2-Propenoic acid, 4-[[(4-benzoylphenoxy)carbonyl]oxy]butyl ester

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 and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-{[(4-benzoylphenoxy)carbonyl]oxy}butyl prop-2-enoate

EC Number:

603-491-5

Cas Number:

131513-00-3

Molecular formula:

C21H20O6

IUPAC Name:

4-{[(4-benzoylphenoxy)carbonyl]oxy}butyl prop-2-enoate

Test material form:

other: liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Weight at study initiation: mean weight of 28.2 g
- Assigned to test groups randomly: no
- Housing: individual housing in Makrolon cages, type Ml
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 3-5 days

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

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

DMSO
- Vehicle/solvent used: DMSO
- Choice of solvent/vehicle:
Due to the insolubility of the test substance in water, 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.

Details on exposure:

For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were evaluated analytically. This analytical investigation was carried out by means of photometry.

Duration of treatment / exposure:

2 treatments at a 24 h interval, samples taken 24 h after the second treatment

Frequency of treatment:

two administrations

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 400, 800 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 600 mg/kg body weight (at 600 mg/kg and 800 mg/kg body weight only a few animals died on the 7th day after the 2nd administration). Evident clinical signs were observed both using male and female animais. However, species specific differences were not observed and thus, only male animais were used for the cytogenetic investigations. Therefore, a dose of 800 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 400 mg/kg and 200 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W..
The two femora were prepared by dissection and removing all soft tissues. After cutting oft 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 3700 (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS. One 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.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5 % Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
Microscopic analysis and determination of the number of polychromatic erythrocytes containing micronuclei and normochromatic erythrocytes containing micronuclei and determination of the the number of small micronuclei (d < DM4) and of large micronuclei (d > DM4) (d = diameter of micronucleus, D = cell diameter).

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test systemn if:
- There was no significant incmease in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft). The number of micronuclei in polychromatic erythrocytes was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. This test was performed one-sided.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Signs of toxicity were observed in all dose groups.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: Deaths were observed down to a dose of 600 mg/kg body weight. Evident clinical signs were observed both using male and female animals.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The test substance did not lead to any increase in the rate of micronuclei.