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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD-Study with insufficient discussions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: granulae
Details on test material:
- Name of test material (as cited in study report): Preventol CI8-100

- Physical state: pale yellow granulae, mild odor
- Analytical purity: 99.83 %

- Purity test date: 05.20.1986
- Lot/batch No.: 909

- Stability under test conditions: stable
- Storage condition of test material: refrigerator
- Other:

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 28 - 42 g
- Assigned to test groups randomly: yes, using a randomisation plan produced by teh Institute of Biometrics, BAYER AG, Wuppertal
- Fasting period before study: no
- Housing: Makrolon cages type I and II, bedding of soft wood granules
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24 °C
- Humidity (%): 50 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
other: oral stomach tube
Vehicle:
polyethylene glycol 400
Details on exposure:
a pilot study indicated 800 mg / kg bw as concentration for the test.
800 mg /kg bw were dissolved in polyethylene glycol 400.
Via stomach tube 5 ml / kg bw were administered.
Frequency of treatment:
once
Post exposure period:
24 to 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
800 mg / kgbw
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
10 animals, 5 male, 5 female
Control animals:
yes
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide is a known clastogen
- Route of administration: oral via stomach tube
- Doses / concentrations: 20 mg / kg bw, dissolved in demineralised water, 10 ml / kg were administered

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:

1. At least one intact femur was prepared from each
sacrificed animal (not pretreated with a spindIe
inhibitor). A suitale instrument was used to
sever the pelvic bones and calf area.
2. The femur was separated from muscular tissue.
3. The lowerleg stump with knee and all attached
soft parts in the distal epiphyseal cartilage
were remtoved by a gentIe pull at the distal end.
4. The proximal end of the femur was opened at its
extreme end with a suitable instrument, e.g.
fine scissors, so that a small opening of the
bone marrow channel became visible.
5. A suitable tube Has filled with sufficient fetal
calf serum.
6. Some serum was drawn from the tube into a suitable
syringe, with thin cannula.
7. The cannula was pushed into the open end of the
marrow cavity.
8. The femur was then completely immersed in the calf
serum and pressed against the wall of the tube,
so that it could not slip off.
9. The contents were then flushed several times, and
the bone marrow passed into the serum as a fine
suspension.
10. FinaIly, the flushing could be repeated from the
other end after it was opened.
11. The tube containing the serum and bone marrow
was centrifuged in a suitable centrifuge at
approximately 1000 rpm for five minutes.
12. The supernatant was removed with a suitable
pipette (e.g. Pasteur pipette) except for a
small resldue.
13. The sediment was mixed until the suspension
was homogeneous.
14. one drop of the viscous suspension was placed
on the thoroughly cleaned slide and spread
with a suitable object, e.g. a slide, so that
the smear could be properly evaluated.
15. The labeled slides were dried overnight.
If fresh smears are to be stained, they must
be dried for a short period with heat.

Staining of smears
The mears were stained automatically with an Ames Hema-Tek
Slide Stainer of Miles company. The slides were then
"destained" with methanol and rinsed with deionized water.
They were then le ft to dry.

METHOD OF ANALYSIS:
the slides were analysed with a light microscope at a magnification of 1000
Evaluation criteria:
ratio of polychromatic to normochromatic erythrocytes
Statistics:
The Preventol CI8-100 group with the highest mean, if this
superceded the negative control mean, and the positive
control were checked by Wilcoxon's non-parametric rank sum
test in respect to the count of polychromatic erythrocytes
with micronuelei and the rate of normochromatic
erythrocytes. A variation is considered statistically
significant if its error probability is below 5% and the
treatment group figure is higher than the negative
control's .
The rate of normochromatic erythrocytes with micronuclei is
examined if the micronucleus rate for polychromatic
erythrocytes was allready relevantly increased. In this case,
the group with the highest mean 1s compared with the
negative control using the one-sided chi 2-test. A variation
wss considered statistically significant if the error
probability is below 5% and the treatment group figure is
higher than the negative control's.

In addition, standard deviations (1s ranges) were calculated
for all the means.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500/750/850/1000 mg/kg bw
- Clinical signs of toxicity in test animals: apathy, reduced motility, unkempt coat, lateral position, abdominal position, cramp, convulsion, rapid breathing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No, 0.9 - 1.3 micronucleated cell per 1000 NCE/PCE
- Ratio of PCE/NCE (for Micronucleus assay): 1.028 - 1.084

Positive control
- Induction of micronuclei (for Micronucleus assay): No, 0.5 / 13.4 micronucleated cell per 1000 NCE/PCE
- Ratio of PCE/NCE (for Micronucleus assay): 1.133

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There is no sign of genotoxicity arising from this study.
In comparison with the negative control, no alteration in the ratio of polychromatic to normochromatic erythocytes was observed.
Further, there is no variation in regard to incidence of micronucleated cells between the negative control and the test groups.
In the positive control group there is a significant change in the incidence of micronuclei compared to the negative control.