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:
13.01. - 10.02.2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP

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)
Version / remarks:
adopted on July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, 4414 Füllinsdorf, Switzerland
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation:
males mean value: 32.7 +/- 3.1 g; 26.2 g +/- 3.0 g
- Housing: individually in Makrolon type I cages
- Diet: standard pelleted laboratory animal diet, ad libitum; food was withheld 18 hrs before treatment
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 20-70%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in corn oil.
Duration of treatment / exposure:
Single treatment.
Frequency of treatment:
The animals received the test item, the vehicle or the positive control substance once each preparation interval.The dosing volume was 10 mL/kg bw.
Post exposure period:
24 or 48 hrs
Doses / concentrations
Remarks:
Doses / Concentrations:
24 h preparation interval: 500, 1000, and 2000 mg/kg bw. and 48 h preparation interval: 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6 NMRI mice for the 24-hour sampling (all dose groups), 3 NMRI mice for the 48-hour sampling (high dose group only)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow smears from femur; erythrocytes (poly- and normochromatic)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The maximum treated dose of 2000 mg/kg bw was used as the high dose. Lower levels were spaced by a factor of 2.


TREATMENT AND SAMPLING TIMES
The animals dosed with corn oil and the test substance were sacrificed by cervical dislocation 24 or 48 h after the second dosing. The animals treated with cyclophosphamide were sacrificed by cervical dislocation 48 hr after dosing. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.

DETAILS OF SLIDE PREPARATION:
A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.

The study was considered valid as the following criteria are met:
- the negative controls are in the range of the historical control data (0.01 - 0.15 %; mean = 0.066 ± 0.032 PCEs with micronuclei).
- the positive controls are in the range of the historical control data (0.91 - 2.975 %; mean = 1.644 ± 0.446 PCEs with micronuclei).
- at least 80 % of animals are evaluable
Statistics:
Statistical significance at the 5 % level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: between 500 and 2000 mg/kg bw (the maximum guideline-recommended dose) was suitable.
- Solubility: soluble in corn oil
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analyzed: no


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.
- Ratio of PCE/NCE (for Micronucleus assay): After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.
- Appropriateness of dose levels and route: yes
- Statistical evaluation: No statistically significant differences in the frequency of erythrocytes containing micronuclei between the solvent control and the dose groups was observed.

Applicant's summary and conclusion