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:
January 7 - March 19, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to OECD test guidelines and GLP Principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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): m-Xylylene diisocyanate (XDI)
- Physical state: colorless transparant liquid

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 7 weeks old
- Weight at study initiation: 217-266.2 g, average 237.4 g
- Assigned to test groups randomly: yes
- Housing: 2 animals housed per cage (3 cages/group). Housed in hanging steel cages with wire-mesh floors.
- Diet: ad libitum to a diet of pellets (MF, Oriental Yeast Co., Ltd.)
- Water: ad libitum Hita City Water Supply (chlorine addition water) via an automatic water supply device.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8-23.9
- Humidity (%): 54.9-64.8
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: The test substance was dissolved at 100 mg/ml in olive oil. The test substance solution at 100 mg/ml in olive oil showed no change in color, exothermic reaction nor gas generation at room temperature within 4 hours after preparation. Therefore olive oil was preferably selected as vehicle.
- Concentration of test material in vehicle: 100 mg/ml was further diluted.
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 042OMS
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulation was prepared on each administration day, stored at room temperature and used within 4 hours of preparation.
5.0 gram of test substance was weighed, vehicle was added and the test substance was dissolved by stirring to make 50 ml of 100 mg/ml tes substance solution. A portion of 50.0, 25.0, 12.5 and 6.25 mg/ml test substance formulation was made.
10 ml/kg was administered.
Duration of treatment / exposure:
Twice
Frequency of treatment:
24 hour interval
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 125, 250, 500, 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: mitomycin C
- Justification for choice of positive control(s): MMC was described as the positive control substance in OECD guideline and the testing facility has background data.
- Route of administration: Intraperitoneal
- Doses / concentrations: 2 mg/kg/day

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the acute oral toxicity study and fourteen day repeated dose oral toxicity study, it was judged that there was no sex difference in toxicity of the test substance. Dead animals were observed on the fourth day of the administration of 1000 mg/kg/day in the 14-d study. Therefore, male rats were used and the highest dose of test substance was 1000 mg/kg/day.

DOSES for observation:
The specimens of the negative and the positive control group and the top three doses were selected for observation. Specimens of 5 animals were taken for analysis.

TREATMENT AND SAMPLING TIMES:
24 hours after second administration animals were euthanized by cervical dislocation. The femur was removed and bone marrow cells were collected with approximately 3 ml of heat inactivated fetal bovine serum into a centrifuge tube, and the tube was centrifuged at 1000 rpm for 5 minutes.

DETAILS OF SLIDE PREPARATION:
A small amount of cell suspension was smeared on a slide glass. The smear specimens were fully air dried and fixed with methanol. In all animals, 2 specimens were prepared per animal. A drop of 40 microg/ml acridine orange solution was dropped onto the specimens, which were then covered with cover glass. Staining was carried out immediately before observation and specimens were observed under a fluorescence microscope.

METHOD OF ANALYSIS:
Two thousand immature erythrocytes (IE) per animal (1000 IE per specimen) were observed and the incidence of the IE with micronuclei (MNIE) per IE, abbreviated as MNIE%, was calculated.
Two hundred total erythrocytes per animal (100 TE per specimen) were observed and the ratio of IE per TE, abbreviated as IE%, was also calculated.
Evaluation criteria:
Study was valid, when the means of the MNIE% in the negative and the positive control groups were within the range of the background data in the testing facility and IE% of each dose of the test substance were not under 20% of negative control.
When MNIE% of the test substance treatment groups increased significantly compared with the negative control group and dose dependency was exhibited, the results for the test substance are judged to be positive.
Statistics:
Judgment of MNIE%: Conditional Binomial test (Kastenbaum and Bowman) was performed. When a significant increase was obtained at a dose, the Cochran-Armitage trend test would be expected to conduct to confirm whether there was a dose dependency.
Judgment of IE%: Bartlett's test for homogeneity of variance (all data except positive control). Because the variances were homogeneous, Williams' test was performed. No significant differences were found, Dunnett's test was performed to compare the mean in the negative control group with that in each dose group. Data from the negative and positive control groups were tested by the F test for homogeneity of vaiance between the groups. Since the result showed homogeneity, Student's t test was performed in order to compare the mean in the negative control group with that in the positive control group.
Statistical tool StatLight was used for mentioned tests.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No animals died before the end of the study. Clinical signs were seen in 1 animal in the 62.5 and 125 mg/kg/day dose group and in 4 animals in the 250 mg/kg/day dose group and in all animals in 500 and 1000 mg/kg/day group, until 1 day after the second administration. Clinical signs included decreased spontaneous locomotion, salivation, aperture respiration and incomplete eyelid opening.
Bodyweight of 2 animals in 250 mg/kg/day group and 1 animal in 1000 mg/kg/day group decreased by 11, 17 and 14%, respectively at 1 day after second administration. Other animals showed body weight within 10 % of starting body weight.

Any other information on results incl. tables

The average of MNIE% in the negative control and positive control groups were 0.17 and 3.47% respectively. MNIE% in te test substance groups was 0.13% at 250 mg/kg/day, 0.11% at 500 mg/kg/day and 0.10% at 1000 mg/kg/day. Thus, no significant increase was noted at any dose of the test substance treatment groups when compared with the negative control group. A significant increase was observed in the positive control group.

IE% in the negative control and positive control groups were 58.0 and 54.4% respectively. IE% at 250 mg/kg/day was 58.7%, at 500 mg/kg/day was 58.3% and at 1000 mg/kg/day was 55.6%. Thus, no significant difference was noted at any doses of the treatment groups and the positive control group compared with the negative control group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The frequency of the micronuclei did not increase by administering the test substance to rats. The positive control showed a significant increase compared to the negative control, it was confirmed that the test was valid.
The exposure of substance to bone marrow cells could not be proved as the IE% did not show significant differences in substance treated compared to negative control. However, doses were set based on dead animals found after 4 days of administration in 14-d repeated dose toxicity study, and toxic symptoms were observed in this study. The potential to induce micronuclei could be evaluated adequately in this study.
Executive summary:

In an in vivo micronucleus study performed according to OECD test guideline 474, XDI was administered orally twice up to 1000 mg/kg bw/day to rats. The frequency of the micronuclei did not increase by administering the test substance to rats. The positive control showed a significant increase compared to the negative control, it was confirmed that the test was valid. The exposure of substance to bone marrow cells could not be proved as the IE% did not show significant differences in substance treated compared to negative control. However, doses were set based on dead animals found after 4 days of administration in 14-d repeated dose toxicity study, and toxic symptoms indicative of systemic exposure were observed in this study. The potential to induce micronuclei could be evaluated adequately in this study. Therefore, it was concluded that XDI had no potential to induce the micronuclei under the present test conditions.