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:
2005-05-23 to 2005-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection - 2004-06-03, 2004-07-19 to 2004-07-22; date of signature 2005-01-06
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH, D-33178 Borchen, Germany
- Age at study initiation: males 5-8 wks; females 7-10 wks
- Weight at study initiation: Males - mean 37.6 g, std dev. 2.8 g. Females - mean 33.9 g, std dev. 1.9 g
- Assigned to test groups randomly: Yes (within sexes)
- Fasting period before study: No data
- Housing: Singly in Makrolon Type I cages with wire mesh top.
- Diet: Pelleted standard diet ad libitum.
- Water: tap water ad libitum.
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-80 %
- Air changes (per hr): No data
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle: Corn oil
- Justification for choice of solvent/vehicle: No data
- Concentrations of test material in vehicle: 50mg/mL, 100mg/mL, 200 mg/mL
- Volume administered: 10 mL/kg bw

Details on exposure:
No data
Duration of treatment / exposure:
No applicable
Frequency of treatment:
Each animal treated once.
Post exposure period:
24 hr and 48 hr.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.
24 hr preparation interval only.
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
24 hr preparation interval only.
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
24 and 48 hr preparation intervals.
No. of animals per sex per dose:
2000 mg/kg bw dose level - 12 males, 12 females.
500 and 1000 mg/kg bw dose levels - 6 males, 6 females.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): The stability of CPA at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hr.
- Route of administration: Oral
- Vehicle: Corn oil
- Dose: 40 mg/kg bw
- Volume administered: 10 mL/kg bw
- Concentration: 4 mg/mL

Examinations

Tissues and cell types examined:
Tissue: Bone marrow.
Cell type: Erythrocyte
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- A pre-experiment for toxicity was initially performed sequencially with doses of 100 mg/kg bw, 500 mg/kg bw and 2000 mg/kg bw. 4 animals were treated per dose.
- A maximum dose of 2000 mg/kg bw for non-toxic test items is standard practice for this type of test.

TREATMENT AND SAMPLING TIMES:
- 24 hr and 48 hr after treatment

DETAILS OF SLIDE PREPARATION:
- Animals sacrificed by CO₂ asphyxiation and were then bled. The femora were removed, ephiphyses were cut off and the marrow flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 G) for 10 mins and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. 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 100× oil immersion objectives. At least 2000 polychromatic erythrocytes (PCEs) were analysed per animal for micronuclei. To describe a toxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed as PCEs per 2000 erythrocytes. The analysis was performed on coded slides
- 10 animals (5 male, 5 female) per test group were evaluated. The 6th animal is a spare in case of mortality.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (see below) can be used as an aid to evaluating the results. However, the primary point of consideration is the biological relevance of those results.

A test item that fails to produce a biologically-relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic.
Statistics:
Non-parametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At > 500 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 500, 2000 mg/kg bw
- Some animals exhibited clinicial signs of toxicity at doses > 500 mg/kg bw. See attached supporting materials for details.

RESULTS OF DEFINITIVE STUDY
- Some animals exhibited clinical signs of toxicity at doses > 500 mg/kg bw. See attached supporting materials for details.
- The mean micronucleus frequencies for all dose groups of the test material were below the vehicle control value
- Statistical evaluation: Since the mean micronucleus frequencies for all dose groups of the test material were below the vehicle control value were not tested for significance. The positive control value was found to be significantly different (Mann-Whitney test, p < 0.0001).
- See attached supporting materials for full details.

Applicant's summary and conclusion

Conclusions:
The test material was found to be not mutagenic in this micronucleus assay (OECD guideline 474).