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
Study period:
August/September 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo 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): Uvinul N 539
- Physical state: light yellowish, liquid/oily
- Analytical purity: 98.3%
- Purity test date: 1992-11-03
- Lot/batch No.: 2070182 ( German version), 505396-70182 ( American version), date of receipt: September 1992
- Stability under test conditions: confirmed
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
male and female NMRI mice
- Source: Charles River GmbH, WIGA, Sulzfeld, FRG
- Age at study initiation: no data
- Weight at study initiation: Animals with a mean weight of about 26 .5 g were used for the study.
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: individually in Makrolon cages , type MI
- Diet (ad libitum): Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG , Kaiseraugst, Switzerland)
- Water (ad libitum): drinking water from bottles
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil (test substance); water (positive control substances)
No further data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Substance formulations were prepared immediately before administration. The concentrations of the test solutions were adjusted to 20 g/100 ml, 10g/100 ml, and 5 g/100 ml for the groups receiving 2000, 1000, and 500 mg/kg bw, respectively
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
sacrifice at 16, 24 and 48 hours after dosing (2000 mg/kg bw)
sacrifice at 24 hours after dosing (other dose groups)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): experienced positive control substance for clastogenicity (chromosome-damaging effects)
- Route of administration: p.o.
- Doses / concentrations: 20 mg/kg bw; vehicle: distilled water

vincristine
- Justification for choice of positive control(s): experienced positive control substance for aneugenicity (spindle poison effects)
- Route of administration: i.p.
- Doses / concentrations: 0.15 mg/kg bw; vehicle: distilled water

Examinations

Tissues and cell types examined:
bone marrow cells (polychromatic and normachromatic erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity the amount of 2000 mg/kg body weight recommended as the highest dose according to the EEC Directive 84/449, B 12, or according to the OECD Guideline No. 474 was survived by all animals. As signs of toxicity only piloerection was observed about 4-5 hours.
Therefore , a dose of 2000 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.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
See table below (Additional remarks)

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W.
- 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 1500 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:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest . for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
Microscopic evaluation
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter )

Slides were coded before microscopic analysis.

OTHER:
Clinical examinations
After the administration of the test substance the animals were examined for any evident clinical signs of toxicity.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG).
The number of micronuclei in polychromatic erythrocytes were analysed.
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 cell s 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 set. 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:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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. Doses of 500 mg/kg , 1000 mg/kg or 2000 mg/kg body weight led to piloerection about 15 minutes after treatment which lasted for about 2 hours. 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.

Microscopic evaluation

Remark: The unit"per mille" used in the original study report has been converted to "per cent" in this freetext due to editorial reasons.

The single oral administration of olive oil in a volume of 10 ml/kg body weight led to 0.13% polychromatic erythrocytes containing micronuclei after the 24 -hour sacrifice interval. After the single administration of the highest dose of  2000 mg/kg body weight, 0.2% polychromatic erythrocytes containing micronuclei were found after 16 hours, 0.15% after 24 hours and 0.13% after 48 hours. In the two lower dose groups rates of micronuclei of about 0.19% (1000 mg/kg group) and 0.13% (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case. 

With 1.54% 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 6.04% 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. 1.48%.

The number of normochromatic erythrocytes (NCEs) 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. An amount of about 0.33% (p<0.05) in the highest dose group must be regarded as an incidental finding and cannot be related to the test substance treatment due to the short sacrifice interval of 24 hours at which the NCEs had already past the sensitive stage at the time of treatment. Furthermore, there is no corresponding finding in polychromatic erythrocytes. Thus, the test substance Uvinul N 538 did not lead to any increase in the rate of micronuclei.

The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d<D/4) did not deviate from the solvent control value at any of the sacrifice intervals. Nor were large micronuclei (d>D/4) observed either in the negative control group or in the three dose groups. No inhibition of erythropoiesis induced by the treatment of mice with Uvinul N 539 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