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:
29-jan-2004 to 23-apr-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SB-322069
- Substance type: White solid
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margrate, Kent
- Age at study initiation: 5 to 8 weeks
- Weight at study initiation: 20 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of seven in solid-floor polypropylene cages with wood-flake bedding
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 120, 60 and 30 mg/ml
- Lot/batch no. (if required): B1011

Duration of treatment / exposure:
Treatment:
Solvent control and highest dose level: 24 and 48 hours
Positive control, low and mid dose: 24 hours
Frequency of treatment:
Once
Doses / concentrations
Remarks:
Doses / Concentrations:
1200, 600 and 300 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Seven animals per dose

Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Accepted and approved by authorities and international guidelines
- Route of administration: orally
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow smears

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium

METHOD OF ANALYSIS:
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal are scored.
- The number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes is counted; these cells are also scored for incidence of micronuclei.

Evaluation criteria:
A positive mutagenic response was demonstrated when statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24- or 48-hour kill times when compared to their corresponding control group.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
The data was analysed following a √(x + 1) transformation using Student’s t-test (two-tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
1.25 mg/kg Intravenous. Since, due to the limited solubility of the test material in 0.9% saline, the maximum dose level was limited to 1.25 mg/kg, where no evidence of toxicity was observed.
1200 and 1000 mg/kg, Intraperitoneal.
- Clinical signs of toxicity in test animals: hunched posture, diarrhoea, pilo-erection and ptosis at 1000 mg/kg and above


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: hunched posture, diarrhoea, ataxia, lethargy and ptosis at and above 300 mg/kg
- Induction of micronuclei (for Micronucleus assay): No statistically significant increases in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their vehicle control groups.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their vehicle control groups. This accompanied with the presence of clinical observations was taken to indicate that systemic absorption had occurred.
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of SB-322069 treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.

The test material was considered to be non-genotoxic.