benzovindiflupyr (ISO); N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March 2009 to 18 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

EC Number:

691-719-4

Cas Number:

1072957-71-1

Molecular formula:

C18H15Cl2F2N3O

IUPAC Name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Details on test material:

- Name of test material (as cited in study report): SYN545192
- Substance type: Technical material
- Physical state: Beige powder
- Analytical purity: 97.0%
_ Analytical method used: HPLC
- Purity test date: 25 February 2009
- Stability under test conditions: Reanalysis date end February 2013
- Storage condition of test material: <30ºC

Test animals

Species:

rat

Strain:

other: Wistar (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 183-205 g males; 147-181 g females
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Two or three (same sex) per cage in suspended polycarbonate cages (61 x 43.5 x 24 cm) with stainless steel tops
- Diet: SDS Rat and Mouse Maintenance Diet No. 1 ad libitum
- Water: Tap water ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5-22.0°C
- Humidity: 29-68%
- Air changes: At least 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 02 March 2009 To: 18 March 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% carboxymethyl cellulose (CMC) with 0.1% TWEEN 80
- Justification for choice of solvent/vehicle: Based on previous experience of dosing SYN545192 in other studies
- Amount of vehicle (if gavage or dermal): Dose volume for control and test item treated animals was a constant 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in 1% Carboxymethyl cellulose (CMC) with 0.1% TWEEN 80 to give the required dosing concentrations, as near to the time of dosing as practicable.

Cyclophosphamide was prepared fresh as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg.

Duration of treatment / exposure:

Dosed at 0 and 24 hours

Frequency of treatment:

2 doses at 0 and 24 hours

Post exposure period:

24 hours after second dose

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 43.8, 87.5, 175 mg/kg/day (males); 0, 75 mg/kg/day (females)
Basis:
nominal conc.

No. of animals per sex per dose:

0 (vehicle controls) =5 males & 5 female, 43.8 and 87.5 mg/kg/day = 5 males, 175 mg/kg/day = 10 males, 75 mg/kg/day = 10 females, positive control = 5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg/day

Examinations

Tissues and cell types examined:

Micronuclei in the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: As limited toxicity information on the test item was available, a preliminary toxicity test was conducted in a step-wise manner using 2 phases up to a dose of 175 mg/kg/day (male) and up to a dose of 100 mg/kg/day (female), in order to determine the maximum tolerated dose the test item.

TREATMENT AND SAMPLING TIMES: Dosed at 0 and 24 hours, sampled at 48 hours.

DETAILS OF SLIDE PREPARATION: Bone marrow from one femur flushed into a centrifuge tube containing 3 mL of a 1:1 mixture of foetal calf serum and 0.8% trisodium citrate in Sorensen's buffer, pH 6.8. Routine tissue culture antibiotics included to prevent microbial growth. The tubes were centrifuged to pellet the cells. All but a few drops of supernatant fluid were discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid.

Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol for ca 5 min and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination. The stained smears were rinsed in 2-3 changes of water and left to air dry. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.

METHOD OF ANALYSIS: The better of the 2 slides was selected and assessed "blind" by the same operator. At least 2000 PCEs per animal were scored for micronuclei and the frequency of MN-PCEs determined.

As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of MNNCEs in mature red blood corpuscles were also recorded. In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic spindle during bone marrow erythropoiesis.

The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.

Evaluation criteria:

Negative: No biologically relevant increases in the numbers of MN-PCE were observed, relative to the concurrent and established historical control frequencies for MN-PCE induction.
Positive: Increase in the number of micronucleated polychromatic erythrocytes (MN-PCE) obtained for one or more of the test item treated dose groups (i.e. an increase greater than 10% over the expected historical control ranges for a group of animals).
Inconclusive: Levels of MN-PCE within any one dose group increased above the established historical control frequencies for MN-PCE induction, but not high enough to meet the criteria for a positive response (i.e. increase up to 10% over the maximum negative control frequency for a group of animals).

Statistics:

The MN-PCE data were transformed by adding one to each individual value before taking the square root. These transformed values were analysed using analysis of variance (ANOVA) techniques for males and females separately. Following the ANOVA, the SYN545192 treated groups were compared to the control using Dunnetts tests at the 5% significance level.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

assessed in preliminary study. MTD 175 mg/kg/day for males and 75 mg/kg/day for females

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING TOXICITY STUDY
- Dose range: 75 mg/kg/day (3 males and 3 females), 100 mg/kg/day (3 females), 175 mg/kg/day (3 males)
- Experimental procedure: Rats dosed orally at 0 h and 24 h. Observed for clinical signs or mortality at frequent intervals (1 min, 30 min, 1 h, 2 h and 4 h) post dosing, then twice daily until the end of the observation period. Surviving animals were subsequently killed.
- Clinical signs of toxicity in test animals: Hunched posture, staggering, subdued behaviour, piloerection and laboured breathing in females at 75 mg/kg/day. Hunched posture, staggering and subdued behaviour in males at 175 mg/kg/day. Severe clinical signs were observed in females at 100 mg/kg/day including: hunched posture, staggering, subdued behaviour, piloerection, laboured breathing, and prostration. Due to the severity of the clinical observations, one female was humanely killed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant micronucleus induction was detected in bone marrow erythrocytes of rats dosed with SYN545192. Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant difference in the ratio of PCE/NCE was seen for SYN545192 treated rats.
- Statistical evaluation: There was no evidence of a difference in the mean number of MN-PCE in the SYN545192 groups compared with the control for either males (p=0.19, p=0.98, p=0.99 for 43.8, 87.5 and 175 mg/kg/day groups respectively) or females (p=0.52). The borderline increases observed in two test item dosed rats are considered to be of no biological significance.

Any other information on results incl. tables

Animal observations: No mortalities. Hunched posture, staggering, laboured breathing, subdued behaviour and piloerection observed in males treated with 175 mg/kg/day and females treated with 75 mg/kg/day.

 

Table 1: Summary of assessment data

Treatment	Sex	No. rats scored	Erythrocytes
			NCEs	PCEs			PCE/NCE ratio (mean±SD)
			No. MN-NCE	PCEs analysed	No. MN-PCEs	% MN-PCE
10 mL 1% CMC with TWEEN 80/kg/day	M	5	5	10015	7	0.07	0.62 ± 0.11
	F	5	5	10008	6	0.06	0.65 ± 0.14
	M/F	10	10	20023	13	0.06	0.64 ± 0.12
43.8 mg SYN545192/kg/day	M	5	5	10012	1	0.01	0.64 ± 0.19
87.5 mgSYN545192/kg/day	M	5	3	10009	9	0.09	0.69 ± 0.18
175 mg SYN545192/kg/day	M	5	8	10016	6	0.06	0.56 ± 0.11
75 mg SYN545192/kg/day	F	5	9	10007	9	0.09	0.55 ± 0.15
50 mg cyclophosphamide/kg/day	M	5	93α	10000	241	2.41 Φ	0.33 ± 0.07
M = male F = female PCE = Polychromatic erythrocytes MN-PCE = Micronucleated PCE NCE = Normochromatic erythrocytes MN-NCE = Micronucleated NCE Φ = Positive response in PCE α = Evident response in NCE

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
SYN545192 did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 175 mg/kg/day in male and 75 mg/kg/day in female Wistar (Han) rats using a 0 h + 24 h oral dosing and 48 h sampling regimen.

Executive summary:

The in vivo genotoxic potential of SYN545192 was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female Wistar (Han) rats following a 0 h + 24 h oral dosing and 48 h sampling regimen. A set of dose range-finder toxicity studies were conducted to establish a suitable dose range for the micronucleus experiment. Concentrations of 175, 87.5 and 43.8 mg/kg/day were tested in males and 75 mg/kg/day in females. Bone marrow samples were taken 48 h after the initial 0 h dose. Two control groups were dosed orally with either the vehicle, 1% carboxymethyl cellulose with 0.1% TWEEN 80 at 10 mL/kg/day, or the positive control agent, 50 mg cyclophosphamide/kg/day.

 

No statistically significant micronucleus induction was detected in bone marrow erythrocytes of rats dosed with SYN545192. Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

 

SYN545192 did not induce micronuclei in bone marrow cells when tested to the maximum tolerated doses of 175 mg/kg/day in male and 75 mg/kg/day in female Wistar (Han) rats using a 0 h + 24 h oral dosing and 48 h sampling regimen.