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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-10-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus assay in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
3-iodo-2-propynyl butylcarbamate
EC Number:
259-627-5
EC Name:
3-iodo-2-propynyl butylcarbamate
Cas Number:
55406-53-6
Molecular formula:
C8H12INO2
IUPAC Name:
3-iodoprop-2-yn-1-yl butylcarbamate
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 32-40 d
- Assigned to test groups randomly: yes
- Housing: individually in wire-mesh cages
- Diet: ad libitum
- Water ad libitum
- Acclimation period: 19 d

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 / 12 hrs dark / hrs light
- controlled climatic conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
- Concentration of test material in vehicle: as required
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was freshly prepared on the day of administration. The dose solutions were prepared on a weight per volume basis. The dose levels were chosen based on the results of a range finding study.
Duration of treatment / exposure:
single application
Frequency of treatment:
see above
Post exposure period:
sampling times: 30, 48 and 72 hrs post treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
660 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 per treatment dose and vehicle control; 5 for positive control
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
femur bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Preliminary range-finding study

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice by cervical dislocation, bone marrow cells were collected from both femurs of each animal by aspiration into 4 mL of 1:1 fetal calf serum/phosphate buffer. The aspirate was centrifuged for five minutes at approximately 1000 rpm. The supernate was decanted and the cells were resiispended in the remaining volume. Bone marrow smears were prepared for each animal by placing one or two drops of the marrow on a microscope slide and spreading it across the slide with another slide. One slide was made for each animal and marked with the animal's identification number. Slides were allowed to air dry over night.

Slide Staining
The cells were fixed in methanol for 15 minutes and rinsed twice in distilled H2O, Fresh stain was prepared by mixing 75 mL of giemsa stain (Harleco) with 175 mL of phosphate buffer (pH 6.8). The cells were stained in this solution for 30 minutes, rinsed once in the same buffer and once in distilled H2O. Slides were air dried and subsequently mounted with glass coverslips.
Erythrocytes that stain blue or with a bluish tint were scored as polychromatic and those staining red or with a red tint were scored as normochromatic. Only cells showing good morphology were scored. Objects within a cell that appeared to be round, darkly stained and in the same focal plane as the cell were scored as micronuclei.

METHOD OF ANALYSIS:
Cytogenetic Analysis
After all slides were prepared, stained, and coverslipped, a code number was temporarily assigned to each animal by a person not involved in the scoring of the slides. The slides were not decoded until all had been analyzed. One thousand polychromatic erythrocytes (PCE) were scored from each mouse for the presence of micronuclei. Slides were analyzed with a high power oil immersion lens (100x). The number of normochromatic erythrocytes (NCE) per 1000 PCE was also recorded.
Statistics:
Upon completion of all scoring, the slides were decoded and the data were entered into the appropriate group for statistical analyses.

The mean number of micronuclei per 1000 PCE was statistically analyzed by Analysis of Variance (ANOVA) with individual group comparisons performed by Dunnett's-t-Test. The number of micronuclei per 1000 PCE for each animal was entered into the statistical program. The analysis was one-tailed at the 99% confidence interval (p < 0.01).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2/15 animals died at 660 mg/kg bw; 9/15 died at 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 160 - 2500 mg/kg bw
- Clinical signs of toxicity in test animals: One animal died (treatment group: 2500 mg/kg bw); Males and females of the two higher dose groups showed greater loss of body weight

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: none
- Ratio of PCE/NCE: 1.2 to 1.7:1 for control animals; approximately 0.6 to 2.1:1 for treated animals

Any other information on results incl. tables

Clinical Observations and Mortality

The number and severity of abnormal observations increased along with increased dose. Two males from Group 4 (660 mg/kg bw) and seven males and two females from Group 5 (2000 mg/kg bw) died while on study.

Body Weights

Most animals, including controls, exhibited slight body weight loss following treatment. Mice administered the highest level (2000 mg/kg bw) showed the largest decreases in body weight.

Cytogenetic Analysis

Results show that no statistically significant increase in the frequency of micronuclei compared to control values was seen in any of the groups treated with the test item.A statistically significant increase in micronucleus frequency (p = 0.0017) was seen in Group 2, the Cyclophosphamide positive control.

Ratio of Polychromatic (PCE) to Normochromatic (NCE) Erythrocytes

Ratio of PCE to NCE ranged from approximately 1.2 to 1.7:1 for control animals and from approximately 0.6 to 2.1:1 for treated animals. Although a large variance was seen between animals of the same group for the ratio of PCE:NCE, animals exposed to the highest level (2000 mg/kg bw) showed lower PCE:NCE ratios than their controls and PCE:NCE ratios decreased with increasing time in the high dose group.Since polychromatic erythrocytes (PCE) are newly formed erythrocytes, it appears that the test material may be producing toxicity in the nucleated stem cells, which would cause a decrease in the number of new erythrocytes produced.

Applicant's summary and conclusion

Conclusions:
The acute oral administration of 200, 660, and 2000 mg/kg body weight of the test item to male and female mice produced no significant increases in the frequency of micronuclei in the polychromatic erythrocyte cells. Toxic effects of the test material were seen in the clinical observations and animal deaths at the the higher dose levels. Therefore, under the conditions of this study, the test item is considered not to be clastogenic at any of the levels tested.
Executive summary:

The study was designed to evaluate the clastogenic potential of the test item as measured by increases in micronuclei production in polychromatic erythrocytes from the bone marrow of Charles River, CD® -7 mice. A single dose of the test material was administered by oral gavage to four groups of 15 male and 15 female mice at levels of 0, 200, 660, and 2000 mg/kg of body weight.

 

Five males and five females from each group were scheduled to be sacrificed at 30, 48 and 72 hours after the single administration of the test material. Results show that no statistically significant increase in the frequency of micronuclei compared to control values was seen for any of the levels that were tested.