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:
From Jun. 2, 1986 to Nov. 3, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Storage condition of test material: room temperature

Test animals

Species:
hamster, Chinese
Strain:
other: random outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 21-32 g (females) and 23-35 g (males)
- Housing: individual caging
- Diet: NAFAG No. 924, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3-4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23°C
- Humidity (%): 52-78%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 ml/kg
Details on exposure:
The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.
Duration of treatment / exposure:
Single dose administered orally.
Frequency of treatment:
Once
Post exposure period:
16, 24, 48 hours after treatment application.
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8/sex/sampling time
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 64 mg/kg in 20 ml/kg 0.5% CMC

Examinations

Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. The highest dose causing no deaths is used as the highest in the mutagenicity test. No deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen as no death occurred at this concentration.

TREATMENT AND SAMPLING TIMES:
16, 24 and 48h after application, 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
Statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at any sampling time.
Statistics:
The significance of difference was assessed by Chi square-test.

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:
RESULTS OF RANGE-FINDING STUDY
In the preliminary tolerability test, no deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen for the micronucleus test as no death occurred at this concentration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.66. In comparison with the negative control (0.11) this value is highly significant (p<0.05).

Any other information on results incl. tables

Table: The effect of the test substance on bone marrow cells of Chinese hamster (arithmetic mean per sex and group).

Sacrifice

Dose

Sex

PCE

NCE

Ratio of PCE/NCE

No. of PCE with MN

% of PCE with MN

16 h

Control

Female

583

417

1.40

2.0

0.20

Male

514

486

1.06

0.8

0.08

TS

Female

650

350

1.86

0.6

0.06

Male

511

489

1.04

1.2

0.12

24 h

Control

Female

595

405

1.47

1.2

0.12

Male

562

438

1.28

1.0

0.10

TS

Female

515

485

1.06

2.0

0.20

Male

508

492

1.03

0.8

0.08

48 h

Control

Female

564

436

1.29

1.8

0.18

Male

561

439

1.28

1.6

0.16

TS

Female

520

480

1.08

0.6

0.06

Male

456

544

0.83

1.0

0.10

Positive control

24 h

Control

Female

595

405

1.47

1.2

0.12

Male

562

438

1.28

1.0

0.10

CPA

Female

514

484

1.06

23.6

2.36

Male

459

541

0.85

29.0

2.96

TS: test substance; PCE: polychromatic erythrocytes; NCE: normochromatic erythrocytes; MN: micronuclei; CPA: cyclophosphamide

Applicant's summary and conclusion

Conclusions:
There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of the test substance as compared with the negative control animals. Under the conditions of this experiment, no evidence of mutagenic effects was obtained in chinese hamster treated with the test substance.
Executive summary:

A micronucleus test in hamster was performed to evaluate any mutagenic effect of the test substance on polychromatic erythrocytes in bone marrow cells in vivo. To that end, the animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. Mutagenic effects present themselves in form of micronuclei in polychromatic erythrocytes in the bone marrow. These micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes which lag behind at anaphase stage during the mitotic process. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. The increase in micronucleated polychromatic erythrocytes shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. In this experiment, the bone marrow smears from animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.66% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.11%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.