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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study, carried out by the Mitsubishi Chemical Safety Institute Ltd. (Japan). Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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): HEMA
- Analytical purity: 99.7%
- Storage condition of test material: cold, dark storage

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 7 weeks old
- Weight at study initiation: 259 to 282 g
- Housing: polycarbonate cages; 5 per cage
- Diet (e.g. ad libitum): pellets; ad libitum
- Water (e.g. ad libitum): flitered tap water; ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 to 23.2 deg C
- Humidity (%): 47.2 to 60 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 2000-11-28 To: 2000-12-03

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Dosing volume: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in water for injection. Then, this solution was diluted with the same solvent. Dosing solutions were administered to rats immediately after preparation.

Duration of treatment / exposure:
2 days
Frequency of treatment:
Twice at 24 hour interval
Post exposure period:
Animals were sacrificed 24 hours after final administration
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg
Basis:

No. of animals per sex per dose:
5/group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: dissolved in water for injection at 1 mg/mL just before use.

Examinations

Tissues and cell types examined:
The femurs were dissected out and the bone  marrow cells were collected.
The animals were weighted just before administration and before  preparation of specimen. The animals were observed once or more per day of administration and  preparation of specimen.
Details of tissue and slide preparation:
The animals in each group were sacrificed 24-hours after the final administration.

Each rat was sacrificed by exsanguination from  the abdominal aorta under anesthethesia with Ravonala (Tanabe Seiyaku  Co.,Ltd. lot no. 07004) and the femurs were dissected out. The bone  marrow cells were collected with PBS(-) (Nissui Pharmaceutical Co.,  Ltd.). The cells were centrifuged at 200 rpm for 5 min and were taken the  supernatant. It wasresuspended in 10 % buffered formalin (Muto Pure  Chemicals,Co., Ltd.) and was centrifuged at 1000 rpm for 5 min. After  centrifugation, the cells were washed twice. Then, the cells were  resusupended in a small amount of 10 % buffered formalin, and was  stocked. The bone marrow suspension was stained with acridine orange, and  was spread on a clean slide glass.

Slides were examined under blind condition and scored under a fluorescent  microscope with B-2 excitation filter. One thousand erythrocytes were  scored from each slide in order to determine the ratio of polychromatic  erythrocytes (PCE's) to the total erythrocytes [PCE's and normochromatic  erythrocytes (NCE's)]. PCEs were further scored up to 2000 cells, the  number of micronucleated PCE's (MNPCE's) in a slide were examined (2  area, Total 2000 cells). PCE's and NCE's were identified according to the  method of Hayashi et al 1.  
Evaluation criteria:
Only when a test substance induced a significant increase in the total number of dose-dependent MNPCEs, the test substance was considered positive in this assay.
Statistics:
For the analysis of the percentage of PCEs, Student's t-test were applied. For the incidence of MNPCEs, tables of Kastenbaum and Bowman were applied.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no significant differences in the incidence of MNPCE's between any treatment group and the negative control group. There were significant increases in the incidences of PCE's between 1000 mg/kg group and the negative control group (p < 0.05).  Clinical signs were not observed with all of test substance groups.  The negative control incidences of MNPCE's among tests was within the range of our laboratory  background data and positive control ones showed remarkable increase.  These findings indicated that the test was conducted appropriately.  This chemical does not induce micronuclei under the test conditions employed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, the test substance did not induce micronuclei.
Executive summary:

A bone marrow micronucleus study in seven week old, Sprague Dawley rats was conducted to assess the mutagenic potential of the test substance. The test substance was administered twice at 24 -hour interval by oral gavage at three doses of 500, 1000 and 2000 mg/kg. Twenty four hours after the final administration bone marrow samples were prepared, and were examined for incidence of micronucleated polychromatic erythrocytes. The test substance did not induce significant increases in the micronucleated polychromatic erythrocytes in any treated groups.