Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
other company data
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material: MCS 2518
- Physical state: colorless liquid
- Analytical purity: 99.8%
- Lot/batch No.: MIC-5041454
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Portage, MI.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes, by a computer-generated randomization scheme.
- Fasting period before study: not specified
- Housing: two per cage prior to dosing and one per cage after dosing
- Diet (ad libitum): Purina Certified Laboratory Rodent Chow No. 5002 (Trademark of Purina Mills Inc., St. Louis, Mo.)
- Water (ad libitum): supplied by the public water system of St. Louis, MO
- Acclimation period: minimum of 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-21.1
- Humidity (%): 40-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-hour light cycle

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 10 mL/kg bw
Details on exposure:
Animals were treated by a single intraperitoneal injection. Animals were sacrificed for micronucleus evaluation (five animals/sex/group) at 24, 48 and 72 hours after dosing.
Duration of treatment / exposure:
Single treatment
Frequency of treatment:
Doses were administered once.
Post exposure period:
up to 72 hours
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
600 mg/kg bw (total dose)
Dose / conc.:
1 200 mg/kg bw (total dose)
No. of animals per sex per dose:
Test group: 15
Vehicle control: 15
Positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide in saline
- Justification for choice of positive control(s): not specified
- Route of administration: intraperitoneal injection
- Doses / concentrations: 10 mL of solution/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow from both femora of each animal were pooled for slide preparation.
For each animal, two scorers each evaluated:
- 500 total erythrocytes for polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)
- 500 PCEs for micronucleated polychromatic e rythrocytes (MN PCEs).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose levels for the main study were selected based on toxicity rangefinding study data. The maximum dose selected for testing in the micronucleus experiment was 1200 mg/kg bw (approximately 69% of the combined calculated LD50 of 1730 mg/kg).

DETAILS OF SLIDE PREPARATION:
All animals were sacrificed by cervical dislocation and their femora were removed. Each bone was opened at the end and the bone marrow was flushed with approximately 2 ml of fetal bovine serum in a centrifuge tube. Bone marrow from both femora of each animal were pooled for slide preparation. The suspension was centrifuged to remove the serum. Portions of the remaining cells were placed on a clean glass microscope slide and a smear was prepared. Two slides were initially prepared for each sample and the remaining cell suspension was stored refrigerated to prepare additional slides if needed. Following preparation of the smears the slides were allowed to air dry overnight. The slides were stained using a HemaTek II slide staining machine and a Wright-Giemsa Stain Pak which includes stain, buffer and rinse solutions.
Evaluation criteria:
To determine whether a statistically significant response in MN PCE frequency is treatment related the following criteria are considered:
- whether there are dose and time-dependent effects that are consistent with a treatment-induced response
- the degree of the response in relation to both concurrent and historical negative and positive control data
Statistics:
Micronucleated PCE frequencies observed for each animal were transformed as the square root prior to analysis (Snedecor and Cochran; 1967, MacGregor et al., 1987). PCE/total erythrocyte ratios were not transformed. A Dunnett's test (one sided) was used for comparison of treatment group and positive control values with vehicle control values (Dunnett, 1955). A critical value of p≤0.05 was used for statistical significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the rangefinding experiments, the tes substance was found to be toxic to male and female CD-1 mice at 2000 mg/kg bw and greater as indicated by clinical signs of toxicity to the treated males and deaths in the treated males and females. Using the binomial method, the combined LD50 was determined to be 1730 mg/kg. Based on these results, a target dose of 1200 mg/kg (approximately 69% of the combined LD50 value) was selected as a maximum dose that would insure a reasonable probability of observing signs of toxicity but allow survival of the treated animals through the 72 hour time point. Two additional lower doses (300 and 600 mg/kg bw) were also selected for testing.

RESULTS OF DEFINITIVE STUDY
No deaths were observed in the treatment or control groups. All animals in the treatment and control groups appeared normal throughout the experiment and no statistically significant decreases in mean body weights compared to control group values were observed. No statistically significant decreases in mean PCE/erythrocyte ratios were observed in any of the test substance treatment groups.
No statistically significant increases in micronucleated PCE frequency were observed for test substance dosed groups at any of the sacrifice times. The positive control (cyclophosphamide) yielded expected positive responses in micronucleated PCE frequency indicating the adequacy of the experimental conditions.

Applicant's summary and conclusion