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

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:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

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: 90/577)
- Lot/batch No.: N 21 (with acetone as carrier)
- Storage condition of test material: 4°C to 6°C
- Physical state: beige powder
- Analytical purity: 98.7% (Reversed-Phase - HPLC with UV-Detection)
- Stability under test conditions: the storage stability was guaranteed over the study period
- Other: the homogeneity of the test substance was confirmed by analysis (Reversed-Phase - HPLC with UV-Detection)

Method

Species / strain

Species / strain / cell type:

other: human lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: “Chromosome medium 1A with PHA" was used as the culture medium; the total volume of the culture medium was 6.0 ml.
Human venous blood was drawn from volunteers aseptically into sterile syringes that contained heparin to prevent clotting. About 0.5 ml of heparinized blood was added to 6.0 ml of culture medium in each centrifuge tube, and the cultures were incubated with closed caps at 37°C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction is prepared according to Ames et al. prepared from at least 5 male Sprague-Dawley rats (200 - 300 g) which receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil -w/v).

Test concentrations with justification for top dose:

0, 10, 20 and 40 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: each dose level was dissolved in each 0.07 ml acetone (as carrier controls with and without 5-9 mix only contained the carrier for the test substance at the same concentration and volume used in the test culture)
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

yes

Remarks:

cell culture medium only

Negative solvent / vehicle controls:

yes

Remarks:

acetone as carrier vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S-9 mix: 0.1 µg mitomycin C/ml culture medium was added in a volume of 0.1 ml (dissolved in aqua dest.); with S-9 mix: 5 µg cyclophosphamide/ml culture medium was added in a volume of 0.1 ml (dissolved in aqua dest.)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: whole blood was added to the culture medium containing PHA and incubated at 37°C for about 48 hours
- Exposure duration: solutions of the test substance, positive control substance or the solvent and 0.2 ml phosphate buffer were then added to the cultures and incubated for about 3 hours of at 37°C
- Expression time (cells in growth medium): 21 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 min in methanol: glacial acetic acid/4 : 1

SPINDLE INHIBITOR (cytogenetic assays): colcemid was added 2 - 3 hours before fixation (the whole duration of incubation was 72 hours) and at the end of the expression time (after cell washing).
STAIN (for cytogenetic assays): 3-5 drops of cell suspension (in fixative) were dropped onto clean, shortly iced microscopic slides, were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (10 ml Giemsa, 190 ml Titrisol pH 7.2) for 10 minutes.

NUMBER OF REPLICATIONS; NUMBER OF CELLS EVALUATED: as a rule, 100 metaphases of each culture for the test substance, negative and solvent controls or 50 cells of each culture for the positive controls were analyzed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; a mitotic index based on 1500 cells/culture was determined for at least the top two doses that yield metaphase cells, for the solvent and negative controls and for the positive controls.

OTHER EXAMINATIONS:
Aneuploidies (hyperploid metaphases) and polyploidies as well as in the number of aberrant cells incl. and/or excl. gaps

Evaluation criteria:

Statistical positivity; preparations were analysed blinded. If only a few metaphases were found, a chromosome analysis was not carried out.

Statistics:

For each group the proportion of metaphases with aberrations were calculated. A comparison of each dose group with the solvent control group was carried out using FISHER’s exact test for the hypothesis of equal proportions. This test was BONFERONI-HOLM corrected for each time point and was performed one-sided.

Results and discussion

Test results

Species / strain:

other: human lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Table 1; no differences regarding aneuploidies (hyperploid metaphases) and polyploidies as well as in the number of aberrant cells incl. and/or excl. gaps between the various dose groups and the negative controls were observed.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

see below (Table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The doses were determined from an appropriate pretest with cultures exposed up to the maximal possible amount of the test article, i.e. 40 µg/ml culture medium. Doses greater than 40 µg/ml dissolved in acetone were not soluble any longer in aqueous medium and thus led to a distinct compound precipitation after instillation into the culture medium. A reduction of the mitotic index was not observed at this dose. Due to the findings of this pretest, the highest doses i.e. 40 µg/ml without S-9 mix and with S-9 mix have to be selected with regard to the solubility of the test substance and not to the mitotic index as required in the OECD guideline method 473.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Analysis of chromosomes (metaphases with aberrations)

 

Treatment conditions	Evaluated test substance concentration (µg/ml)	Number of aberrant cells (percentage) and corresponding mitotic index
		Aberrant cells incl. gaps and mitotic index relative to untreated controls				Aberrant cells excl. gaps
		- S-9 mix		+ S-9 mix		- S-9 mix	+ S-9 mix
		Aberrant cells	Mitotic index	Aberrant cells	Mitotic index
Untreated control	0	10 (5%)		5 (2.5%)		1 (0.5%)	3 (1.5%)
Carrier control (acetone)	0	9 (4.5%)	100%	7 (3.5%)	100	1 (0.5%)	2 (1.0%)
Test substance	40	10 (5%)	74.7%	5 (2.5%)	124.5%	4 (2%)	5 (2.5%)
	20	9 (4.5%)	88.0%	12 (6.0%)	120.4%	4 (2%)	5 (2.5%)
	10	9 (4.5%)	94.7%	11 (5.5%)	110.2%	1 (0.5%)	1 (0.5%)
Positive control	0.1 – 5.0	26 (26%)	108.0%	29 (29%)	61.2%	18 (18%)	25 (25%)