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:
1985-04-04 to 1985-05-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study close to Guideline with acceptable restrictions; Non GLP but QA-statement included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
no
Remarks:
but QA statement included
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, insoluble in water
- Analytical purity: commercial grade

Test animals

Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: females: 6-10 weeks, males: 4-9 weeks
- Weight at study initiation: females: 22-30 g, males: 20-29 g
- Housing: individual caging
- Diet: NAFAG No.924
- Water: tap water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 40-44
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
0.5% sodium carboxymethylcellulose (CMC) in water
Details on exposure:
The preparation was administered orally to 3 groups of 6 female and 6 male animals each. Treatment consisted of daily one application on 2 consecutive days. 24 h after the second application the animals were sacrificed by dislocation of the cervical vertebrae.

a) Test substance: 1250, 2500 and 5000 mg/kg bw in 20 mL/kg 0.5 % aqueous solution of sodium carboxymethylcellulose (CMC).
b) Cyclophosphamide (ENDOXAN®): 128 mg/kg bw in 20 mL/kg 0.5 % CMC (positive control).
c) 20 mL/kg bw 0.5 % CMC (negative control)
Duration of treatment / exposure:
One daily application on 2 consecutive days.
Frequency of treatment:
daily
Post exposure period:
24 h after the second application the animals were sacrificed
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1250, 2500 or 5000 mg/kg bw in 20 mL/kg bw 0.5 % aqueous solution of sodium carboxymethylcellulose
Basis:

No. of animals per sex per dose:
The preparation was administered orally to three groups of 6 female and 6 male animals each.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 128 mg/kg bw in 20 mL/kg bw 0.5 % CMC, two consecutive daily treatments

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:
Results from a previously performed tolerability test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 h after the second application the animals were sacrificed by dislocation of the cervical vertebrae and bone marrow was harvested from the shafts of both femurs

DETAILS OF SLIDE PREPARATION:
In a sicliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogenous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40 % for 20 min. After being rinsed in methanol 55 % for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered:
a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.
Statistics:
The significance of difference was assessed by ChiSq -test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (Tolerability test)
- Dose range:
Part 1.: 5000, 1000 and 200 mg/kg bw
Part 2.: (optional) 5000, 2500 and 1250 mg/kg bw
In this experiment the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay, together with further two doses, diminishing by a factor of 0.5.

RESULTS OF DEFINITIVE STUDY
In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 8.08, whereas the negative control yielded a percentage of 0.08. The difference is highly significant (p< 0.05).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this experiment, treatment of groups of Chinese hamsters with the test substance did not cause increased amounts of nucleus anomalies when compared to controls. Therefore the test substance is considered as not clastogenic when tested in hamsters.
Executive summary:

In order to assess the test article's potential to cause clastogenic effects on somatic interphase cells in vivo, female and male Chinese hamsters were treated by gavage with the test substance once daily on two consecutive days at dose levels of 1250, 2500 or 5000 mg/kg body weight. A control group was treated according to the same dosing scheme with the vehicle alone (0.5% CMC). The animals were sacrificed 24 h after the second application and smears from the bone marrow were made. The bone marrow smears from animals treated with the various doses showed no significant increase from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 8.08% cells with anomalies of nuclei. This is significantly different from the controls (0.08%) treated with the vehicle alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.