Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 24, 2010 to January 25, 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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): E-Y110
- Physical state: Orange powder, solid
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: November 4, 2015
- Storage condition of test material: Room temperature, in a dark place, tight container

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: 288 to 322 g
- Assigned to test groups randomly: yes, based on body weights to give homogeneous distribution of body weights among the groups.
- Fasting period before study: not indicated
- Housing: Polycarbonate cages and bedding, three animals/cage
- 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): 22.0-22.7
- Humidity (%): 53.5-63.0
- Air changes (per hr): 6 to 20
- 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 (not reported in this study, but used in other studies)
- Dosing volume: 20 ml/kg bw/day
- Lot/batch no. (if required): K0C77
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation was performed under a UV-cut light. The test substance formulations were prepared just before each administration. The test substance was weighed: 2.09 and 4.19 g. Vehicle was mixed to the test substance to get a final volume of 40 ml and concentrations of 50 and 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:
once
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000 and 2000 mg/kg/day
Basis:
actual ingested
via gavage
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine (DMN) for 2-hr post treatment group
2-acetylaminofluorene (2-AAF) for 16 hr post treatment group
- Justification for choice of positive control(s): The substance is commonly used as a positve control in UDS tests and is recommended in the applied guideline.
- Route of administration: gavage
- Doses / concentrations: 10 mg/kg/day for DMN and 50 mg/kg for 2-AAF

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, only compound colored stool and chromaturia were noted. Therefore the highest dose was set at 2000 mg/kg/day and the lower dose was set at 1000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
After the post observation period, 2-h or 16-h, specimens were prepared from control groups, and the substance treated groups. Hepatocytes were isolated.

DETAILS OF SLIDE PREPARATION:
Cells were fixed with acetic acid-ethanol (1:3 v/v) solution by the double layer method. Slides with fixed hepatocytes were coated, placed in a dark refrigerator for 7 days for radioactive exposure, after which the slide was dipped in developmental solution. Development was terminated by acetic acid and fixated.

METHOD OF ANALYSIS:
Among three specimens prepared for each animal, 2 slides were examined microscopically, 100 hepatocytes/2slides. Number of net grains and hepatocytes indicating 5 net grains or more (counted as cells in repair) were calculated.

OTHER:
Evaluation criteria:
Study acceptable:
- cell viability of all animals in the negative control groups indicated 50% or more
- individual data of net grains in the negative control groups was within the historical control data
- results of the positive control groups satisfied the criteria for positive response.
Test substance was judged as positive when the mean number of net grains in a group treated, was 5 or more as well as the mean percentage of cells in repair was 20% or more.
Statistics:
Not conducted.

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
- Response: Mean number of net grains and the mean percentages of cells in repair were comparable to those in the negative control groups: <5 net grains and <20% of cells in repair.
- Appropriateness of dose levels and route: All the viabilities of hepatocytes from the animals in the negative control groups, test substance and positive control groups were more than 80% in the 2-h and 16-h post treatment groups, which indicates that the study is acceptable and the hepatocytes were isolated in a proper manner.
- Statistical evaluation: none

Any other information on results incl. tables

No differences in body weight among all the groups of animals. Compound colored stool was observed in the cages of the 2000 mg/kg group at 15 hr after administration. No notable changes in the other groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
E-Y110 did not induce UDS in rat livers and did not have a potential to induce DNA damage in vivo under the conditions of this study (OECD 486).
Executive summary:

E-Y110 was assessed in an in vivo/in vitro unscheduled DNA synthesis test using male rats, 8 weeks old, according to OECD test guideline 486, to examine its ability to induce a DNA damage in vivo by the UDS in rat hepatocytes as an index to assess its genotoxic potential. 3 animals per group received via oral gavage 0, 1000 and 2000 mg/kg. Hepatocytes were isolated from the livers of treated animals at 2 or 16 h after the administration. The incidences of UDS in the cultures were detected by autoradiography. The mean numbers of net grains and percentages of cells in repair in the E-Y110 treated groups were comparable to those in the negative control group and were less than 5 net grains and less than 20% of cells in repair. Negative and positive control groups showed responses as expected, showing the validity of the study. It was concluded that, E-Y110 did not induce UDS in rat livers and did not have a potential to induce DNA damage in vivo under the conditions of this study.