Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
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 1250, 2500 or 5000 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
Remarks:
but QAU statment included.
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2',3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]propionohydrazide
EC Number:
251-156-3
EC Name:
2',3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]propionohydrazide
Cas Number:
32687-78-8
Molecular formula:
C34H52N2O4
IUPAC Name:
3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N'-[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]propanehydrazide
Details on test material:
- Physical state: white powder
- Analytical purity: 100%
- Stability under test conditions: not determined
- Storage condition of test material: room temperature

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
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: 6-10 weeks (females), 4-9 weeks (males)
- Weight at study initiation: 20-27 g (females), 22-30 g (males)
- Housing: single housing
- Diet: Standard diet: NAFAG No.924, ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 49-60
- Photoperiod: 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachid oil (20 mL/kg bw)
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once daily for two consecutive days
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 128 mg/kg bw in 20 ml/kg Arachid oil.

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The oral LD50 was found to be >5000 mg/kg bw in Chinese hamsters of either sex (cf. Lab.Report: GU 2, dated March 15, 1983)

TREATMENT AND SAMPLING TIMES:
Single daily dosing of animals for 2 days. 24 h after last dosing, the bone marrow was prepared.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 mL 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:
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 test substance were examined microscopically. 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.
Evaluation criteria:
Statistic significant difference of examined cell types in control and test group.
Statistics:
The significance of difference was assessed by CHI-Square test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
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 bw) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 7.2, whereas the negative control yielded a percentage of 0.17. 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

0.2

 

 

 

 

0.2   

 

2

F

0.2

 

 

 

 

0.2   

 

3

F

0.1

 

 

 

 

0.1   

 

4

M

 

 

 

 

 

0.0   

 

5

M

0.2

 

 

 

 

0.2   

 

6

M

0.3

 

 

 

 

0.3   

Cyclophosphamide, 128 mg/kg bw

1

F

7.7

0.3

1

0.3

 

9.3   

 

2

F

6.5

0.2

1.5

0.8

0.1

9.1   

 

3

F

7.9

0.7

0.3

0.1

 

9.0   

 

4

M

4.9

0.2

0.7

0.1

0.1

6.0   

 

5

M

3.9

 

0.4

0.4

 

4.7   

 

6

M

4.6

0.2

0.3

0.1

 

5.2   

Test substance, 1250 mg/kg bw

1

F

 

 

 

 

0.0   

 

2

F

0.1

 

 

 

 

0.1   

 

3

F

0.1

 

 

 

 

0.1   

 

4

M

 

 

0.1

 

0.1   

 

5

M

 

 

 

 

0.0   

 

6

M

0.1

 

 

 

 

0.1   

Test substance,2500 mg/kg bw  1  F  0.1          0.1   
   2  F            0.0   
   3  F            0.0   
   4  M  0.1          0.1   
   5  M            0.0   
   6  M            0.0   
 Test substance, 5000 mg/kg bw  1  F  0.1          0.1   
   2  F            0.0   
   3  F  0.2          0.2   
   4  M            0.0   
   5  M  0.1          0.1   
   6  M  0.1          0.1   

Applicant's summary and conclusion

Conclusions:
The test substance did not show any genotoxic potential under the test conditions chosen.
Executive summary:

The test article's clastogenic potential was determined in a nucleus anomaly test in Chinese hamsters. The test substance was administered by gavage to 6 male and female Chinese hamsters. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg bw 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 observed for anomalies. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. Thus, the test substance did not show any genotoxic potential under the test conditions chosen.