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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2014
GLP compliance:
yes (incl. QA statement)

Test material

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WS400128, batch no. 0001591644
- Expiration date of the lot/batch: February 2020
- Purity test date: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS:

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd
- Age at study initiation: ca. 41-48 days old
- Weight at study initiation: 29-34 g
- Assigned to test groups randomly: yes
- Housing: group housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 0, 50, 100, 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: mixing of WS400128 with corn oil
Duration of treatment / exposure:
The test item was administered on two ccasions approx. 24 hours apart.
Frequency of treatment:
two times
Post exposure period:
18-24 hours after the second dose
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:
6 males per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
bone marrow smears previously prepared from mice administered Mitomycin C (12 mg/kg bw/day) were stained and coded along with the bone marrow smears prepared in the study summarised .

Examinations

Tissues and cell types examined:
bone marrow from femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary study at max. dose of 2000 mg/kg/day in male and female mice

DETAILS OF SLIDE PREPARATION:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature in the dark until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

METHOD OF ANALYSIS: fluorescence microscopy
Evaluation criteria:
Acceptance Criteria
The following criteria were applied for assessment of assay acceptability:
The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
Concurrent positive control or scoring controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
A maximum tolerated dose or maximum feasible dose has been achieved.
Adequate number of cells and doses have been analysed.
Each treated and control group should include at least 5 analysable animals.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative, if in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and d) Bone marrow exposure to the test item(s) occurred.
Evidence of exposure of the bone marrow to a test item may include a depression of the polychromatic to normochromatic erythrocyte ratio or measurement of the plasma or blood levels of the test item. In case of intravenous administration, evidence of exposure is not needed. Alternatively, ADME data, obtained in an independent study using the same route and same species can be used to demonstrate bone marrow exposure. Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in the polychromatic erythrocytes of the test species.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see attached "Table 2 Individual animal data" for details of results.

Applicant's summary and conclusion