Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 22 to July 18, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1996
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
Repeat administration of 10 ml/kg bw test item in corn oil via intraperitoneal injection with 24 hour interval
Frequency of treatment:
Two administrations, 24 hours apart; two sampling times of 24 and 48 hours per dose
Post exposure period:
For each concentration (high, medium, low) and for the solvent control, a post exposure period of 24 or 48 hours was used.
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 males and 5 females per group; two groups per dose (sampled at (a) 24 hours or (b) 48 hours)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Reference substance: cyclophosphamide dissolved in 0.9 % (w/v) NaCl in Milli-RO water
- Administration: 50 mg salt/kg bw as a single 10 ml/kg bw intraperitoneal injection

Examinations

Tissues and cell types examined:
Erythrocytes from the femoral bone marrow
Details of tissue and slide preparation:
Both femurs of each animal were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was f'lushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted slides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were airdried, fixed for 5 minutes in 100 % methanol and air-dried overnight. Two slides were prepared per animal (one for each femur).
The slides were automatically stained using the "Wright-stain-procedure" in an 'Ames' HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
All slides were randomly coded before examination. An adhesive label with N0T0X study identif ication number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 × for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 ×. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
a) The positive control substance induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
At the 24 hours sampling time, the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in the male animals treated with 500 mg/kg body weight and at the 48 hours sampling time in the female animals treated with 500 mg/kg body weight was statistically significantly (P=0.05) increased. Since, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of all other test substance treated groups, the increases just statistically were significant (P=0.05) and moreover the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes were within historical control data range, therefore, these increases were not considered biologically relevant.
The animals of the treated groups showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of the test item on the erythropoiesis.

Applicant's summary and conclusion

Conclusions:
Not mutagenic in the micronucleus test under the described experimental conditions.