Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Chromosomal breakage
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and appropriate guidelines
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
not relevant
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
E-AF098T (Batch No. T# 176-3, Lot No. 474-0306-17), a white powder. Stored at room temp, dry, closed container.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Source: Harlan, Frederick, Maryland
Young adult CD-1 (ICR) BR mice
Number of animals and sex: In the dose range-finding test 3 males and 3 females used for each dose group.
In the micronucleus assay- Since no relevant differences in toxicity between sexes were observed in the dose range-finding study, only males were used (5 males for each dose group).

Age at initiation of treatment- approximately 8 weeks old

Weight range at initiation of the test: 34.0 t0 39.6g and 24.3 to 26.6g for the males and females respectively.

Husbandary: The animals were housed in sanitary polycarbonate cages containing Sani-Chips Harwood Chip Laboratory bedding.
Room temperature and relative humidity: 17.8 to 26.1oC and 30 to 70%, respectively. The air handling controls was set up at least 10 air changes/hour. Light: 12 hr artificial light 12 hr dark.
Accommodation: Individually in labelled cages
Acclimatisation period was at least 5 days before start of the treatment. The animals were separated by gender, up to 5 animals per cage during acclimation.
Identification: By ear tag.
Diet- PMI Certified Rodent Diet #5002 ad libitum thorought the day.
Water- Tap water was supplied to each cage ad libitum.

No contaminants were known to be present in the diet or water at levels that might interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
A single oral gavage dose administration.
Dosing volume didn't exceed 20 ml/kg body weight.

PREPARATION OF DOSING SOLUTIONS: In solubility testing in corn oil, the test item was observed to form a white, opaque, slightly viscous, heterogenous suspention at approximately 200mg/mL and a white, opaque, slightly viscous, homogenus suspension at approximately 100mg/mL.
Prior to dosing, the top stock of the test item was prepared by adding the appropriate volume of the vehicle, corn oil, to a pre-weighed quantity of the test item and mixed, forming a homogenous suspension. The top-stock was homogenized for 5-10 min to aid in solubilization. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.

The target dose levels in the micronucleus assay were 500, 1000 and 2000 mg/kg body weight as well.


Duration of treatment / exposure:
Bone marrow was extracted at 24 and 48 hours after dosing.
Frequency of treatment:
A single oral gavage dose administration.
Post exposure period:
All animals were examined immediately after dosing, approximately 1 hour after dosing and at least daily for the duration of this assay for signs of clinical
toxicity and/or mortality.

The animals were sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained.
Post exposure period:
Negative Control (vehicle= corn oil): 24h and 48h.
Positive Control (Cyclophosphamide (CP) 80 mg/kg): 24h
Test Item: 500mg/kg: 24h, 1000mg/kg: 24h, 2000mg/kg: 24h and 48h
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
500 mg/kg, 1000 mg/kg, 2000 mg/kg
No. of animals per sex per dose:
The dose range-finding study was designed on the basis of information provided by the sponsor.
The target doses were: 500, 1000 and 2000 mg/kg body weight.
The daily observations of toxic signs and/or mortality data were used to estimate the higest appropriate dose level (maximum tolarated dose) for the micronucleus assay.

For the "Dose range-finding test": 3 males and 3 females were examined per each dose
For the "Micronucleus assay": 5 males were examined per each dose
Control animals:
yes, concurrent vehicle
Positive control(s):
5 males were dosed with 80 mg/kg body weight Cyclophosphamide (CP) and were sacrified 24h after dose gavage

Examinations

Tissues and cell types examined:
Bone Marrow was extracted from the hind limb bones of all survived animals.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range-finding study was designed on the basis of information provided by the sponsor. The target doses
were: 500, 1000 and 2000 mg/kg body weight. The daily observations of toxic signs and/or mortality data were used to estimate the higest
appropriate dose level (maximum tolarated dose) for the micronucleus assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The hind limb bones were removed for marrow extraction from all
surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

DETAILS OF SLIDE PREPARATION: Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed, stained and protected by mounting with coverslips.

METHOD OF ANALYSIS: Slides prepared from the bone marrow collected from 5 animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

Evaluation criteria:
Assay Acceptable Controls: The vehicle control group mean must lie within the historical control range and will usually be less than 0.4% micronucleated PCEs.
There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control deta.
The criteria for a positive response is the ditection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test item that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the biological relevance of the results are also considered in the final evaluation.
Statistics:
See attached document.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All animals in all dose groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. All animals in
the vehicle and positive control groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint.

The test item did not induce signs of clinical toxicity in the animals at all doses treated.
The test item did not induce statistically significant increases in micronucleated PCEs at any of the doses examined. In addition, the test item was not cytotoxic to the bone marrow at any of the dose levels examined.

The vehicle control group had approximately 0.15% micronucleated PCEs and the group mean was within the historical control range.
The positive control induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard deviation of 3.8 ± 0.75.

Any other information on results incl. tables

While statistical higher values were observed for PCE:NCE ratios in the test item dose group at 500 mg/kg, these higher values were evaluated as variations and were not considered to be biologically significant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item, E-AF098T, was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Executive summary:

The test item, E-AF098T, was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.