Registration Dossier

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:
2005
Report date:
2005

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Glycerol, ethoxylated, esters with acrylic acid
EC Number:
500-322-6
EC Name:
Glycerol, ethoxylated, esters with acrylic acid
Cas Number:
144086-03-3
Molecular formula:
UVCB substance
IUPAC Name:
[(uraniovanadio)methyl]borane
Details on test material:
- Name of test material (as cited in study report): Glycerin3EOTA
- Physical state: Liquid, beige, turbid
- Analytical purity: > 99%
- Lot/batch No.: GK0561/160
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period is guaranteed until June 15, 2005 as indicated by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Roomn temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks (according to the information from the breeder)
- Weight at study initiation: ca. 29 g
- Assigned to test groups randomly: yes
- Housing: Makrolon cages
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days


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: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available
- Concentration of test material in vehicle: 5, 10 or 20g/100mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
24 (all treatments), 48 h (control and high dose)
Frequency of treatment:
once
Post exposure period:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
80-98% of the theoretical values were found analytically during preparation
No. of animals per sex per dose:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
The following positive controls, both dissolved in purified water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight:
- Cyclophosphamide (CPP): 20 mg CPP/kg body weight for clastogenic effects
- Vincristine Suiphate (VCR): 0. 15 mg VCR /kg body weight for aneugenic effects

Examinations

Tissues and cell types examined:
Bone marrow preparation
The two femora of the animals sacrificed by cervical dislocation 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 which was at 37°C (about 2 ml/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 µl fresh FCS.
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.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
Staining of the slides
The slides were stained in eosin and methylene blue (modified May-Gruenwald solution or Wrights solution) for about 5 minutes.
After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Gorbit-Balsam.
Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.


Assessment criteria
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILGOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The single oral administration of olive oil in a volume of 10 ml/kg body weight led to 1.3‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.7‰ after the 48-hour sacrifice interval.
After the single administration of the highest dose of 2000 mg/kg body weight, 1.9‰
polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.7‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.6‰ (1000 mg/kg group) and 1.8‰ (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
With 13.3‰ the positive control substance cyclophosphamide for clastogenicity led to the expected clear increase in the number of polychromatic erythrocytes containing mainly small micronuclei.
With 40.4‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 8.4‰.
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.
No inhibition of erythropoiesis induced by the treatment of mice with Glycerin3EOTA 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.

Any other information on results incl. tables

Substance Dose (mg/kg) post exposure period (h) total PCEs PCE with micronuclei, total (‰) PCE with small micronuclei (‰) PCE with large micronuclei (‰) total NCEs NCE with micronuclei (‰)
vehicle solvent 24 10000 1.3 1.3 0.0 3598 0.3
vehicle solvent 48 10000 1.7 1.7 0.0 5736 0.3
test substance 500 24 10000 1.8 1.8 0.0 4587 2.2
test substance 1000 24 10000 1.6 1.6 0.0 4065 2.2
test substance 2000 24 10000 1.9 1.9 0.0 3245 0.9
test substance 2000 48 10000 1.7 1.7 0.0 4079 0.7
CPP 20 24 10000 13.3 13.2 0.1 4178 1.2
VCR 0.15 24 10000 40.4 32.0 8.4 5851 0.5
PCE = polychromatic erythrocyts (2000 per animal were scored for micronuclei)
NCE = normochromatic erythrocyts
* = significantly different from the control

Applicant's summary and conclusion