Benzoic acid, 4-[[[4-[(1-oxo-2-propenyl)oxy]butoxy]carbonyl]oxy]-, 2-methyl-1,4-phenylene 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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: young adult animals (male animals approx. 8- 12 weeks, female animals approx. 14-18 weeks)
- Weight at study initiation: mean weight 28.47 g
- Fasting period before study: the animals were given no feed at least 16 hours before administration, but water was available ad libitum.
- Housing: Makrolon cages type MIII, in groups of 5. The animais were identified using cage cards. No bedding in the cages
- Diet, ad libitum: standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water, ad libitum: drinking water from bottles.
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air-conditioning guaranteed a range of 20 - 24 degrees
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 hours (6.00 am - 600 pm/ 6.00 pm - 6.00 am)

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the insolubility of the test substance in water; DMSO had been demonstrated to be suitable in the in vivo micronucleus test (historical data available).
- Concentration of test material in vehicle: 12.5 g/100 ml, 25 g/100 ml and 50 g/100 ml, respectiveliy for the 3 dose groups

Duration of treatment / exposure:

2 days

Frequency of treatment:

once daily

Post exposure period:

- animals of the vehicle control and the dose groups: samples of bone marrow were collected 24 hours after the last treatment.
- animals of the positive control groups: samples of bone marrow were collected after 24 hours after the unique treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
484, 964, and 1912 mg/kg bw (actually injected in 4 ml DMSO: 500, 1000 and 2000 mg/kg bw)
Basis:
analytical conc.
HPLC

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR)
- Route of administration: ip
- Doses / concentrations: 20 mg CPP in 10 ml solution and 0.15 mg VCR in10 ml solution

Examinations

Tissues and cell types examined:

bone marrow (from femora)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived following two treatments with the highest recommended dose according to the OECD Guideline. No symptomatic differences were observed between the male and female animals. Thus, only male animals were used for the cytogenetic investigations. Therefore, dose of 500, 1000 and 2,000 mg/kg body weight were selected for the present study.

DETAILS OF SLIDE PREPARATION:
- 2 femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mI/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.
- 1 drop of this Suspension was dropped onto clean microscopic sildes. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- The sildes were stained in eosin and methylene blue solution for 5 minutes, 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):
In general, 2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes. An alteration of this ratio indicates that the test substance actually reached the target Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4) (d = diameter of micronucleus, D = cell diameter)
- Slides were coded before microscopic analysis.

OTHER:
Clinical examinations: the animals were examined for any evident clinical signs of toxicity in each treatment group.

Evaluation criteria:

-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.
- An alteration of the polychromatic to normochromatic erythrocytes ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- 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.
- 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 was observed; 2) 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 system if: 1) there was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies; 2) 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.
- A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection and irregular respiration were observed in all tested animals during the 4 subsequent hours to each application, but were completely reversible afterwards.

Vehicle controls validity:

valid

Negative controls validity:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: limit test; 2000 mg/kg bw
- Solubility: yes, in DMSO
- Clinical signs of toxicity in test animals: as clinical signs only piloerection and irregular respiration were observed

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): within the vehicle control range
- Ratio of PCE/NCE (for Micronucleus assay): within the vehicle control range

Any other information on results incl. tables

Genotoxic effects:

- Vehicle control treatment led to 0.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.

- The highest dose administration (2000 mg/kg bw) led to 0.9‰ polychromatic erythrocytes containing micronuclei.

- In the two lower dose groups, rates of micronuclei of about 0.9‰ (1,000 mg/kg group) and 0.6‰ (500 mg/kg group) were detected.

- With 18.6‰ the positive control substance CPP (clastogenicity) led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.

- With 76.3‰, the positive control VCR (induction of spindle poison) also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with an amount of large micronuclei (9.4‰).

- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.

- Thus, the test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d >= D1/4) observed either in the negative control group or in the 3 dose groups.

- No inhibition of erythropoiesis, induced by the treatment of mice with the test substance 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.

Clinical examinations:

- The administrations of the vehicle was tolerated by all animals without any signs or symptoms.

- The administration of the test substance led irregular respiration and piloerection.

- No evident signs of toxicity were observed in the positive control animals.