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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Fuellinsdorf
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: mean weight 39.6 ± 3.0 g
- Housing: single
- Cage type: Makrolon Type II, with wire mesh top (Ehret GmbH, Emmendingen)
- Diet (ad libitum): pelleted standard diet
- Water (ad libitum): tap water
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The test material was diluted in deionised water and was administered at a volume of 10 mL/kg b.w. The vehicle control animals were administered deionised water at the same volume.
Duration of treatment / exposure:
24 and 48 hours, respectively.
Frequency of treatment:
once
Doses / concentrationsopen allclose all
Dose / conc.:
37.5 mg/kg bw (total dose)
Dose / conc.:
75 mg/kg bw (total dose)
Dose / conc.:
150 mg/kg bw (total dose)
No. of animals per sex per dose:
6
Control animals:
yes
Positive control(s):
Cyclophosphamide (CPA), Dosing: 40 mg/kg bw, Volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow.
Details of tissue and slide preparation:
DOSE SELECTION:
The active ingredient content of the test substance was approximately 75%. Therefore, the doses used in this study and reported presently are
expressed in terms of this indicated content. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test substance, the vehicle or the positive control substance once ip. Six males were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test substance. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

PREPARATION OF THE ANIMALS
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded . A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.


ANALYSIS OF CELLS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes
(PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes
was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs . The analysis was performed with coded
slides.
Evaluation criteria:
A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, which clearly exceeds the negative control range or a relevant positive response for at least one of the test points.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance was evaluated by means of the non-parametric Mann-Whitney test (Krauth J., Annals of Math. Stat., 42: 1949-1956, 1971).
However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
150 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Summary of the Micronucleus Test Results:

Test group

Period

Dose

(mg/kg bw)

PCEs with

micronuclei

range

PCE/ NCE

deionised water

24 h

0

0.60

0 – 4

2000/ 1937

CPA

24 h

40

16.90

23 – 55

2000/ 1760

Protectol HT

 

24 h

37.5

0.20

0 – 1

2000/ 2053

24 h

75.0

0.50

0 – 3

2000/ 1792

  24 h

150.0

1.50

1 – 8

2000/ 2368

deionised water

48 h

0

0.40

0 – 4

2000/ 1880

Protectol HT

48 h

 

150.0

0.80

0 – 3

2000/ 2535

CPA: Cyclophosphamide

Applicant's summary and conclusion