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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
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment. No Purity of test substance available. Only one sampling time.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Principles of method if other than guideline:
Three Chinese hamsters per sex were treated each with 1250, 2500, and 5000 mg/kg bw test substance by gavage. Treatments were performed once daily on 2 consecutive days. Cyclophosphamide was used as a positive control substance, and a vehicle control was used as negative control. 24 h after the second treatment the animals were sacrificed and the bone marrow was prepared for scoring. 1000 bone marrow cells were scored per animal, and the following parameters were investigated: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells.
GLP compliance:
no
Remarks:
QAU statement available
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
EC Number:
265-512-0
EC Name:
Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
Cas Number:
65140-91-2
Molecular formula:
C17 H29 O4 P. 1/2Ca
IUPAC Name:
calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]
Details on test material:
Purity: no data

Test animals

Species:
hamster, Chinese
Strain:
other: Random Outbred Strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-10 weeks (females), 4-9 weeks (males)
- Weight at study initiation: 21-27 (females), 23-29 g (males)
- Housing: individual caging
- Diet: NAFAG, No. 924
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 61-70
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle: 0.5% Carboxymethylcellulose + 0.1% Tween 80
- Amount of vehicle: 20 mL/kg
- Manufactorer: CMC: Hercules Comp. U.S.A., Tw 80: Fluka AG, Switzerland.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once daily on 2 consecutive days
Post exposure period:
24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 128 mg/kg bw in vehicle

Examinations

Tissues and cell types examined:
Bone marrow.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment once daily on 2 consecutive days. Sampling 24 h after second treatment.

DETAILS OF SLIDE PREPARATION:
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 air-dried. Three hours later, the slides were stained in undiluted May-Gruenwald solution for 2 min then in May-Gruenwald 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 Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
Scoring of the slides.
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 of TK 11 578 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 in each dose-group and 1 female of the intermediate dose group died.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
One male animal each of the low-dose, the intermediate-dose and the high-dose group and one female animal of the intermediate dose group died in the course of the experiment. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control (for details see table 1 "Any other information on results").
By contrast, the positive control (cyclophosphamide, 128 mg/kg bw) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 11.4, whereas the negative control yielded a percentage of 0.08. 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 animals

Sex of animals

Single Jolly-Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythroblasts

Micronuclei in leucopoietic cells

Polyploid cells

Totals

Control

1

F

 

 

 

 

 

0.0

 

2

F

 

 

 

0.1

 

0.1

 

3

F

0.2

 

 

 

 

0.2

 

4

M

0.1

 

 

 

 

0.1

 

5

M

0.1

 

 

 

 

0.1

 

6

M

 

 

 

 

 

0.0

Cyclophosphamide, 128 mg/kg bw

1

F

12.0

2.9

1.9

0.3

 

17.1

 

2

F

10.9

3.1

1.7

0.3

0.1

16.1

 

3

F

4.2

0.7

0.9

0.1

 

5.9

 

4

M

6.8

1.3

0.5

0.2

0.1

8.9

 

5

M

8.9

0.7

0.7

0.3

 

10.6

 

6

M

6.7

1.9

0.8

0.4

 

9.8

Test substance, 1250 mg/kg bw

1

F

 

 

 

 

 

0.0

 

2

F

0.1

 

 

 

 

0.1

 

3

F

 

 

 

 

 

0.0

 

4

M

0.5

 

 

 

 

0.5

 

5

M

0.1

 

 

 

 

0.1

 

6

M

 

 

 

 

 

0.0

Test substance, 2500 mg/kg bw

1

F

0.1

 

 

0.1

 

0.2

 

2

F

0.3

 

 

 

 

0.3

 

3

F

0.1

 

 

 

 

0.1

 

4

M

 

 

 

 

 

0.0

 

5

M

0.3

 

 

 

 

0.1

 

6

M

 

 

 

 

 

0.0

Test substance, 5000 mg/kg bw

1

F

0.1

 

 

 

 

0.1

 

2

F

0.3

 

 

 

 

0.3

 

3

F

0.1

 

 

 

 

0.1

 

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
It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters.
Executive summary:

In a nucleus anomaly test groups of 3 male and 3 female Chinese hamsters were treated with 1'250, 2'500 and 5'000 mg/kg bw to (gavage) for 2 consecutive days and sacrificed 24 h later. One male animal each of the low-dose, the intermediate-dose and the high-dose group and one female animal of the intermediate dose group died in the course of the experiment. No induction of nuclear anomalies and/or chromosomal aberrations was observed in bone marrow cells in any of the dose groups. 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 11.4% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (0.5% CMC + 0.1% Tween 80) alone. In conclusion, the test article is considered to be not mutagenic in this assay.

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