Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-07 to 2009-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
No effect on study; slight problem with analysis of test concentrations
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: 20090226
Purity: 99.38%

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc., 143-1 Sangdeawon-Dong, Jungwon-Gu, Seongnam-Si, Gyeonggi-Do, Korea)
- Age at study initiation: Approx. 7 wks
- Weight at study initiation: Males 29.7-34.0 g; females 23.5-27.5 g
- Assigned to test groups under following basis: Using algorithm of Path/tox system v 4.2.2, so that body weights of each group will have similar weight distribution
- Fasting period before study:
- Housing: ≤ 10 (during acclimation period) and ≤ 6 (during dosing) animals per polycarbonate cage (240 mm width × 390 mm length × 180 mm height), including bedding.
- Diet: Ad libitum, pelleted diet
- Water: ad libitum, filtered, UV-radiated tap water
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature 22 ± 3 °C
- Humidity: 50-60 %
- Air changes: 10-20 times per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: Corn oil
- Justification for choice of solvent/vehicle: Sponsor indicated that test substance was insoluble in water. Solubility tests were conducted in DMSO, distilled water, corn oil. The test material was soluble in corn oil, up to the highest tested concentration of 200 mg/mL, but not in either of the other solvents. Therefore, corn oil was chosen as the solvent/vehicle.
- Volume of administered vehicle and test material: 10 mL/kg bw
Details on exposure:
RANGE-FINDING STUDY
- 4 males and 4 females were dosed with test material at 250, 500, 1000, 2000 mg/kg bw for 2 consecutive days.
- Based on the results of the range-finding study, three dose levels were selected as 500, 1000, 2000 mg/kg bw.

PREPARATION OF DOSING SOLUTIONS:
Duration of treatment / exposure:
2 d
Frequency of treatment:
Daily
Post exposure period:
Approx. 24 hrs
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
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 male, 6 female per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: Cyclophosphamide
- Justification for choice of positive control: Widely used in this type of assay
- Route of administration: Intraperitoneally
- Dose: 70 mg/kg bw
- Volume: 10 mL/kg bw
- Frequency of administration: Single administration

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Based on range-finding study

DETAILS OF SLIDE PREPARATION:
- Animals were sacrificed by CO₂ gas inhalation at 24 hrs after final administration. Bone marrow preparations were made according to Schmid (1975), and 2 slides of the cell suspensions per animal were made.
- Bone marrow cells were collected into 3 mL of foetal bovine serum using a syringe with a 23G needle, centrifuged at approx. 1000 rpm for 5 mins.
- After removing the supernatant, a small drop of viscous suspension was smeared onto clean microscopic slides.
- Preparations were air dried and fixed by submerging in absolute methanol for 5 mins.
- Fixed slides were stained as follows:
-- May Grünwald stain 3 mins
-- May-Grünwald stain (1:1 dilution) 2 mins
-- Giemsa stain (1:6 dilution) 10 mins
- Stained slides were rinsed with distilled water, dried and mounted with a mountant.

METHOD OF ANALYSIS:
- Slides with good staining were coded and examined by microscopy at 1000× magnification
- Small round or oval-shaped bodies with erythrocytes with a size of approx. ¹⁄₅ to ¹⁄₂₀ of the diameter of a PCE were regarded as micronuclei.
- Attention was given to the discriminate micronuclei from artefacts.
- Results were expressed as number of MNPCEs in 2000 PCEs.
- The mean number of MNPCEs ± standard deviation was calculated for each treatment group.
- The PCE/(PCE+NCE) ratio, indicating cytotoxicity to haematopoietic system was calculated by counting 500 cells.

OTHER:
- Mortality and external appearance of animals was checked and recorded daily during the study period.
- During the 24 hr post exposure period there were 3 sets of observations
- Bodyweights were measured on the day of reception, grouping, dosing and autopsy.
Evaluation criteria:
- The results would have been considered positive if there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs at at least one dose level.
- The study would have been accepted if PCE/(PCE+NCE) ratios were all >0.1, and the number of MNPCEs in the vehicle control group was within range of the laboratory's historical background data.
Statistics:
Performed according to Lovell et al. 1989 with minor modification, with a significance threshold of p < 0.05
- Test for differences in numbers between PCEs between treated group and vehicle control group: Kruskal-Wallace H-test and Dunn's rank sum test.
- Test for differences in numbers of PCEs between positive control group and vehicle control group: Mann-Whitney U-test
- Tests for differences of PCE/(PCE+NCE) ratio between treated group and vehicle control group: arcsine value of each datum was subjected to Fisher's ANOVA test and Dunnett's test.
- Test for differences of PCE/(PCE+NCE) ratio between positive control group and vehicle control group: arcsine value of each datum was subjected to Student's t-test
- Comparison of bodyweight of animals: Bartlett's test, Fisher's ANOVA if p > 0.05
- Dose-response: Cochran-Armitage trend test
- Vehicle control and test item groups: Pearson's χ² test, and Fisher's exact test if p < 0.05
- Test for dose-response: Cochran-Armitage trend test if p < 0.05 in the above analyses.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: None

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei per 2000 PCEs:
-- Vehicle control: Male 1.00; female 0.83
-- 500 mg/kg bw: Male 1.17; female 0.50
-- 1000 mg/kg bw: Male 1.00; female 1.17
-- 2000 mg/kg bw: Male 1.33; female 1.33
-- CPA: Male 50.17; female 43.17
- Ratio of PCE/NCE:
-- Vehicle control: Male 0.57; female 0.55
-- 500 mg/kg bw: Male 0.60; female 0.56
-- 1000 mg/kg bw: Male 0.57; female 0.57
-- 2000 mg/kg bw: Male 0.56; female 0.55
-- CPA: Male 0.44; female 0.44

- Statistical evaluation:
-- There were no statistically-significant increases in the number of MNPCEs at any dose of the test article compared to the vehicle control group
-- There was a significantly significant increase in the number of MNPCEs in the positive control groups (p < 0.01 in both males and females)
-- There was no statistically significant differences in the PCE/(PCE+NCE) ratio between vehicle control and article-treated groups
-- A statisticaly significant difference was observed in the PCE/(PCE+NCE) ratio between positive control and vehicle control groups (p < 0.01, in both males and females)
- Clinical signs: No clinical signs or behavioural alterations were noted during the observation period.
- Bodyweights: There were no statistically-significant differences in the mean body weights of test-item treated groups and positive control groups compared to those of the vehicle control.

VALIDITY OF TEST
- The test was considered valid as the PCE/(PCE+NCE) ratios were all > 0.1, and the number of MNPCEs in the vehicle control group was in the range of historical control data.

DOSING ANALYSIS
- The dosing concentrations were determined to be 105.50 % to 108.28 % of the target concentrations and the concentrations of test material were stable.

Applicant's summary and conclusion

Conclusions:
The test susbtance was assessed for genotoxicity according to OECD Guideline 474. Under the conditions of the study the oral adminsitration of the test substance at doses up to 2000 mg/kg did not induce an increase in the incidence of micronucleated polychromatic erthrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucelus test.