Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

27 Jan 2006 to 27 Feb 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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Method

Target gene:

his- (S. typhimurium) and trp- (E.coli) strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : phenobarbital/β-naphthoflavone induced rat liver
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 is prepared from male rats (Sprague-Dawley) induced with 80 mg/kg bw phenobarbital and 100 mg/kg bw β-naphthoflavone (3 mL of phenobarbital and β-naphthoflavone in total) combined with 7 mL Sucrose-Tris-EDTA buffer and 20 mL cofactor solution. The cofactor solution was prepared as a single stock solution of Na2HPO4 (150mM), KCl (49.5mM), glucose-6-phosphate (7.5mM), NADP (Na salt) (6mM) and MgCl2 (12mM) in sterile double deionised water and adjusted to a final pH of 7.4.
- Quality controls of S9: Positive control substances are tested to confirm the activity of the S9-mix.

Test concentrations with justification for top dose:

100, 200, 500, 1000, 2500, 5000 μg/plate

Vehicle / solvent:

- Solvent used: dried dimethylsulphoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: Daunorubicin hydrochloride: 1.0 μg/plate in TA98 (without metabolic activation), 2-Aminoanthracene: 1.0 μg/plate in TA100 and TA98; 2.0 μg/plate in TA1535 and TA1537; and 20 μg/plate in WP2 (pKM101) (with metabolic activation)

Details on test system and experimental conditions:

DOSING PREPARATIONS
All test and positive control substance dosing preparations were prepared as close to the time of culture treatment as possible and are dosed at a dosing volume of 100 μL/plate (apart from in the pre-incubation experiment, where the volume is reduced to 20 μL/plate).

EXPERIMENTAL PERFORMANCE
Bacterial cultures were prepared from frozen stocks by incubating for 10-12 hours at 37°C in a shaking incubator. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent and positive controls;
- 500 μL S9 mix or phosphate buffer;
- 100 μL Bacteria suspension;
- 2 mL Overlay agar containing 50 μM histidine or tryptophan as appropriate.
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix and 100 μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After the agar was set the plates were incubated upside down for 3 days at 37° C in the dark.
For each strain and dose level, including the controls three plates were used.
Following incubation all plates were counted using an automated colony counter adjusted appropriately to permit the optimal counting of mutant colonies.

CYTOTOXICITY
Following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test substance, and that the positive controls had responded as expected. Except where prohibited by the density of the precipitate the number of revertant colonies on each plate was counted using an automated adjusted appropriately to permit the optimal counting of mutant colonies. Plates that were obviously contaminated were recorded as such without being scored.

PRE-INCUBATION PROTOCOL
The assay procedure was as for the plate-incorporation protocol described above, except that a) each compound/ solvent dose was added in 0.02 mL volumes, with the total volume made up to 0.1 mL with phosphate buffered saline; b) before adding the top agar, each compound/strain group of containers were placed on an orbital shaker for 60 minutes (at 37°C).

DATA PRESENTATION
The data reported included individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration. Historical values of negative controls obtained with each strain without and with microsomal activation were listed in a separate table, which contains mean values of controls as well as standard deviations and acceptable minimal and maximal values of spontaneous revertants for each bacterial strain.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
a) a significant, dose-related increase in the mean number of revertants is observed;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations

CRITERIA FOR A NEGATIVE RESPONSE
A negative result in a (valid) individual experiment is achieved when:
a) there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

CRITERIA FOR VALID TEST DATA
Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable;
b) the positive control data show acceptable increases;

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli, other: WP2 pKM 101

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Precipitation occurred at concentrations of 500-5000 μL/0.1mL. Incomplete background lawns were reported only at a concentration of 200 μL/0.1mL on the E. coli WP2 (pKM101) plates.

MUTAGENICITY
In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.
The positive controls for each experiment induced the expected responses indicating the strains were responding satisfactorily in each case.

Applicant's summary and conclusion

Conclusions:

The test material gave a negative (non-mutagenic) response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2 (pKM101) and WP2 uvrA (pKM101) in both the presence and absence of S9-mix.

Executive summary:

The test material was evaluated in a bacterial reverse mutation assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2 (pKM101) and WP2 uvrA (pKM101) in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). The investigations were performed with concentrations of 100, 200, 500, 1000, 2500, 5000 μg/plate in all experiments. This study was conducted in accordance with OECD TG 471 following GLP principles.

In two independent experiments, the test material did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the strains used, either in the presence or absence of S9-mix. The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

The test material gave a negative (non-mutagenic) response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2 (pKM101) and WP2 uvrA (pKM101) in both the presence and absence of S9-mix.