kresoxim-methyl (ISO);methyl (E)-2-methoxyimino-[2-(o-tolyloxymethyl)phenyl]acetate

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:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

mouse

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

EC Number:

604-351-6

Cas Number:

143390-89-0

Molecular formula:

C18 H19 N O4

IUPAC Name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

Details on test material:

- Name of test material (as cited in study report): Reg. No. 242 009 (test substance number: 91/180-2)
- Lot/batch No.: N 36 (III c 1); date of manufaturing: 1991-10-23
- Storage condition of test material: approx. Refrigerator (protected from light)
- Physical state: lightbrown powder
- Analytical purity: 94.3% (Reversed-Phase - HPLC with UV-Detection analysed 1993-04-26/27)
- Stability under test conditions: the storage stability was guaranteed over the study period (stability in DMSO/water: stable for at least 4 hours)
- Other: the homogeneity of the test substance was confirmed by analysis (Reversed-Phase - HPLC with UV-Detection)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, FRG
- Age at study initiation: healthy young adult animals
- Weight at study initiation: mean weight of 28 g
- Assigned to test groups randomly: yes
- Fasting period before study: ot reported
- Housing: before the start of the treatment the animals were transferred to Makrolon cages, type M I, and housed individually under the same conditions until the end of the test.
- Diet (e.g. ad libitum): Standardized pelleted feed (Kliba Haltungsdiat, Klingentalmuhle AG, CH-4303 Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water
- Acclimation period: for the duration of about one week the animals were housed in groups of 5 separately according to sex in fully air-conditioned rooms in which central air conditioning.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose); 0.5% CMC formulation
- Justification for choice of solvent/vehicle: suspension with a concentration of 20 g/100 ml (2000 mg/kg dose group), 10 g/100 ml (1000 mg/kg dose group) and 5 g/100 ml (500 mg/kg dose group)
- Concentration of test material in vehicle: the test substance to be administered per kg body weight was suspended in an 0.5% CMC formulation.
- Amount of vehicle (if gavage or dermal): 10 ml/kg

Details on exposure:

IP application: first test group were given 2000 mg/kg bw (10 ml/kg bw of a suspension at 20 g/100 ml; second test group 1000 mg/kg bw (10 ml/kg bw of suspension at 10 g/100 ml); and third test group 500 mg mg/kg bw (10 ml/kg bw of suspension at 5 g/100 ml)

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

16, 24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg bw
Basis:
analytical conc.
actually injected

No. of animals per sex per dose:

5 the negative control and test substance group and 2/3 in the positive control group (Table 1)

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; 20 mg cyclophosphamide (10 ml test solution); 0.15 mg vincristine (10 ml test solution)
- Route of administration: IP
- Doses / concentrations: the control animals were sacrificed 24 hours after administration of the solvent. The control animals were treated at different times so that it was always possible to prepare control animals simultaneously for all the different sacrifice intervals of the test animals.

Examinations

Tissues and cell types examined:

femoral bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity the amount of 2000 mg/kg body weight recommended as the highest dose according to the EEC Directive 84/449, B 12., was survived by all animals but led to clinical signs of toxicity (see below). Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
See Table 1

DETAILS OF SLIDE PREPARATION:
- The two femora were prepared from the animals, and all soft tissues were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37 0C (about 2 ml/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- Staining: The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded: (1) Number of polychromatic erythrocytes. (2) Number of polychromatic erythrocytes containing micronuclei. The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested. (3) Number of normochromatic erythrocytes. (4) Number of normochromatic erythrocytes containing micronuclei. The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals. (5) Ratio of polychromatic to normochromatic erythrocytes. This ratio indicates an influence of the test substance specifically on the bone marrow. (6) Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) (d = diameter of micronucleus, D = cell diameter) The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis

OTHER:
- Clinical examinations
After the administration of the test substance the animals were examined for any evident clinical signs of toxicity.

Evaluation criteria:

The mouse micronucleus test is to be considered valid if the following criteria are met: (1) the quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 1000 polychromatic erythrocytes; (2) the positive control chemical induced a significant increase in the number of cells containing micronuclei. The test chemical is to be considered positive in this assay if the following criteria are met: (1) a dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals. (2) The proportion of cells containing micronuclei exceeded the values of the concurrent negative control range. A test substance is generally considered negative in this test system if: (1) there was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.

Statistics:

A statistical evaluation was not necessary to perform. The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

see below (and Table 2)

Toxicity:

yes

Remarks:

See below: Clinical signs

Vehicle controls validity:

valid

Negative controls validity:

other: results are within the range of the historic controls from the same testing facility

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: administered as suspension in 0.5% CMC
- Clinical signs of toxicity in test animals: signs of toxicity such as irregular respiration, piloerection, squatting posture, apathy, poor general state and closed eyelids within 5 hours after treatment were observed

RESULTS OF DEFINITIVE STUDY
The single intraperitoneal administration of a 0.5% CMC formulation in a volume of 10 ml/kg body weight led to 2.3% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
After the single administration of the highest dose of 2000 mg/kg body weight, 2.8% polychromatic erythrocytes containing micronuclei were found after 16 hours, 2.2% after 24 hours and 2.0% after 48 hours.
In the two lower dose groups rates of micronuclei of about 2.0% (1000 mg/kg group) and 1.9% (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
With 25.6% the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing mainly small micronuclei at a dose level of 20 mg/kg body weight.
With 97.8% the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 15.0%.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

Thus, the test substance Reg. No. 242 009 did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the solvent control value at any of the sacrifice intervals.
No inhibition of erythropoiesis induced by the treatment of mice with Reg. No. 242 009 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Any other information on results incl. tables

Clinical signs:

The single intraperitoneal administration of the carrier in a volume of 10 m1/kg body weight was tolerated by all animals without any signs or symptoms.

A single intraperitoneal dose of 2000 mg/kg body weight led to irregular respiration, pi1oerection and in few cases, to squatting posture, apathy and closed eyelids about 30 minutes after administration; the general state of the animals was poor. Some of these signs were observed for about 4 hours after treatment.

About 30 minutes after treatment of the animals with doses of 1000 mg/kg or 500 mg/kg irregular respiration, pi1oerection and squatting posture were observed which lasted for about 30 minutes.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Table 2: Analysis of the polychromatic and monochromatic erythrocytes (mean values)

 

Treatment group	Dosis	Sacrifice interval	Polychromatic erythrocytes in a total of 1000 cells examined				Normochromatic erythrocytes
			Cells with MN d<¼ D	Cells with MN d≥¼ D	Total micronucleus		Total number of cells	Cells with MN
					Number	%
Vehicle control	0	24	2.3	0	2.3	2.3	445.0	1.4
Test substance	2000	16	2.6	0.2	2.8	2.8	479.3	0.8
	2000	24	2.1	0.1	2.2	2.2	485.2	1.2
	2000	48	1.8	0.2	2.0	2.0	485.0	0.8
	1000	24	1.9	0.1	2.0	2.0	485.1	0.6
	500	24	1.9	0	1.9	1.9	501.6	1.1
Cyclophosphamide	20	24	25.4	0.2	25.6	25.6	446.0	0.6
Vincristine	0.15	24	82.8	15	97.8	97.8	576.2	2.2
MN: Micronucleus; d: diameter of the micronucleus; D: diameter of the cell