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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-09-28 to 2001-03-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 19, 2000
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Crj: CD(SD)
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Germany
- Age at study initiation: 12 - 14 weeks
- Weight at study initiation: mean 224.8 g
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program
- Fasting period before study: no
- Housing: Makrolon cages, separately according to sex.
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland) was available ad libitum.
- Water: Drinking water from bottles was available ad libitum.
- Acclimation period: at least 4 days

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

Administration / exposure

Route of administration:
- Vehicle used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was selected as the vehicle.
- Concentration of test material in vehicle: 5-20 g/100 mL
- Amount of vehicle: 10 ml/kg
Details on exposure:
Male and female animals per test group were given N-(2-Hydroxyethyl)morpholin dissolved in purified water at dose levels of 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg bw . Treatment consisted of two intraperitoneal administrations. The volume administered was 10 mL/kg body weight. All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the 1st administration .
Duration of treatment / exposure:
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.
Frequency of treatment:
Post exposure period:
24 hours
Doses / concentrations
Doses / Concentrations:
500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg bw
nominal conc.
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control: The stability of CPP is well-defined under the selected conditions, since this positive control article is a well-defined clastogen.
- Route of administration: intraperitoneally
- Doses / concentrations: 20 mg/kg bw


Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived following two treatments with 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline. The clinical signs such as staggering, tremor and squatting posture were only observed after the 2nd administration, however, showing no distinct symptomatic differences between the male and female animals. Therefore, a dose of 2,000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

The bone marrow was prepared according to the method described by SCHMID, W. and SALAMONE, M . et al.
- The 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 filled with fetal calf serum (FCS) which was at 37°C (about 3 ml/femur).

In general, 2000 polychromatic erythrocytes (PCE) from each of,the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters were 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.

Clinical examinations: The animals of all test groups were examined for any evident clinical signs of toxicity.
Feed analysis: The feed used in the study was assayed for chemical and microbial contaminants.
Water analysis: The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and the Department of Water Chemistry and the Technical Services of BASF Aktiengesellschaft as well as for microorganisms by a contract laboratory.
Bedding analysis: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
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.
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft). The number of micronuclei in polychromatic erythrocytes was arralyzed . A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians . 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 given. This test was performed one-sided.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
In the untreated control group 1 .5 ‰ micronucleated polychromatic cells were observed. The two intraperitoneal administrations of purified water in a volume of 10 mL/kg body weight led to 1 .8 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval. After two administrations of the highest dose of 2000 mg/kg body weight, 1 .6 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours. In the two lower dose groups, rates of micronuclei of about 1 .7 ‰ (1000 mg/kg group) and 1 .8 ‰ (500 mg/kg group) were detected. An 29.7 ‰ increase in polychromatic erythrocytes was observed in animals treated with cyclophosphamide, the positive control agent. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.

Thus, the test substance N-(2-Hydroxyethyl)morpholin did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing either small micronuclei (d < D/4) or larger micronuclei (d >= D/4) did not deviate from the value of the negative controls (untreated control, vehicle control). A slight inhibition of erythropoiesis, induced by the treatment of rats with N-(2-Hydroxyethyl)morpholin, the top was detected at dose of 2000 mg/kg body weight.
According to the results of the present study, there are thus no differences in the frequency of erythrocytes containing micronuclei between the negative controls (untreated control and vehicle control) and the 3 dose groups (500 mg/kg, 1,000 mg/kg and 2,000 mg/kg). Thus, under the experimental conditions chosen here, the test substance N-(2-Hydroxyethyl)morpholin has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

The two intraperitoneal administrations of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. The two administrations of the test substance led to evident signs of toxicity. The single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight did not cause any evident signs of toxicity.

Applicant's summary and conclusion