Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 October 2007 to 05 November 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 9580
- Substance type: Clear, colorless liquid
- Physical state: Liquid
- Analytical purity: 99%
- Purity test date: 11/15/2006
- Lot/batch no.: CM 16F176
- Expiration date of the lot/batch: 29 February 2008
- Storage condition of test material: At room temperature in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation: 168.8-175.8 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Overnight
- Housing: 5 animals per cage in polycarbonate (type MIV) cages
- Diet (e.g. ad libitum): SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany, ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days prior to the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 38-80
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From:02 October 2007 To: 05 November 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: None
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test article was dosed unchanged (undilluted)
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg body weight
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
A single dose was administered by oral gavage.
Frequency of treatment:
A single dose was administered.
Post exposure period:
24 hours after administration (one group) and 48 hours after administration (second group).
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
5 males per group
Control animals:
other: Control animals received Milli-Q water by oral gavage
Positive control(s):
Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow smears (femur) were examined from test and control group animals.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: Bone marrow was harvested 24 hours after administration (one group) and 48 hours after administration (second group).
DETAILS OF SLIDE PREPARATION: Slides were stained using Wright stain, dipped in xylene, and embedded in Pertex before being mounted with a coverslip.
METHOD OF ANALYSIS: The number of micronucleated polychrmatic erythrocytes was determined by counting and differentiating the first 1000 erythrocutes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
The test article is considered positive if it induced a biologically as well as statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no cytotoxicity
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: No abnormal signs
- Evidence of cytotoxicity in tissue analyzed: None

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No changes in the number of micronecleated polychromatic erythrocytes was observed in test animal bone marrow smears
- Induction of micronuclei (for Micronucleus assay): The positive control (cyclophosphamide) animal smears induced a statistically significant increase in the number of micronucleated erythrocytes
- Ratio of PCE/NCE (for Micronucleus assay): A ratio was not reported, but no change in the number of micronecleated polychromatic erythrocytes was observed in test animal bone marrow smears

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based on the results of the study, the test article is not clastogenic in the micronucleus assay.
Executive summary:

The potential clastogenicity of the test article was evaluated in the micronucleus test. The study was conducted according to OECD GLP (1997), US FDA GLP, and US EPA GLP regulations. The test method was based on EEC Directive 2000/32/EC, Part B.12 (June 8, 2000) and OECD Guideline 474 (July 21, 1997). The test material was administered as received. Five male rats per group were administered Milli-Q water (negative control), 50 mg/kg cyclophosphamide (CP, positive control) or the test article at 2000 (two group) mg/kg via oral intubation. Bone marrow was harvested at 24 hours (one test article group and negative control) and 48 hours (one test article group and positive control) after dosing. All animals were normal immediately after dose administration. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of negative control animals was within the historical range and CP (positive control) induced a significant increase in the number of micronucleated polychromatic erythrocytes indicating that the assay was valid. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test article. Animals treated with the test article showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the negative controls which indicated there was no toxic effect on the erythropoiesis. Based on the results of this test, the test article is not clastogenic.