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:
December 2, 2010 to January 6, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD test guidelines and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
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): E-C104
- Physical state: Blue powder, solid
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: July 7, 2015
- Storage condition of test material: Room temperature, in a dark place

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 285 to 316 g
- Assigned to test groups randomly: yes, based on body weights to give homogeneous distribution of body wieghts among the groups.
- Fasting period before study: not reported
- Housing: Polycarbonate cages, two or three animals/cage, bedding for experimental animals and draining boards placed on the bedding.
- Diet (ad libitum): Pellet diet (MF, Oriental Yeast Co., Ltd., analysed and contaminants determined to be within acceptable limits established by the testing facility,
- Water (ad libitum): Tap water filtered through a filter (5 microm) and irration with UV light, analysed twice a year, and determined to be within acceptable limits established by the testing facility.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2-23.0
- Humidity (%): 53.7-65.3
- Air changes (per hr): 10 to 30
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water for injection (Japanese Pharmacopoeia)
- Justification for choice of solvent/vehicle: see other studies
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared just before each administration. The test substance was weighed: 0.660, 1.32, 2.64 and 5.28 g. Vehicle was mixed to the test substance to get a final volume of 50 ml and concentrations of 12.5, 25, 50, 100 mg/ml respectively to a final volume of about 80% of vehicle. Formulations were mixed with a magnetic stirrer, final volume was adjusted with the vehicle.
Frequency of treatment:
twice at a 24 hour interval
Post exposure period:
24 hour after final dosing
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 and 2000 mg/kg/day
Basis:
actual ingested
via gavage
No. of animals per sex per dose:
5 males only
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): The substance is commonly used as a positve control in micronucleus tests and is recommended in the applied guideline.
- Route of administration: gavage
- Doses / concentrations: 20 mg/kg/day

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In the acute oral toxicity study, at 2000 mg/kg no mortality was observed, compound colored stool and chromaturia were noted. Therefore the highest dose was set at 2000 mg/kg/day and the lower doses were set with a common ratio of 2.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
After the post observation period, specimens were prepared from control groups, and three highest dose groups of the test substance (since no animals died in any test substance group). Bone marrow cells were collected.

DETAILS OF SLIDE PREPARATION:
Cell suspension was stained with acridine orange and spread on a slide for microscopic observation.

METHOD OF ANALYSIS:
Bone marrow cell specimens wer observed under a fluorescence microscope with B excitation filter in a blind manner. 1000 erythrocytes/animal inlcuding both immature and mature erythrocytes were examined to determine the % of immature erythrocytes (IMEs) among total erythrocytes. A total of 2000 IMEs were examined for the number of micronucleated cells (MNIMEs).

OTHER:
Evaluation criteria:
Positive: test substance significantly increased the number of MNIMEs as compared to the negative control group with a dose dependency.
Statistics:
MNIMEs: Kastenbaum and Bowman method was applied to compare number of MNIMEs in treatment group with negative control.
IMEs: All data, except positive control, were tested by Bartlett's test for homogeneity of variance among the groups. Since variance was homogeneous, Williams' test was applied to determine significant difference between test group and negative control. No significant difference occurred, and thus Dunnett's test was performed to compare the mean values between test group and negative control. Data from positive control group was compared with negative control by the F test for homogeneity of variance and as the variance was homogeneous the Student's t-test was applied to compare the mean values of the two groups.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit concentration of 2000 mg/kg/day
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
MNIMEs per 10000 (2000x5 animals) IMEs were 13, 14, 13 and 7 at 0, 500, 1000, 2000 mg/kg/day, respectively.
No significant increase in the incidence of MNIMEs in all test substance groups compared to the negative control group and no statistically significant difference in the % of IMEs between the test groups and the negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): see attached tables.
- Appropriateness of dose levels and route: see rationale for dose selection above.
- See attached tables for more information.

Any other information on results incl. tables

No differences in body weight among all the groups of animals. Compound colored stool was observed in 1/5, 3/5 and 5/5 animals in the 500, 1000 and 2000 mg/kg/day group, respectively. Loose stool was observed in 2/5 animals in the 2000 mg/kg/day group at 3 hours after the first dosing.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
E-C104 did not induce micronucleated erythrocytes in bone marrow and did not have a genotoxic potential to induce chromose aberrations in vivo under the conditions of this study.
Executive summary:

E-C104 was assessed in an in vivo micronucleus test in male rats, 8 weeks old, according to OECD test guideline 474, to examine its ability to induce micronucleated erythrocytes in the bone marrow as an index for assessing its genotoxic potential to induce chromosome aberrations in vivo. 5 animals per group received via oral gavage 0, 250, 500, 1000 and 2000 mg/kg twice at a 24 hour interval and bone marrow celld were collected 24 hours after the final administration. Cell specimens did not show a statistically significant difference between the negative control and the test substance groups in hte number of MNIMEs/10000 IMEs nor in the %IMEs. The negative control groups showed results within the range of the historical data. Significant increases in MNIMEs were present in the positive control group, confirming the validity of the study. It was concluded that E-C104 did not induce micronucleated erythrocytes in rat bone marrow under the conditions of this study and E-C104 did not have a genotoxic potential to induce chromosome aberrations in vivo.