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:
From Nov. 23, 1987 to May 24, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Only 1000 erythrocytes scored per animal.
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): TK 12599

Test animals

Species:
hamster, Chinese
Strain:
other: random outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Scientific name: Cricetulus griseus
- Weight at study initiation:
Tolerability test: 26-32 g (females) and 30-35 g (males)
Mutagenicity test: 22-33 g (females) and 24-35 g (males)
- Assigned to test groups randomly: yes, selected by random numbers generated by computer.
- Housing: individual caging
- Diet (e.g. ad libitum): NAFAG No. 924
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3-4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 46-62%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 0.5%
- Amount of vehicle (if gavage or dermal): 20 ml/kg


Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
5000 mg/kg bw in 20 mL/kg vehicle
Duration of treatment / exposure:
Single dose administered orally.
Frequency of treatment:
Once
Post exposure period:
16, 24, 48 hours after treatment application.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
Main test
No. of animals per sex per dose:
8/sex/sampling time
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 64 mg/kg in 20 ml/kg 0.5% CMC

Examinations

Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. The highest dose causing no deaths is used as the highest in the mutagenicity test. No deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen as no death occurred at this concentration.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Evaluation criteria:
Statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at any sampling time.
Statistics:
The significance of difference was assessed by Chi square-test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times.

There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg of TK 12 599 (IRGACURE 184) as compared with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg, sampling time 24 hours) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.47. In comparison with ,the negative control (0.11) this value is highly significant (p <0.05).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No evidence for a clastogenic effect in Chinese hamster bone marrow cells was obtained after treatment by gavage with a single dose of 5000 mg/kg.