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 vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated 24 March 2009. Experimental phase 02 April to 25 May 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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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
- Physical state: Beige powder
- Analytical purity: 97.0% (by HPLC)
- Purity test date: 25 February 2009
- Expiration date of the lot/batch: End February 2013
- Storage condition of test material: <30°C

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 [phenobarbital i.p. (80 mg/kg) and β-naphthoflavone p.o. induced]

Test concentrations with justification for top dose:

0.34, 0.60, 1.83, 2.5, 3.1, 3.20, 5.0, 5.5, 7.5, 9.6 and 10.0 µg/mL (chromosomal aberration analysis)
3.1, 5.5, 9.6, 16.9, 29.5, 51.7, 90.5, 158.3, 277.1, 484.9, 848.6 and 1485.0 µg/mL (cytogenetic tests)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: chosen due to its solubilisation properties and lack of toxicity to the cells

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9, 30 µg/mL in experiment 1; 15 µg/mL in experiment 2

Controls 3

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9, 825.0 µg/mL in experiment 1; 770.0 µg/mL in experiment 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substances at appropriate concentrations.

TREATMENT:
- Exposure time 4 hours: About 70 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 LL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium and cultured until preparation.
- Exposure time 22 hours (without S9 mix): About 70 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with complete medium (with 10 % FCS) containing the test item without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5% CO2 (94.5% air).

SPINDLE INHIBITOR (cytogenetic assays): Approximately 3 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.2 µg/mL.

CULTURE HARVESTING: The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37°C for 20-25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.

STAIN (for cytogenetic assays): The cells for evaluation of cytogenetic damage were stained with Giemsa.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 (test groups)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: Two independent cytogenetic experiments were carried out using ranges of SYN545192 concentrations.
- The highest applied concentration in this study (1485.0 µg/mL of the test item, approx. 3.7 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Evaluation criteria:

The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46±1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid: A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of the historical control data.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05)

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation was seen in cultures treated with SYN545192 at concentrations of 51.7 µg/mL (without S9) and 29.5 µg/mL (with S9) and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The highest treatment concentration in this study, 1485.0 µg/mL (approx. 3.7 mM) was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

In Experiment I, in the absence of S9 mix, precipitation of the test item in the culture medium was observed with 51.7 µg/mL and above and with 29.5 µg/mL and above in the presence of S9 mix.

No relevant increase in the osmolarity or pH value was observed (e.g. Exp. I: solvent control: 375 mOsm, pH 7.3 versus 373 mOsm and pH 7.4 at 1485.0 µg/mL).

In all experimental parts cytotoxicity was observed at the highest evaluated concentration. In Experiment I, the mitotic index was reduced to 38.7% (without S9 mix) and 47.8% (with S9 mix). In Experiment II, a reduction to 40.7% (without S9 mix) and 39.1% (with S9 mix) was observed.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5–3.5% aberrant cells, excluding gaps) exceeded the solvent control values (1.0–1.5% aberrant cells, excluding gaps) but were within the range of the laboratory’s historical solvent control data (0.0–4.0 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

In both experiments, either EMS (770 or 825 µg/mL) or CPA (15 or 30 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table 1: Summary of results of the chromosomal aberration study with SYN545192

Experiment	Preparation interval	Test item concentration µg/mL	Mitotic indices in % of control	Aberrant cells
				Including gaps*	Aberrant cells in % excluding gaps*	With exchanges
Exposure period 4 hours without S9
I	22 hrs	DMSO 0.5% (v/v)	100.0	2.0	1.5	0.0
		EMS 825.0	105.0	8.0	8.0s	0.0
		SYN545192 3.1	68.1	3.5	2.5	0.0
		5.5	50.0	1.0	0.5	0.0
		9.6	38.7	4.0	3.5	0.0
Exposure period 22 hours without S9
II	22 hrs	DMSO 0.5% (v/v)	100.0	2.0	1.0	0.0
		EMS 770.0	44.9	17.0	17.0s	5.0
		SYN545192 0.34	76.0	1.0	1.0	0.0
		0.60	66.0	1.0	1.0	0.0
		1.83	52.9	2.0	1.5	0.0
		3.20	40.7	1.5	1.5	0.0
Exposure period 4 hours with S9
I	22 hrs	DMSO 0.5% (v/v)	100.0	1.5	1.5	0.0
		CPA 30.0	39.3	13.5	13.0s	2.0
		SYN545192 3.1	103.5	1.0	1.0	0.0
		5.5	61.2	3.0	2.5	0.0
		9.6	47.8	1.5	1.5	0.0
!!	22 hrs	DMSO 0.5% (v/v)	100.0	1.0	1.0	0.0
		CPA 15.0	23.2	8.0	8.0s	1.5
		SYN545192 2.5	74.5	2.0	1.0	0.0
		5.0	70.0	1.0	1.0	0.0
		7.5	55.0	1.0	1.0	0.0
		10.0	39.1	2.0	2.0	0.0

*Inclusive cells carrying exchanges

Experimental groups evaluated for cytogenetic damage are shown in bold characters

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions reported, the test item SYN545192 did not induce structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation.

Executive summary:

This in vitro assay was performed to assess the potential of SYN545192 to induce structural chromosomal aberrations in the absence and the presence of an exogenous metabolic activation system. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations. The highest applied concentration in this study was 1485.0 Lg/mL of the test item, approx.3.7 mM.

In all experimental parts cytotoxicity was observed at the highest evaluated concentration. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, SYN545192 is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation when tested up to cytotoxic concentrations.