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: Comparable to guideline study with acceptable restrictions ( 1000 erythrocytes/animal examined for MN)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(1000 erythrocytes/animal examined for MN)
Principles of method if other than guideline:
The weight variation of the test animals is not reported.
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: organic
- Physical state: solid
- Analytical purity: >98 %
- Stability under test conditions: The stability of the test substance throughout the study period had been proven analytically.
- Storage condition of test material: 4°C

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH WIGA, Sulzfeld, Germany
- Weight at study initiation: mean weight of 25.2 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5 % CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration of test material in vehicle: 5, 10 and 20 %
- Application volume: 20 mL/kg bw
Duration of treatment / exposure:
16 h (4000 mg/kg bw), 24 h (0, 1000, 2000, 4000 mg/kg bw and positive control), 48 h (4000 mg/kg bw)
Frequency of treatment:
single oral administration
Post exposure period:
The animals were sacrificed 16, 24 and 48 hours after the treatment, respectively.
- Necropsy: At the end of the experiment, the animals were sacrificed and, after removal of the two femora for bone marrow preparation, necropsied and examined for gross-pahtological changes of the internal organs.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Dose / conc.:
4 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw
- Application volume: 10 mL/kg bw

Examinations

Tissues and cell types examined:
- Bone marrow was obtained by femora preparation
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: (preliminary test)
In the determination of the acute oral toxicity all animals survived the administered dose of 4000 mg/kg body weight. A volume as high as 20 mL/kg body weight had to be selected in order to be able to administer this amount.
Doses higher than 4000 mg/kg bw suspended in an 0.5% CMC formulation led to a viscous mass which could no longer be administered. Therefore, 4000 mg/kg body weight, which led to irregular respiration 15 minutes after administration for about 4 hours, was selected as the highest dose in the present cytogenetic investigations.

TREATMENT AND SAMPLING TIMES:
16 h (4000 mg/kg bw), 24 h (0, 1000, 2000, 4000 mg/kg bw and positive control), 48 h (4000 mg/kg bw)
DETAILS OF SLIDE PREPARATION:
The two femora were prepared from the animals sacrificed by cervical dislocation, and all soft parts were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube in reciprocal direction using a cannula filled with fetal calf serum which was at room temperature (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was pipetted off except for a few drops, and the precipitate was resuspended. 1 drop of this suspenision was dropped onto clean microscopic slides in each case using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
Staining: eosin and methylene blue solution for 5 minutes. Then Giemsa solution for 12 minutes after washing in dest. water for 2-3 min. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

Evaluation criteria:
As a rule, 1000 polychromatic erythroeytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded:
(1) Number of polychromatic erythrocytes; (2) Number of polychromatic erythrocytes containing micronuclei. The increase in the micronucleus rate in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.
(3) Number of normochromatic erythrocytes; (4) Number of normochromatic erythrocytes containing micronuclei. The rate of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and serves as a certain control value. There may, however, be a substance-induced increase in the number of micronuclei in normocytes with an increase in the duration of the sacrifice intervals.
(5) Ratio of polychromatic to normochromatic erythrocytes. This ratio indicates a general activity of the test substance on the bone marrow.
(6) Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4) (d = diameter of micronucleus, D = cell diameter).
The size of micronuclei possibly allows conclusions to be drawn with regard to the mode of action of the test substance (clastogenic effect, spindle poison effect).
Statistics:
Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual dose groups concerning the rate of micronuclei in polychromatic erythrocytes: first, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups, and, second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON). The relative frequencies of cells with micronuclei per animal were used as a criterion of the rank determination for the U test. The two tests were calculated at the levels of 95% and 99%. Significances were marked with * (95%) and with ** (99%). A sequential statistical test was used to answer the question of a possible change of the characteristic in terms of time within a dose group ("test for homogeneity in terms of time"). On the basis of the hypothesis saying that the relative frequencies of the characteristic are identical at all times, the relative frequencies which reject this hypothesis at the level of 95% or 99% were determined successively. Significances were again marked with * (95%) or with ** (99%).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Clinical signs of toxicity in test animals: About 15 minutes after administration doses of 4000 mg/kg body weight led to irregular respiration which lasted for 3 - 5 hours. An amount of 2000 mg/kg and 1000 mg/kg body weight did not induce any evident signs of toxicity.
Animals which were administered the 0.5% CMC formulation or the positive control substance cyclophosphamide did not show any clinical signs of toxicity.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei and ratio of PCE/PNEC (for Micronucleus assay): summary see table below

Any other information on results incl. tables

Induction of Micronuclei in bone marrow cells

Group

Substance

Dose

(mg/kg b.w.)

sex

Post exposure period

(h)

Number of NCEa

PCE/NCE

ratio

Cells with micronuclei

in PCE

[‰]

in NCE

[‰]

I

vehicle

aqua dest.

males/

females

24

3218

3.10

1.7

1.55

II

test substance

4000

males/

females

24

3166

3.16

1.5

0.95

III

test substance

2000

males/

females

24

3624

 2.76

0.8+

2.76

IV

test substance

1000

males/

females

24

3080

3.25

1.4

0.65

V

positive control cyclophosphamid

40

males/

females

24

3461

2.89

**/++/30.0

2.89

II/16

test substance

4000

males/

females

16

3140

3.18

1.6

0.64

II/48

test substance

4000

males/

females

48

3811

2.62

1.8

2.36

 

 

 

 

 

 

 

 

 

PCE = polychromatic erythrocytes (1000 were scored for micronuclei)

 

 

 

 

NCE = normochromatic erythrocytes / 10000 PCE

 

 

 

 

a= number of NCEs observed when scoring 10 000 PCEs

 

 

 

 

Fischer-Yates Test: significance 99% (**)

 

 

 

 

U Test: significance 95% (+); significance 99% (++)

 

 

 

 

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control (solvent control) or in the various dose groups at any of the sacrifice intervals. The significance found in the intermediate dose group in the rank test is due to the slightly decreased number of polychromatic erythrocytes containing micronuclei in this group and is of no biological relevance.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative