Registration Dossier

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
Difference in treatment schedule (12 week treatment, 3 times a week), No positive control
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
hamster, Chinese
Strain:
other: Cricetulus griseus
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: female: 23-30 g, male: 22-34 g
- Diet: NAFAG No. 924
- Water: Tap water ad libitum
- Acclimation period: Air conditioned room

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 °C
- Humidity (%): 50-72 %
- Photoperiod: Illuminated for 12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Polyethylene glycole 400
- Concentration of test material in vehicle: 250, 500, 1000 and 2000 mg/kg in 20 mL/kg PEG 400
Details on exposure:
Treatment schedule: The preparation was administered orally to groups of 6 female and 6 male animals each, thrice weekly for twelve weeks. Six hours after the last application the animals were sacrificed by dislocation of the cervical vertebra.
Duration of treatment / exposure:
12 weeks
Frequency of treatment:
thrice weekly
Post exposure period:
no
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males
6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
none

Examinations

Tissues and cell types examined:
Bone marrow from the shafts of both femurs.
Cell types examined: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronucleiin leuocpoietic cells, polyploid cells.
Details of tissue and slide preparation:
PREPARATION OF BONE MARROW:
The animals were sacrificed six hours after the last application. From the bone marrow smears were made.Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous 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 airdried. Three hours later, the slides were stained in undiluted May-Griinwald solution for 2 min then in May-Griinwald 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 are left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying the slides were cleared in Xylol and mounted in Eukitt.

DETAILS OF SLIDE PREPARATION:
The slides of three female and three male animals per group 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) micronucleiin leucopoietic cells, e) polyploid cells.
Statistics:
The significance of difference was assessed by X^2 test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
Of the twelve animals in the control group one female and one male animal died during the seventh week of the experiment. Of the animals treated with the lowest dose (250 mg/kg) one female and one male animal died during the second week of treatment. In the group treated with 500 mg/kg one male animal of the twelve animals died by the sixth week. Of the twelve animals in the group given 1000 mg/kg of the test substance all survived. In the group treated with the highest dose (2000 mg/kg) two male animals died during the second and the sixth week respectively of the treatment and one male animal died during the seventh week. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control.

Any other information on results incl. tables

 

Number of animals

Sex of females

Single Jolly bodies

Fragments of nuclei
in erythrocytes

Micronuclei in
erythroblasts

Micronuclei in
leucopoietic cells

Polyploid cells

Total

Control

1

m

 

 

 

 

 

0.0

(PEG 400)

2

m

0.1

 

 

 

 

0.1

 

3

m

0.1

 

 

 

 

0.1

 

4

f

 

 

 

 

 

0.0

 

5

f

0.2

 

 

 

 

0.2

 

6

f

0.1

 

 

 

 

0.1

Test item

1

m

0.1

 

0.1

 

 

0.2

(250 mg/kg)

2

m

0.1

 

 

 

 

0.1

 

3

m

 

 

 

 

 

0.0

 

4

f

 

 

0.3

 

 

0.3

 

5

f

0.1

 

 

 

 

0.1

 

6

f

0.1

 

 

 

 

0.1

Test item

1

m

 

 

 

 

 

0.0

(500 mg/kg)

2

m

0.1

 

 

 

 

0.1

 

3

m

0.2

 

 

 

 

0.2

 

4

f

 

 

 

 

 

0.0

 

5

f

0.1

 

 

 0.1

 

0.2

Test item

6

f

 

 

 

 

 

0.0

(1000 mg/kg)

1

m

 

 

 

 

 

0.0

 

2

m

0.1

 

 

 

 

0.1

 

3

m

0.2

 

 

 

 

0.2

 

4

f

0.1

 

 

 

 

0.1

 

5

f

 

 

 

 

 

0.0

 

6

f

0.1

 

 

 

0.1

0.2

Test item

1

m

 

 

 

 

 

0.0

(2000 mg/kg)

2

m

 

 

 

0.1

 

0.1

 

3

m

0.1

 

 

 

 

0.1

 

4

f

 

 

 

0.1

 

0.1

 

5

f

0.4

 

 

 

 

0.4

 

6

f

0.1

 

 

 

 

0.1

Applicant's summary and conclusion

Conclusions:
The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.