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:
supporting study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard guideline study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation:
- Weight at study initiation:male: 37,1g ; female: 28,5g
- Assigned to test groups randomly: yes

- Housing: groups of 5 in makrolon cage type 3
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%):50+/-20

- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sesame oil
Details on exposure:
Animals were treated twice at an interval of 24 hours
Duration of treatment / exposure:
2 days
Frequency of treatment:
1/day for 2 days
Post exposure period:
24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
other: concurrent, positive control
Positive control(s):
cyclophosphamide; c
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw.

Examinations

Tissues and cell types examined:
bone marrow from the femur
Details of tissue and slide preparation:
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
Evaluation
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
A one-sided Wilcoxon-Test (4, 5] was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose-relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.

Criteria for a positive response
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Pooled result for both sexes

Dose

Time

Animals

Poly

Poly/Ery

Poly/Ery

Poly with MN

Poly with MN

Poly with MN

Mut. Index

 

 

 

counted

Mean

SD

mean

%

SD

 

0 (neg Ccontrol)

 

10

2000

0,49

0,07

1,30

0,1

0,05

1,0

2000

 

10

2000

0,45

0,10

1,80

0,1

0,07

1,4

50 (pos control)

 

10

2000

0,47

0,04

53,30

2,7

0,79

41,0*

 

*=significantly different from control (p<0,05)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described.
Executive summary:

Mice were treated twice with 2000 mg of the test item per kg body weight to study the induction of micronuclei in bone marrow cells. All animals survived after treatment.

No signs of toxicity were observed. The dissection of the animals revealed no test substance related findings.

The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.

The incidence of micronucleated polychromatic erythrocytes in the dose group of the test item was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed.

The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

CONCLUSION

The results lead to the conclusion that the test item did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.