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Toxicological information

Genetic toxicity: in vivo

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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:
1992-04-28 to 1992-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Directive 84/449/EEC
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
other: Bor: NMRI (SPF Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Bor: NMRI (SPF Han)
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 28-41 g
- Assigned to test groups randomly: yes
- Housing: The females were kept in groups of a maximum of three mice in Macrolon type I cages. Males were kept singly in type I cages. Bedding of soft wood granules, type S 8/15 was used. The animals were identified by cage and picric acid markers. The wood granules was spot-checked for contaminants at regular intervals.
- Diet: Altromin 1324 Standard diet was available ad libitum.
- Water: Drinking water. Tap water was available ad libitum.
- Acclimation period: at least one week
- Other: The breed's state of health was regularly spot-checked for the major specific pathogens. Only healthy animals without symptoms were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5-23 °C
- Humidity: 43-48 %
- Air changes: ca. 10 times per hour
- Photoperiod: 12 hours of electrical light daily (6.00 to 18.00 hours), about 500 lux

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The test substance was dissolved in polyethylene glycol 400.
Details on exposure:
single treatment
Duration of treatment / exposure:
The substance was administered once.
Frequency of treatment:
once
Post exposure period:
48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
15 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Each group comprised ten mice, five females and five males.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: intraperitoneal
- Dose: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Erythrocytes (evaluation of micronucleated polychromatic erythrocytes) in bone marrow of male and female mice
Details of tissue and slide preparation:
Preparation of specimens:
Schmid's method was used to produce the smears.
At least one intact femur was prepared from each sacrified animal (not prepared with a spindle inhibitor). A suitable instrument was used to sever the pevic bones and lower leg. The femur was separated from muscular tissue.
The lower-leg stum, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument. A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a suitable syringe with a thin canula. The canula was pushed into the open end of the marrow cavity. The femur was then completelyimmersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off. Thecontents were then flushed several times and the bone marrow was passed into the serum as a fine suspension. Finally the flushing might be repeated from the other end, after it had been opened. Thetube containing the serum and bone marrow was centrifuged at ca. 1000 rpm for 5 minutes. The supernatant was removed with a pipette. The sediment was mixed to produce a homogeneous suspension. One drop of a viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear. The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.

- Staining of smears: The smears were stained automatically with the Ames HemaTek Slide Stainer fro the Miles Company.
- Covering of smears: The smears were transferred o a holder, A cuvette was filled with xylene, into which the holder was immersed of ac. 10 minutes. The slides were remoned singly tobe covered. A small amount of covering agent was taken from a bottle with a suitable object and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.
Evaluation criteria:
Coded slides were evaluated using a light microscope at a magnifiaction of about 1000. Micronuclei appear as strained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts be varying the focus.
Normally, 1000 polychromatic erythrocytes were couted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. It is expedient ti establish the ratio of polychromatic to nomochromatic erythrocytes for two reasns:
1. Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alternation of this ratio may show that the test substance actually reaches the target.
Therefore, the number of normochromatic erythrocytes per 1000 polychromatic ones was notes. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. a relevent treatment-related elteration of the ratio polychromatic to nomochromatic reythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
The test substance with the highest mean and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5 % and the treatment group figure was higher than that of the negative control.
The rate of normochromatic reythrocytes containing micronuclei was examined if the micronulcear rate for polychromatic reythrocytes eas already relevent increased. In this case, the group with the highest mean was compared with the neagative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5 % and the treatmant group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The animals treated with the test substance showed symptoms of toxicity after administration. One of fourty animals died before the end of the test due to the acute intraperitoneal toxicity of 15 mg/kg test substance.
Vehicle controls validity:
valid
Negative controls validity:
other: Yes, based on literature data and laboratories' own experience
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The selection of the test substance dose was based on the pilot test, in which groups of five animals, including both females and males, were intraperitoneally adminstered 10 mg/kg, 17.5 mg/kg, 25 mg/kg and 100 mg/kg test substance.
The following symptoms were recorded for up to 48 hours: apathy, roughened fur, staggering gait, spasm and difficulty in beathening. In addition, 1 of 5 animals died in the 25 mg/kg group and all animals died in the 100 mg/kg group. Based on these results, 15 mg/kg test substance was chosen as MTD for this test.

RESULTS OF DEFINITIVE STUDY
Toleration by the animals:
The following sompound-related symptoms until sacrifice were obcerved after single intraperitoneal administration of 15 mg/kg:
apathy, staggering gait, spasm, sternal recumbency, twitching and difficulty in breathing. Their feeding behaviour was normal. One of the 40 animals died during the test period, due to the acute toxicity of 15 mg/kg test substance.
Animals of the negative control showed apathy and spasm for up to four hours after treatment. Therefater their external appearance and physical activity remained unaffected. Their feeding behaviour was normal. No symptoms were recorded for the positive control. No animals died in the control groups.

Microscopic evaluation:
There were no relevant variations between males and females. Therefore, thes were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test substance, being 1000:1096 (1s=223) in the negative control, 1000:1692 (1s=1119) in the 16 hours group, 1000:1449 (s=457) in the 24 hours group and 1000:884 (1s= 292) in the 48 hours group. week but relevant variations were thus noted. However, statistically significant differences were not found.
No biologicaly importantor statistically significant variations existed between the negative control and the group treated with test substance, with respect to the incidence of micronucleated polychromatic reythrocytes.
Further, no biologically significant variations between the negative control and the test substance groups in the number of micronucleated normochromatic erythrocytes were found, since normochromatiic reythrocyes originated from polychromatic ones.
- The positive controlcaused a clear increase in the number of polychromatic reythrocytes with micronuclei. The incidence of micronucleated cells was 18.4/1000 (1s=9.4), which represents a biologically relevent increase in comparision with the negative control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg test substance in the microncleus test on the mouse, i.e. in a somatic test system in vivo.
Executive summary:
The test substance was tested in a mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. Possible clastogenic effect to the chromosomes of bone-marrow erythroblasts of the test substance were investigated in male and female mice.

The treated animals received a single intraperitoneal administration of either test substance or cyclophosphamide (positive control). The femoral marrow of groups treated with test substance was prepared 16,24 and 48 hours after administration. All negative and positive control animals were sacrified after 24 hours. The doses of test substance and positive control were 15 and 20 mg/kg bw, respectively. The animals treated with test substance showed symptoms of toxicity after administration. One of the forty animals died before the end of the test due to the acute inraperitoneal toxicity of 15 mg/kg test substance. There was weekly altered ratio between polychromatic and normochromatic erythrocytes.

The results with the test substance gave no relevant indications of clastogenic effects after single intraperitoneal treatment with 15 mg/kg. The known mutagen and clastogen, cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw. The number of micronucleated polychromatic erythrocytes increased to a biologically relevant degree.

The number of microncleated normochromatic erythrocytes did not increase relevantly in any of the groups. The test substance was judged to be not clastogenic in vivo.