Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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): Monoethanolamine, 94/221
- Physical state: colorless liquid
- Analytical purity: 99.5 %
- Production date: 1994-05-03
- Lot/batch No.: continuous production, Tank B705
- Storage condition of test material: room temperature, N2-conditions

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Wiga, Sulzfeld, Germany
- Weight at study initiation: mean 27 g
- Assigned to test groups randomly: yes, under following basis: computorized
- Fasting period before study: none
- Housing: Makrolon cages, individually
- Diet: standardized pellet feed, Kliba, Klingentalmuehle, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: about 1 week


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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: [water]
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved at dose levels of 375, 750 and 1500 mg/kg immediately before administration
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single application
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
375, 750 and 1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males, 5 females used for each dose level of the test substance;
5 animals in total were used for each positive control substance
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide or vincristine
- Route of administration: orally or ip
- Doses / concentrations: 20 mg/kg (cyclophosphamide) or 0.15 mg/kg (vincristine)

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Pretests for dose selection
In a pretest for the determination of the acute oral deaths were observed down to a dose of 1,750 mg/kg body weight whereas 1,500 mg/kg body weight were survived by all animals. Therefore, a dose of 1,500 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 750 mg/kg and 375 mg/kg body weight were administered as further doses.

The substance to be administered per kg body weight was dissolved in purified water.

- Test group 3 was given 375 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 3.75 g/100 ml.
- Test group 4 was given 750 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 7.5 g/100 ml.
- Test groups 5 and 6 were given 1,500 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 15.0 g/100 ml.


Preparation of, the bone marrow
The bone marrow was prepared according to the method described by Schmid, W (1976,1977).
- The two femora were prepared from the animals, and all soft tissues were removed.
- 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 which was at 37°C (about 2 ml/femur )
- The suspension was mixed thoroughly with a pipette, centrifuged at 1,500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

Staining of the slides
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam.
Evaluation criteria:
Microscopic evaluation
In general, 1,000 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 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

The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normochromatic erythrocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes. This ratio indicates an influence of the test substance specifically on the bone marrow.
- 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.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). 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 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 printed with the group means in the tables. This test was performed one-sided. This analysis was done separately for each sex and combined for both sexes.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 females of the 48h 1500 mg/kg bw dosing group died
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF PRETEST FOR DOSE SELECTION
In a pretest for the determination of the acute oral deaths were observed down to a dose of 1,750 mg/kg body weight whereas 1,500 mg/kg body weight were survived by all animals.

RESULTS OF DEFINITIVE STUDY
The single oral administration of purified water in a volume of 10 ml/kg body weight led to 1.7 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.4 ‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 1,500 mg/kg body weight, 1.2 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.3 ‰ after 48 hours.

In the two lower dose groups rates of micronuclei of about 2.1 ‰ (750 mg/kg group) and 1.0 ‰ (375 mg/kg group) were detected after a sacrifice interval of 24 hours in
each case.

With 11.4 ‰ the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing mainly small micronuclei at a dose level of 20 mg/kg body weight.

With 112 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e . 27.2 ‰.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus, the test substance Monoethanolamine did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals.
A slight inhibition of erythropoiesis induced by the treatment of mice with Monoethanolamine was detected at the highest dose of 1,500 mg/kg body weight both after a sacrifice interval of 24 hours and 48 hours.

CLINICAL EXAMINATIONS
The single oral administration of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms. In the treatment groups the following signs of toxicity were observed:
375 mg/kg: Piloerection after about 30 minutes; no symptoms any longer after 3 hours.

750 mg/kg: Piloerection, squatting posture and irregular respiration after about 30 minutes - 2 hours. Piloerection was observed even the day after treatment.

1,500 mg/kg: Piloerection, squatting posture and irregular respiration after about 30 minutes - 5 hours; the general state of the animals was poor. Piloerection was still observed the day after treatment and 4 animals were found dead.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Any other information on results incl. tables

According to the results of the present study, there are thus no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (375 mg/kg, 750 mg/kg and 1,500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). Thus, under the experimental conditions chosen here, the test substance Monoethanolamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative