methyl (2E)-[2-(chloromethyl)phenyl](methoxyimino)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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Remarks:

BASF AG, Experimental Toxicology and Ecology

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): 520 F-Chlormethyloximether
- Physical state: Solid, dark green
- Analytical purity: 97.2 g/100 g
- Lot/batch No.: 31196-182
- Stability under test conditions: The stability of the test substance throughout the study period was not determined analytically. However, the recharacterization was performed in Jun 2007, indicating a stability of about 3 years after the date of manufacture.
- Storage condition of test material: Room temperature (avoid temperature above 50°C)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Mean weight at study initiation: 30.0 g
- Housing: single
- Diet (e.g. ad libitum): Standardized pelleted feed
- Water (e.g. ad libitum): Drinking water from bottles
- Acclimation period: at least 5 days


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Amount of vehicle (if gavage or dermal): To achieve a better volume for administration (DMSO is used up to 4 mL/kg body weight only) the DMSO solution was filled up to 10 mL/kg body weight with corn oil and mixed thoroughly

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance to be administered per kg body weight was dissolved in DMSO (4 mL/kg bw) and than emulsified in corn oil (up to 10 mL/kg bw). To achieve a homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. All test substance formulations were prepared immediately before administration.

Frequency of treatment:

twice

Post exposure period:

24 h

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP), vincristine sulfate (VCR)
- Route of administration: orally (CPP) or intraperitoneally (VCR)
- Doses / concentrations: 20 mg/kg bw for CPP, 0.15 mg/kg bw for VCR

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, following twice administration with 2000 mg/kg body weight, recommended as the highest dose according to the OECD Guideline, all animals (male and female) survived. The clinical signs observed were hunched posture and eyelid closure. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. 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): The animals of the vehicle control groups and the dose groups were treated twice orally (gavage) with a volume of 10 mL/kg body weight of the test substance with a 24-hour interval between both administrations. Animals of the positive control groups were treated only once. All animals were sacrificed 24 hours after the last treatment, respectively.


DETAILS OF SLIDE PREPARATION: The bone marrow was prepared according to the method described by Schmid ( In: Hollaender, A. (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New York, 1976) and Salamone et al. (Mut. Res., 74, 347 - 356, 1980).


METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei: The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
• Ratio of polychromatic to normochromatic erythrocytes
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to determine statistically significant differences between the vehicle control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- The two oral administrations of the vehicle mixture DMSO/corn oil (2:3) in a volume of 10 mL/kg body weight led to 0.7‰ polychromatic erythrocytes containing micronuclei.
- After two administrations of the highest dose of 2000 mg/kg body weight, 1.2‰ polychromatic erythrocytes containing micronuclei were found.
- In the two lower dose groups, rates of micronuclei of about 1.2‰ (1000 mg/kg bw group) and 1.1‰ (500 mg/kg bw group) were detected in each case.
- The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (13.6‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
- Vincristine sulfate, a spindle poison, produced a 45.8‰ increase in micronuclei in polychromatic erythrocytes. A significant portion increase, 11.3‰ was attributable to large micronuclei.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups.

CLINICAL EXAMINATIONS
- The two oral administrations of the vehicle in a volume of 10 mL/kg body weight were tolerated by all animals without any signs or symptoms.
- The administration of 500, 1000 and 2000 mg/kg body weight of the test substance led to clinical signs of toxicity (hunched posture and eyelid closure). In addition, one animal of the lowest dose group died unexpectedly 24 hours after the first administration.
- Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine sulfate in a dose of 0.15 mg/kg body weight caused any signs of toxicity.

Any other information on results incl. tables

Results of all groups for polychromatic and normochromatic erythrocytes

Dose group	Total number of		MN (o/oo) in
	PCE’s	NCE’s	PCE’s	NCE’s
Vehicle control	10000	3894	0.7	1.0
500 mg/kg bw	8000	2128	1.1	0.5
1000 mg/kg bw	10000	4565	1.2	1.3
2000 mg/kg bw	10000	3080	1.2	0.3
CPP, 20 mg/kg bw	10000	4211	13.6*	0.9
VCR, 0.15 mg/kg bw	10000	4294	45.8*	0.2

 

Results of all groups for polychromatic erythrocytes: differentiation between small and large micronuclei

Dose group	Total number of PCE’s	Cell (o/oo) with
		MN (d < D/4)	MN (d≥D/4)
Vehicle control	10000	0.7	0.0
500 mg/kg bw	8000	1.1	0.0
1000 mg/kg bw	10000	1.2	0.0
2000 mg/kg bw	10000	1.2	0.0
CPP, 20 mg/kg bw	10000	13.6*	0.0
VCR, 0.15 mg/kg bw	10000	45.8*	11.3*

p≤0.01

Under the experimental conditions chosen here, the test substance 520 F- chlormethyloximether has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.