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, 1985 - May 6, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP study with sufficient details acceptable for assessment.

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:
yes
Remarks:
1000 instead of 2000 cells scored per animal
Principles of method if other than guideline:
Treatment consisted of one daily dose (gavage) of 1000, 2000 or 4000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were analyzed for nucleus anomalies resulting from chromosomal damage.
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: Commercial grade

Test animals

Species:
hamster, Chinese
Strain:
other: random outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln.
- Weight at study initiation: females: 20-34 g, males: 22-34 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet (e.g. ad libitum): Standard diet; NAFAG No.924.
- Water: Tap water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 42-44
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5% aqueous solution of sodium carboxymethylcellulose (CMC)
- Amount of vehicle (if gavage or dermal): 20 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No details.
Duration of treatment / exposure:
Treatment consisted of one daily dose on each of two consecutive days.
Frequency of treatment:
Once/day
Post exposure period:
24 h after the second application, the animals were sacrificed by dislocation of the cervical vertebrae.
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 2000, 4000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
- In the tolerability test: 2
- in the mutagenicity test: 6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral/gavage
- Doses / concentrations: 128 mg/kg in 20 ml/kg bw 0.5% CMC

Examinations

Tissues and cell types examined:
Bone marrow was harvested from the shafts bf 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. In this experiment the dose of 4000 mg/kg was determined as the highest applicable in the mutagenicity assay,, together with further two doses, diminishing by a factor of 0.5.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts bf both femurs. 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 polishedcover 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.
Evaluation criteria:
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 chi-square test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
One male animal of the 4000 mg/kg-dose group died in the course of the experiment.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
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 8.12, whereas the negative control yielded a percentage of 0.03. The difference is highly significant (p< 0.05),

Any other information on results incl. tables

Table 1: Percent of cells with anomalies of nuclei, 24 h after second application of test substance.

 

Number of animal

Sex of animals

Single Jolly-Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythroblasts

Micronuclei in leucopoietic cells

Polyploid cells

Totals   

Vehicle control

1

F

0.0

 

2

F

0.0

 

3

F

0.0

 

4

M

 0.2

 

0.2

 

5

M

0.0

 

6

M

0.0

Cyclophosphamide, 128 mg/kg bw

1

F

3.7

1.4

1.8

0.1

7.0

 

2

F

6.8

1.2

0.8

8.8

 

3

F

5.1

2.3

1.9

0.1

9.4

 

4

M

8.6

1.2

2.0

0.3

12.1

 

5

M

4.4

0.7

1.1

0.1

6.3

 

6

M

3.7

0.7

0.3

0.3

0.1

5.1

Test substance, 1000 mg/kg bw

1

F

0.1

 

0.1

 

2

F

 

0.0 

 

3

F

 

0.0

 

4

M

0.1

0.0

 

5

M

 

0.0

 

6

M

 

0.0

Test substance, 2000 mg/kg bw  1  F          0.1
   2  F  0.1        0.1
   3  F          0.0
   4  M          0.0
   5  M          0.0
   6  M  0.1        0.1
 Test substance, 4000 mg/kg bw  1  F  0.1        0.1
   2  F  0.2        0.2
   3  F  0.2        0.2
   4  M  0.2        0.2
   5  M  0.1        0.1
   6  M  0.1      0.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this experiment, no evidence of mutagenic effects were noted in Chinese hamsters treated with the test substance.
Executive summary:

The test substance was evaluated for mutagenicity in a Micronucleus test in Chinese hamsters. Treatment consisted of one daily dose of 1000, 2000 or 4000 mg/kg on each of two consecutive days. The test substance was applied orally by stomach tube. The animals were sacrificed 24 h after the second application and bone marrow smears prepared. The bone marrow smears from animals treated with the various doses of the test substance showed no significant difference 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 8.12% cells with anomalies of nuclei. This is significantly different from the controls (0.03%) treated with the vehicle (0.5% CMC) alone. Therefore, the test substance was considered to be not mutagenic under the presented experimental conditions.