Methyl (2E)-2-cyano-3-(2,2-difluoro-1,3-benzodioxol-4-yl)prop-2-enoate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl (2E)-2-cyano-3-(2,2-difluoro-1,3-benzodioxol-4-yl)prop-2-enoate

EC Number:

700-526-7

Cas Number:

1333488-95-1

Molecular formula:

C12H7F2NO4

IUPAC Name:

methyl (2E)-2-cyano-3-(2,2-difluoro-1,3-benzodioxol-4-yl)prop-2-enoate

Test animals

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-9 weeks for phase I, 5-6 weeks for phase II
- Housing: mobile rat cage racks, environmental enrichment and nesting material provided
- Diet: ad libitum
- Water: ad libitum
- Acclimation: yes, time period not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes per hour: 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14.09.2005 To: 28.11.2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% w/v carboxymethylcellulose in 0.1% polysorbate 80

Details on exposure:

EXPERIMENTAL DESIGN:
Phase I: confirmation of suitability of limit dose 2000 mg/kg bw
Phase II: main micronucleus test (dosing of test group, vehicle control group and positive control group)

PREPARATION OF DOSING SOLUTIONS:
A stock suspension of the test material was prepared in the vehicle. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. Dosing volume was 10 mL/kg bw.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

single oral dose

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
other: test material in vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
20 mg/kg bw
Basis:
other: positive control cyclophosphamide in vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0 mg/kg bw
Basis:
other: vehicle control

No. of animals per sex per dose:

Test group: 10
Vehicle control group: 10
Positive control group: 5
(All groups consisted of males only)

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
The positive control substance was applied as a solution in sterile double deionised water.

Examinations

Tissues and cell types examined:

Bone marrow, immature erythrocytes

Details of tissue and slide preparation:

TISSUE PREPARATION:
Animals were killed by over-exposure to halothane followed by cervical dislocation. All animals treated with the test material were examined internally for abnormalities to organs/tissues.

SLIDE PREPARATION:
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed with saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). The conditioned brush was dipped into the marrow canal and two smears were painted on an appropriately labelled clean, day microscope slide. This procedure was repeated to give 4 smears of marrow per slide.
The slides were air dried and fixed in solvent (methanol) for at least 10 minutes. The slides were dipped into phosphate buffer, then stained with a solution of acridine orange (0.125 mg/mL) for 1 minute, then placed in fresh phosphate buffer for 10 minutes and then again in fresh buffer for a further 15 minutes. After staining the slides were air-dried and wet mounted in buffer prior to analysis.

SLIDE ANALYSIS:
Slides were coded and scored blind. 2000 immature erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity by couting the ratio of immature to mature erythrocytes in a sample of 1000 erythrocytes.

Evaluation criteria:

VALIDITY CRITERIA:
- vehicle control group mean should be within, or close to, the historical control range
- positive control group should show an appropriate increase in the incidence of micronucleated immature erythrocytes, compared to vehicle control values

CRITERIA FOR DATA EVALUATION:
- negative result if no significant increase in the incidence of micronucleated immature erythrocytes above concurrent vehicle control incidences or if a significant increase in the incidence of micronucleated immature erythrocytes above the concurrent vehicle control incidences but within the laboratory historical vehicle control range
- positive result if a significant increase in the incidence of micronucleated immature erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences
- an incidence of micronucleated immature erythrocytes which is significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant increases in the incidence of micronucleated immature erythrocytes, compared to the vehicle control values, were in any rats treated with the test item up to the maximum tolerated dose at either of the sampling times investigated.

The sensitivity of the test system was clearly demonstrated by the marked increases in the frequencies of micronucleated immature erythrocytes induced by the positive control substance cyclophosphamide.

Examination of the internal organs showed one animal had gas distened intestines.

Any other information on results incl. tables

 

Group	Treatment	Dose	Mean incidence of MIE/1000 IE ± SD
			24 h	48 h
1	Vehicle Control	10 mL/kg bw	2.3 ± 2.8	0.6 ± 0.4
2	Positive Control	20 mg/kg bw	61.5 ± 25.2	-
3	Test item	2000 mg/kg bw	0.7 ± 0.6	1.1 ± 0.7

IE: immature erythrocytes

MIE: micronucleated immature erythrocytes

SD: standard deviation

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under conditions tested, the test item is not clastogenic in the rat bone micronucleus test.