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: Guideline study (OECD 474), GLP compliant

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): Triisopropanolamin
- Analytical purity: 92.0 %
- Lot/batch No.: 10-4852
- 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,
- Weight at study initiation: 27.5 g (mean)
- Housing: individually
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmühle AG, Kaiseraugst, Switzerland) ad libitum
- Water: botteled tap water ad libitum



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

Administration / exposure

Route of administration:
oral: gavage
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 / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CCP); vincristine (VCR)

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: pretests

DETAILS OF SLIDE PREPARATION: The bone marrow was prepared according to the method described by Schmid, W. One drop of cells was dropped onto clean microscopic slide. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. 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:
In general, 1000 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 normocytes 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.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG ). 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. 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:
Oral administration of the test substance led to irregular respiration and piloerection at all three dose levels after about 30 minutes.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Polychromatic and normochromatic erythrocytes:

    24 h           48 h                                                                
Dose (mg/kg) Total No. of PCE´s NCE´s/PCE´s  MN in PCE´s (‰) MN in NCE´s (‰)  Total No. of PCE´s NCE´s/PCE´s  MN in PCE´s (‰) MN in PCE´s (‰)                                                        
control  10000  4928  2.4  4.1  10000  4419  2.7  2.9                                                        
500  10000  4641  3.2  3.2  -  -                                                        
1000  10000  5404  2.0  2.4  -  -  -  -                                                        
2000  10000  5708  3.4  5.1  10000  6389  2.3  3.8                                                        
CCP 20  5000  2875  21.4*  5.9  -  -  -                                                        
VCR 0.15  5000  5054  112.6* 14.2  -  -  -  -                                                        

*: p<0.01

CCP: cyclophosphamide

VCR: vincristine

The oral administration of the test substance led to irregular respiration and piloerection at all three dose levels after about 30 minutes. In the dose groups of 1,000 mg/kg and 2,000 mg/kg squatting posture was additionally observed. After 2-4 hours these signs were not observed any longer.

The single administration of the vehicle in a volume of 10 ml/kg body weight led to 2.4‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 2.7‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 2,000 mg/kg body weight, 3.4‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 2.3‰ after 48 hours. In the two lower dose groups rates of micronuclei of about 2 ‰ (1,000 mg/kg group) and 3.2‰ (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 21.4‰ the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing mainly small  micronuclei at a dose level of 20 mg/kg body weight. With 112.6‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei cntaining polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 15.6‰. 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 Triisopropanolamin 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. No inhibition of erythropoiesis induced by the treatment of mice with Triisopropanolamin was detected. The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups .

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative