Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: White powder
- Physical state: solid
- Analytical purity: no data
- Purity test date: no data
- Lot/batch No.: H42265
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD (USA)
- Age at study initiation: approximately 6 to 8 weeks old
- Weight at study initiation: male 28.5 - 32.5 g, 20.5 - 25.7 g
- Assigned to test groups randomly: random
- Fasting period before study:
- Housing: Mice of the same sex were housed up to five per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 °F
- Humidity (%): 50 - 20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2003-07-28 To:2009-09-01

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
The test arcle-vehicle mixture, the vehicle alone, or the positive control was administered by a single intrperitoneal injection at a dose volume of 20 mUkg. Intraperitoneal injection was selected to maximize delivery of the test arcle to the test system. All mice in the experimental and control groups were weighed immediately before dose administration, and the dose volume was based on individual body weight. Mice were observed afer dose adnistration for clinical signs of toxicity.
Duration of treatment / exposure:
24h (all dose groups)
48h (high dose group)
Frequency of treatment:
single
Post exposure period:
24h (all dose groups)
48h (high dose group)
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide was dissolved in sterile distiled water at a concentrtion of 2.5 mg/mL for use as the positive control.

Examinations

Tissues and cell types examined:
Bone marow cells (polychromatic eryocytes, PCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finder study.

DETAILS OF SLIDE PREPARATION
At the scheduled sacrifice times, five mice per sex per tratment were sacrificed by CO2 asphyxation. Imediately following sacrifce, the femurs were distaly exposed, cut just above the knee, and the bone marow was aspirated into a syringe contaig fetal bovine serum. The bone marow cells were trasferrd to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. Two slides were prepard from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS
To contrl for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic eryrocytes per animal were scored for the presence of micronuclei. The number of micronucleated nonnochromatic erythrocytes in the field of 2000 polychromatic eryrocytes was enumerated for each animaL. The proportion of polychromatic eryocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
All conclusions were based on sound scientific judgement; however. as a guide to interpretation of the data, the test arcle was considered to induce a positive response if a dose-reponsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p < 0.05, Kastenbaum-Bowman Tables) at any sampling time.
Statistics:
Kastenbaum-Bowman tables

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection was seen in male and female mice at 1000 and 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the pilot toxicity study, two male mice each were exposed to the test article at a dose of 1, 10, 100, or 1000 mg/kg body weight while five male and five female mice were exposed to 2000 mg/kg. No mortity was observed during the course of the study and only piloerection was seen in male mice at 2000 mg/kg. In the absence of mortality in the pilot study, the high dose for the micronucleus test was set at 2000 mg/kg.

RESULTS OF DEFINITIVE STUDY
The definitive micronucleus study consisted of seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with the test item at a dose of 500, 1000, or 2000 mg/kg and were euthanized 24 hour after treatment. Animals in other two groups were treated either with the negative control or the test item at a dose of 2000 mg/kg and were euthanized 48 hours afer treatment. No mortality was observed in any male or female mice in the micronucleus study while piloerection was seen in male and female mice at 1000 and 2000 mg/kg. Bone marow cells (polychromatic eryocytes, PCEs), collected 24 and 48 hours after tratment, were examined microscopically for the presence of micronuclei (MPCEs/2000 PCEs/animal). No appreciable reduction in the ratio of polychromatic eryhrocytes to total eryhrocytes was
observed in the test article-treated groups relative to the vehicle control groups suggesting that the test arcle did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in maIe or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman Tables).

Applicant's summary and conclusion