Registration Dossier

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:
2000
Report Date:
2000

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:

- Lot/batch No.: 98/390-5
- Expiration date of the lot/batch: March 15, 2000

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd.; Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 43.6 g (SD ± 2.4 g), females mean value 32.8 g (SD ± 1.8 g)
- Assigned to test groups randomly: yes
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 18 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
Duration of treatment / exposure:
24 h or 48 h
Frequency of treatment:
single application
Post exposure period:
24 h or 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
48 h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (40 mg/kg bw)

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
As estimated in the pre-experiments 100 mg 3-dimethylaminopropylamine per kg b.w. was suitable, i.e suitable to the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
A small drop of the 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.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was deterrnined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and biologically relevant positive response.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The mean numbers of normochromatic erythrocytes at the highest dose (100 mg/kg b.w.) of the test item was clearly increased
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 150, 250 and 500 mg/kg bw
- Clinical signs of toxicity in test animals: death (150 mg/kg bw onwards)
- Harvest times: 24 h and 48 h

Any other information on results incl. tables

Summary of Micronucleus Test Results:

 Test group  Dose: mg/kg bw  Sampling time (h)

 PCEs with micronuclei (%)

 Range*  PCE / NCE
 Vehicle  24  0.145  0 - 6  2000 / 1802
 Test item  25  24  0.055  0 - 5  2000 / 1965
 Test item  50  24  0.080  0 - 5  2000 / 1738
 Test item 100   24  0.160  0 - 8  2000 / 2673
Cyclophosphamide  40  24  1.530  12 - 42  2000 / 2180
Vehicle  0  48  0.07  0 - 5  2000 / 1794
 Test item  100  48  0.04  0 - 3  2000 / 2067

*: Number of micronucleated PCEs

The mean numbers of normochromatic erythrocytes at the highest dose (100 mg/kg b.w.) of the test item was clearly increased (2673 and 2067 NCEs per 2000 PCEs for the 24 and 48 h sampling times, respectively) as compared to the mean values of NCEs of the vehicle controls (1802 and 1794 NCEs per 2000 PCEs for the 24 and 48 h sampling times, respectively). This indicates a test item specific toxic effect in the bone marrow of the treated mice. At all other doses and sampling times no relevant changes in the NCE/PCE ratio were observed. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 3-Dimethylaminopropyl were below or near to the values of the vehicle control groups.

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion