Desmedipham

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Mouse bone marrow micronucleus assay

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985-02-11 to 1985-03-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Desmedipham

EC Number:

237-198-5

EC Name:

Desmedipham

Cas Number:

13684-56-5

Molecular formula:

C16 H16 N2 O4

IUPAC Name:

ethyl 3´-phenylcarbamoyloxycarbanilate

Test material form:

solid: particulate/powder

Details on test material:

The test material is Desmedipham.

Specific details on test material used for the study:

Physical appearance light beige coloured fine powder.
Storage The test article and a reserve sample were stored at room temperature, in the dark.

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Standard laboratory species/strain used for this type of study, with background data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: males: 6 weeks, females: 7 weeks
- Weight at study initiation: males: 26-37 g, females: 26-34 g
- Assigned to test groups randomly: yes, Animals mere randomized into the different groups after delivery using a random algorithm.
- Housing: Groups of 6 in Makrolon Type 3, with wire mesh top and granulated softwood bedding.
- Diet: Pelleted standard mouse diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 7 days under test conditions, with veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12 per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 2% carboxymethylcellulose, (CMC) in distilled water.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A suspension was prepared immediately before application by adding the test article to distilled water containing CMC (2%).
Homogeneity of the suspension was maintained during application using a magnetic stirrer.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

At 24-, 48- and 72-hour intervals after treatment, six mice per sex and group were sacrificed for examination. The first five animals of each sex were evaluated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg bw

Remarks:

Negative Control (2% carboxymethylcellulose, sodium salt (CMC) in distilled water)

Doses / concentrations 2

Dose / conc.:

5 000 other: mg/kg bw

Remarks:

Desmedipham

No. of animals per sex per dose:

18/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (reference mutagen)
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose was based on acute oral toxicity data. The acute oral LD50 performed with mouse is greater than 5000 mg/kg body weight. The 5000 mg/kg body weight dose was used in this study as the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: The animals were dosed once, at 24 -, 48 -, and 72 -hour intervals after treatment, six mice per sex and group were sacrificed for examination, bone marrow was collected

DETAILS OF SLIDE PREPARATION:
All mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue.
The proximal end of the femur was cut with scissors. The needle of a plastic syringe containing 0.2 ml calf serum was inserted into the proximal part of the marrow canal which was closed at the distal end. The femur was submerged in 1.5 ml calf serum in a labeled centrifuge tube. The bone marrow cells mere dispersed in the calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized Pasteur pipette. A small drop of the marrow serum suspension was smeared on the slide, which was identified by project code and the animal number and allowed to dry overnight. Two slides per animal mere prepared. The following day, the smears mere stained using the panoptic stain method developed by Pappenheim

METHOD OF ANALYSIS:
From each animal 1000 polychromatic erythrocytes (PCE) were scored under the microscope for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), based on scoring 1000 erythrocytes per slide, measured the toxicity of desmedipham.

Evaluation criteria:

The frequencies of micronuclei of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution mas applied. Estimation and testing were performed by maximum likelihood method. If a test article produced no statistically significant and reproducible positive response at any one of the test points, it was considered non-mutagenic in this system.

Statistics:

Statistical evaluation of the data was made using a regression model assuming a Poisson distribution and the maximum likelihood method.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Sedation was observed in all treated animals for at least 6 hours after dosing.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

See the Results' tables attached in "Overall remarks, attachments"

Applicant's summary and conclusion

Conclusions:

After a single dose of 5000 mg desmedipham /kg bw by gavage, no significant increase of micronucleated polychromatic erythrocytes in the bone marrow was observed in either male or female treated groups at either 24, 48 or 72 hours post-dose.

Executive summary:

This in vivo study with mice was performed to assess the potential mutagenic activity, induced by DESMEDIPHAM, through damage to the chromosomes or to the mitotic apparatus, according to Schmid (1) with the modifications of Salomone et al.(2).  The experiment was performed with three groups, each containing 18 males and 18 females, for a total of 108 mice.  The negative control groups received the test article vehicle, i.e. 2% carbaoxymethylcellulose sodium salt in distilled water. The test groups received 5000 mg/kg body weight as the maximum tolerated dose of the test article. The positive control groups received 50 mg/kg body weight cyclophosphamide dissolved in 0.9% saline solution.  All animals were given a single application of 20 ml/kg body weight by gavage. Twenty-four (24), 48 and 72 hours after treatment, six mice per sex and group were sacrificed and bone marraw was removed from the femora for examination.  The first five animals were used for evaluation. One thousand polychromatic erythrocytes per animal were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was used to assess the toxicity of the test article by counting a total of 1000 erythrocytes.  Data was statistically analyzed by means of a regression model assuming a Poisson distribution. Estimation and testing were performed by maximum likelihood method.  Sedation was observed in all test article-treated animals for at least 6 hours after application.  After single application of the test article at 5000 mg/kg body weight by gavage, no significant test article-related increase of micronucleated polychromatic erythrocytes was observed in either male and female treated groups, when compared with corresponding negative control groups. These results were found at the three examination times, 24, 48 and 72 hours post-application, respectively. The positive control groups, which received cyclophosphamide, exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes and thus validated, the test.  In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced no chromosome mutations by chromosome-breaking activity or by damage to the mitotic apparatus. Therefore, DESMEDIPHAM is not considered to be mutagenic in this in vivo mouse micronucleus assay.