Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
18 September 1996 and 18 October
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(Drug Approval and Licensing Procedures in Japan, 1992 ISBN 4-8407-1419-3 C3047.)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1592U89UAQ succinate
- Molecular formula (if other than submission substance): Please see Attachment 1.
- Molecular weight (if other than submission substance): Please see Attachment 1.
- Structural formula attached as image file (if other than submission substance): Please see Attachment 1.
- Batch number: #94/5977-045
- Storage: Raw drug: Dry, cool place (15 - 20°C)
- Storage: Prepared compound: Suspensions of the test material will be prepared at the start of the study. The suspensions will be placed in a glass bottle and refridgerated at 4°C after use but allowed to warm to room temperature before next use.


Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Male and female CD-1 mice were received from Charles River Breeding Laboratories, Inc. (Raleigh Facility) on 6 September 1995.
Upon receipt, animals were housed in suspended wire cages in the Toxicology Animal facility. All mice were fed Agway® Prolab® 3000 R-M-H Certified Pellets and were allowed tap water ad libitum.
All mice were acclimated to the controlled environment for 12 days prior to dosing.
Date of Birth: Males: 12 August 1995; Females: 8 August 1995
Body weight range: Male: 24.5 - 32.1g; female: 20.2 - 26.3g.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71 +/- 2°F
- Humidity (%): 50 +/- 10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: Methylcellulose
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 0.5%
- Amount of vehicle (if gavage or dermal): 10.0 mL/kg/day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the appropriate concentrations of the test material for dosing were prepared prior to first dosing. Suspensions were stirred to give uniform mixing of the test compound. Calculations were entered in a Burroughs Wellcome official laboratory notebook or designated sheets.
Concentrations were verified by High Performance Liquid Chromatography at the beginning of the study.
Duration of treatment / exposure:
Three days
Frequency of treatment:
Three daily oral doses of the test material for groups 1-5 and 7-11 and a single oral dose for group 6 (positive control).
Post exposure period:
24 hours after final dose.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Group 2
Basis:
analytical conc.
250 mg/kg/day
Remarks:
Doses / Concentrations:
Group 3
Basis:
analytical conc.
500 mg/kg/day
Remarks:
Doses / Concentrations:
Group 4
Basis:
analytical conc.
750 mg/kg/day
Remarks:
Doses / Concentrations:
Group 5
Basis:
analytical conc.
1000 mg/kg/day
No. of animals per sex per dose:
Five animals of each sex per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral
- Doses / concentrations: 10.0 mL/kg at a concentration of 7.5 mg/mL

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes: polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
Processing of Bone Marrow Cells and Preparation of Slides
The bone marrow cells were processed according to standard tcchniques for micronucleus analysis. Immediately following sacrifice, both femurs of each
mouse were exposed, separated from surrounding tissue, and rinsed free of any blood with Dulbecco's phosphate buffered saline. Two to three drops of a
solution of 50% fetal bovine serum in 1% (hypotonic) sodium citrate was dropped into the marrow canal using a #000 sable hair brush dipped in the solution.
The brush wetted with the same solution was then inserted into the marrow canal, and gently rotated to mix the marrow and to create a cell suspension. Four samples for each femur were streaked on each of two microscope slides. Slides from each animal were inscribed in duplicate with animal number, sex, group, and study number. Slides were air-dried for 1-4 days before fixing and staining.

Slide Staining
Slides were fixed in methanol for 5 minutes and allowed to air dry. Slides were then stained in 0.24 mM acridine orange in Hank's Balanced Salt Solution (pH 6.8) for approximately 3 minutes, rinsed in distilled or deionized water 3 times for 1-3 min each time, and air-dried. Slides were coverslipped before scoring using 1-2 drops Hank's Balanced Salt Solution.
Evaluation criteria:
The test article is assessed for its ability to produce an increased incidence of micronucleated polychromatic erythrocytes.
The final assessment of the test article is made by correlating the frequencies of micronucleated polychromatic erythrocytes to the doses inducing them.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
positive
Remarks:
1000 mg/kg/day
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Antemortem Observations and Measurements
Clinical Signs
No animal deaths or clinical signs of toxicity were noted.

Body Weights
No treatment-related changes were noted.

Analysis of Micronuclei
Bone marrow was taken from groups 1-6. Since no treatment-related toxicity was observed, the 3 highest dose levels (500,750, and 1,000 mg/kg/day) and
the vehicle and positive control groups were analyzed. The % PCEs was based on analysis of 1000 erythrocytes per animal except in the vehicle control
and the test material-treated males where 2000 erythrocytes were scored. The number of micronucleated PCEs (MN-PCEs) was based on analysis of
2000 PCEs per animal except in the vehicle control and the test material-treated males where 4000 PCEs were scored. An additional 2000 PCEs were scored for males because of the high variability in the number of MN-PCEs observed in the highest dose group (1000 mg/kg/day). For the second scoring, additional slides which were not counted in the first scoring were used for most animals unless insufficient numbers of cells were present; all vehicle control and test material-treated groups for male mice were recoded prior to scoring. Data for the individual animals are shown in Tables 2 and 3 in Attachment 2.
Data are summarized by dose, sacrifice time and sex in Tables 4 (males) and 5 (females) (please see Attachment 2). The dose group average of MN-PCEs ±the standard deviation, normalized to 1000 PCEs, is given for each sex and sacrifice time.

For males only, there was a statistically significant difference between the group treated with 1000 mg/kg/day of test material and vehicle controls for the number of MN-PCEs. The mean number of MN-PCEs per 1000 PCEs was 3.9 ±1.96 in males dosed with 1,000 mg/kg/day as compared with 1.7 ± 1.28 for male vehicle control animals. For females there were no statistically significant differences between the test material-treated groups and vehicle controls for the number of MN-PCEs. For the percent PCEs [% PCEs = No. of PCEs x 100/(No. NCEs + PCEs)] there were no statistically significant differences between the test material-treated groups and vehicle controls.
For both sexes, there was a statistically significant (p < 0.05) increase in MN-PCEs of the positive control groups, cyclophosphamide (75 mg/kg) compared to the vehicle controls. The average numbers of MN-PCEs found in the male and female positive control animals were 36.1 and 54.6 per 1000 PCEs, respectively (Tables 4 and 5, please see Attachment 2). The vehicle control animals averaged 1.7 and 1.5 MN-PCEs per 1000 PCEs for males and females, respectively.
Mean percent PCE values for each animal are also summarized in Tables 4 and 5 (please see Attachment 2). Mean percent PCE values for vehicle control groups were 42.2 and 40.0
for males and females, respectively.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females.
Executive summary:

The test material induced a slight increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the highest dose, 1000 mg/kg/day, in male mice in the micronucleus assay; no increase was observed in females. One group of 5 male and 5 female mice was administered three daily oral doses of a vehicle control, and four groups of the same size received three daily oral doses of 250,500,750, or 1000 mg/kg of the test material. In addition, one group of 5 male and 5 female mice were dosed with a single oral dose of 75 mg/kg cyclophosphamide as the positive control. All mice were sacrificed at 24 hours after the final dose. Micronucleus analysis was performed on the three highest dose levels along with the vehicle and positive controls.

Under the conditions of this study, there was a slight, but statistically significant increase in the number of micronucleated PCEs observed in males treated with 1000 mg/kg/day of the test material as compared to the vehicle controls; the mean value for the control males was 1.7 micronucleated PCEs per 1000 PCEs and the corresponding value for males treated with 1000 mg/kg/day was 3.9. At lower doses of the test material in males and at all closes in females, there were no statistically significant differences between the test material-treated groups and vehicle controls. For the percent PCEs there were no statistically significant differences between the test material-treated groups and vehicle controls.

The mean plasma concentration for animals exposed to 1000 mg/kg/day was 42.0 and 72.1 µg/mL free base for males and females, respectively.