	7-30
	sodium azide	2.0	699.4	277.8
E. Colie WP2uvrA	Negative control		20.2	4.5	8-40
	4-nitroquinoline-N-oxide	1.0	501.7	323.4
TA 98	Negative control		19.1	4.4	12-50
	2-nitrofluorene	10.0	1287.5	308.9
TA 1537	Negative control		7.9	2.6	3-20
	9-amino acridine	150.0	2158.9	583.3

Table 2. Historical data positive and negative control in presence of metabolic activation.

Strain	Substance	µg/plate	Mean	StDev	Acceptable range
TA 100	Negative control		142.2	25.8	70-220
	2-aminoanthracene	2.5	1768.8	544.2
TA 1535	Negative control		12.6	3.2	7-35
	Cyclophosphamide	400.0	349.6	126.1
E. Colie WP2uvrA	Negative control		22.3	5.2	8-50
	2-aminoanthracene	50.0	1109.7	277.1
TA 98	Negative control		35.4	5.7	20-70
	2-aminoanthracene	2.5	1759.9	470.3
TA 1537	Negative control		12.0	4.3	5-30
	2-aminoanthracene	5.0	204.3	82.4

Applicant's summary and conclusion

Conclusions:

In this GLP compliant study, performed according to OECD 471, the test substance and its metabolites did not induce gene mutations in the tested strains of S. typhimurium and E. coli

Executive summary:

In this GLP compliant OECD 471 study (a reverse gene mutation assay in bacteria), the test substance in dimethylsulphoxide (DMSO) was tested on four histidine-auxotrophic strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate. Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor 1254 induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting with an Artek counter and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments. In the range finding test, normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. In the main study, the test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. Therefore, it was concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.