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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-METHYLACETAMIDE, 98/392-1
- Physical state: Colorless solid
- Analytical purity: 99.82% (GC)
- Lot/batch No.: Tank 143
- Date of manufacturing: September 24, 1998
- Stability: The stability of the test substance at room temperature in the vehicle water has been determined analytically.
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH.
- Mean weight at study initiation: 28.3 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages type MIII, in groups of 5 during acclimatization of about 3-5 days. Before the start of the treatment
the animals were transferred to Makrolon cages, type Ml, and housed individually.
- Diet (e.g. ad libitum): Standardized pelleted feed (Kliba Haltungsdiaet, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles were available ad libitum.
- Acclimation period: 3-5 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Details on exposure:
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived following two treatments with 2,000 mg/kg body weight recommended as the highest dose according to the EEC Directive 92/69, B 12. and to the OECD Guideline 474. As clinical signs only irregular respiratory was observed both using male and female animals. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2,000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1,000 mg/kg and 500 mg/kg body weight were administered as further doses.

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered twice per kg body weight was dissolved in purified water and given by intraperitoneal injektion.
- The low dose group was given 500 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 5.0 g/100 ml.
- The intermediate dose group was given 1,000 mg test substance/kg body weight or 10 mI/kg body weight of a solution with a concentration of 10.0 g/100 ml.
- The top dose group was given 2,000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 20.0 g/100 ml.
All test substance formulations were prepared immediately before administration.

TREATMENT:
The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment. Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.

Duration of treatment / exposure:
twice at a 24 hour interval
Frequency of treatment:
twice
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:

No. of animals per sex per dose:
5 (only males)
Control animals:
yes
Positive control(s):
cyclophosphamide (CPP); vincristine (VCR)
- Justification for choice of positive control(s): Ihe stability of CPP and VCR is well-defined under the selected conditions, since both
positive control articles are welI-defined clastogens and aneugens respectively.
- Route of administration: dissolved in purified water, administered to male animals once, intraperitoneally, each in a volume of 10 ml/kg body weight.
- Doses / concentrations: 20 mg cyclophosphamide/10 ml test solution, 0.15 mg vincristine/10 ml test solution

Examinations

Tissues and cell types examined:
bone marrow: polychromatic erythrocytes (PCE), normochromatic erythrocytes (NGE)
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula fihled with fetal calf serum which was at 37°C (about 2 mI/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 pI fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using sildes with ground edges, the preparations were dried in the air and subsequently stained.

The sildes were stained in eosin and methylene blue solution for 5 minutes (May Gruenwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

ANALYSIS:
In general, 2,000 PCE from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The NGE which occur are also scored. The following parameters are recorded:
* Number of polychromatic erythrocytes
* Number of polychromatic erythrocytes containing micronuclei.
The increase in the number of micronuclei 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.
* Number of normochromatic erythrocytes
* Number of normochromatic erythrocytes containing micronuclei
* Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
* 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 may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
* A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.
* The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test System if:
* There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
* The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal med ans. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <= 0.05, ** for p <= 0.01) were printed with the group means in the tables.
This test was performed one-sided.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
N-Methylacetamid did not lead to any relevant increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes
containing small micronuclei (d < D/4) did not deviate from the vehicle control value. Nor were large micronuclei (d> D/4) observed either in the negative control group or in the three dose groups of N-Methylacetamid.

A slight inhibition of erythropoiesis, induced by the treatment of mice with N-Methylacetamid, was detected at a dose of 2,000 mg/kg body weight.

The statistical significances observed at 1,000 mg/kg and 2,000 mg/kg body weight are due to the very low spontaneous rate in the vehicle control group and, furthermore, the values found at these doses are well within the range of the historical control values [water (i.p.) mean 1.6 (min 1.1 - max 2.3)].

There are no signs of clinical symptoms.

Any other information on results incl. tables

Induction of Micronuclei in bone marrow cells

 

 

 

 

Substance

Dose

(mg/kg b.w.)

sex

post exposure period (h)

Number of NCEa

Cells with micronuclei

Ratio PCE/NCE

in PCE [‰]

in NCE [‰]

vehicle

aqua dest.

Males

24

4390

0.4

1.1

0.46

test substance

500

Males

24

3285

1

1.8

0.61

test substance

1000

Males

24

3632

2.3*

2.5

0.55

test substance

2000

Males

24

6761

2.5**

1.6

0.30

positive control cyclophosphamid

20

Males

24

2840

21.4**

3.9

0.70

positive control vincristine

0.15

Males

24

5407

70.4**

3.1

0.37

 

 

 

 

 

 

 

 

PCE = polychromatic erythrocytes (2000 were scored for micronuclei)

 

 

 

NCE = normochromatic erythrocytes

 

 

 

 

 

 

 

 

 

 

 

 

 

a= number of NCEs observed when scoring 10 000 PCEs

 

 

 

 

 

 

 

 

 

 

 

 

WILCOXON TEST (ONE-SIDED) *:p<= 0.05. **: p<= 0.01

 

 

 

 

A PAIRWISE COMPARISON OF EACH DOSE GROuP WITH THE VEHICLE CONTROL GROUP

 

Applicant's summary and conclusion