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:
2002-05-13 to 2002-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
8 June 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, 33178 Borchen, Gartenstraße 27, Postfach 1161, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: mean 177 g
- Assigned to test groups randomly: yes, under following basis: according to a random list which had been provided by the Rando 96 program developed and used at Merck KGaA, Darmstadt.
- Housing: individually, Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm)
- Diet: ad libitum, standard diet (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 49 - 52
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Miglyoloil 812 (10 mL/kg body weight)
Duration of treatment / exposure:
Dosing of the animals of different groups was staggerred over one to three day intervals to allow for sufficient sample preparation time.
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
316 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Male: 100 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 316 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide: 16.5 mg CPA were dissolved in 10 mL Aqua pro injectione directly before use

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose given in the present study was selected to produce signs of toxicity but no mortality. In a preliminary dose-finding experiment 6 male and 6 female rats treated intraperitoneally with 2000 mg/kg bw, showed clear toxic effects and two male and one female rat died. In a second dose-finding experiment, 5 male and 5 female rats treated intraperitoneally with 1000 mg/kg bw showed clear toxic effects (ptosis, dyspnea, diarrhea, prone position and locomotor disturbance) but not mortality. For these reasons, the dose of 1000 mg/kg body weight was selected as the highest doses for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of VlO = 3.16.

TREATMENT AND SAMPLING TIMES
Dosing of the animals of different groups was staggerred over one to three day intervals to allow for sufficient sample preparation time.
For the highest test item dose groups (1000 mg/kg) preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the lower dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment.

DETAILS OF SLIDE PREPARATION:
Immediately after the rats had been killed by CO2, one femur of each animal was dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum (Biochrom, Berlin, Germany) with the aid of a syringe, and suspended in the serum. This suspension was filtered through cellulose according to Romagna (1988) and centrifuged for 5 min at 150 x g. The sediment was then resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension.
After 3 hours of drying, the slides were stained according to a modified Giemsa-staining method described by Gollapudi and Kamra (1979) using Giemsa's solution (Merck, Darmstadt, Germany) with Weise buffer solution (Merck, Darmstadt, Germany) and mounted in Entellan (Merck, Darmstadt, Germany).

METHOD OF ANALYSIS:
A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250x). Round particles with about 1/20 - 1/5 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. They were differentiated from granules by thorough examination at different optical levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic. For determination of the quotient of normochromatic to polychromatic erythrocytes, both erythrocyte stages were screened for micronuclei and counted separately up to a total of 1000 erythrocytes per animal. Then counting was limited to polychromatic erythrocytes. Normochromatic erythrocytes with micronuclei, however, observed during scoring were registered also. Thus, based on this value and on the quotient, the number of micronucleated normochromatic erythrocytes per 1000 could be extrapolated. After termination of the microscopic evaluation, the data were loaded into a computer-aided program.
Evaluation criteria:
A positive effect in this test system is defined by the occurrence of mean MN-PCE values of a treatment group which are statistically significantly higher than those of the actual negative control. A prerequisite for this is, however, that these values are above those predetermined as historical negative controls of our laboratory (Historical data).
An indispensable prerequisite for evaluating the results of such investigations is the occurrence of significant positive effects in the actual positive control group.
A test material showing no positive effect in the main study is defined as a non- mutagen in this test system. In this case the study is terminated.
If a positive effect in a single test group occurs (i.e. dose-independently), a repeat experiment has to be considered. In case that no positive effects occur in that experiment the test material is defined as a non-mutagen. The single positive effect of the first experiment is interpreted as a randomly occurring event of no biological significance.
A test material is defined as mutagenic in this system if dose-related and/or single, reproducible (in independent experiments) positive effects occur. Establishment of dose-dependent effects of the test material is preferable. For this reason, if a positive effect occurs in a study in which a single, limit dose of 2000 mg/kg has been appli-cated, 3 different test material doses have to be administered in the supplementary experiment. The above mentioned criteria for a negative or positive test result apply for this experimental design like wise.
If borderline cases occur, the decision on further procedures should be based on a scientific evaluation of all available results including the toxicokinetic data.
Statistics:
Descriptive statistics
For all groups, mean values were calculated of the following parameters:
NCE/PCE - number of normochromatic erythrocytes (NCE) / number of polychromatic erythrocytes (PCE) / animal
MN-NCE - number of micronuclei-containing cells / 1000 NCE / animal
MN-PCE - number of micronuclei-containing cells / 1000 PCE / animal
For the parameter body weight the mean values and the relative body weight gains to the preceding mean values were calculated.

For further statistical analysis the number of micronuclei-containing polychromatic erythrocytes (MN-PCE), normochromatic erythrocytes (MN-NCE) and the quotient of NCE/PCE per animal was used.

Pairwise comparison
Each treatment group was compared to the negative control.
For comparisons of micronuclei-containing polychromatic and normochromatic erythrocytes, the exact Mann-Whitney-test was used against one-sided alternatives. The p-values (exact significance one-sided) of these comparisons are presented.
For comparisons of the quotient of NCE/PCE, the Dunnett's t-test was used against two-sided alternatives. The p-values (significance) of these comparisons are presented for those dose groups that showed a higher mean value than the negative control.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 1000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a preliminary dose-finding experiment 6 male and 6 female rats treated intraperitoneally with 2000 mg/kg bw, showed clear toxic effects and two male and one female rat died. In a second dose-finding experiment, 5 male and 5 female rats treated intraperitoneally with 1000 mg/kg bw showed clear toxic effects (ptosis, dyspnea, diarrhea, prone position and locomotor disturbance) but not mortality.

RESULTS OF DEFINITIVE STUDY
2000 polychromatic erythrocytes per animal, using 5 males per group, were evaluated. The negative control (solvent) values were all in the expected range predetermined as historical controls of the laboratory. For the positive control, a statistically significant increase in polychromatic erythrocytes with micronuclei was established (p < 0.01). No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed. For the number of normochromatic cells with micronuclei, calculated per 1000 normo-chromatic erythrocytes by means of the above mentioned quotient, no increase was observed.
Quotient normochromatic /polychromatic erythrocytes: No relevant treatment-related variation was observed
A relevant treatment-related decrease in body weight, in comparison to the solvent control animals, was not observed.

Applicant's summary and conclusion

Conclusions:
No increase in micronucleated polychromatic erythrocytes was observed in this micronucleus test in male rats after a single intraperitoneal administration of the test item. According to the predetermined criteria for interpretation of results the substance is not mutagenic in this test system.
Executive summary:

A micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines and the OECD Guideline for Testing of Chemicals No. 474.

The substance was given once intraperitoneally by gavage to male rats, at doses of 100, 316 and 1000 mg/kg body weight. Rats of the negative control group received the solvent alone, i.e. an intraperitoneal dose of 10 mL Miglyoloil / kg body weight. The animals of the positive control group were treated with an oral dose of 16.5 mg cyclophosphamide/kg body weight.A total of 30 animals was used.

Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the highest dose groups (1000 mg/kg) preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the lower dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. For microscopic investigation one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micro-nucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.

The highest test material dose induced clear clinical signs of toxicity. No relevant treatment-related decrease in body weight (in comparison to the solvent control) occurred and no relevant variation was observed for the quotient normochromatic : polychromatic erythrocytes.

The mean numbers of polychromatic erythrocytes with micronuclei for the negative control (solvent) were all in or very close to the expected range predetermined by historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.

No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

The substance was not mutagenic in the micronucleus test in male rats under conditions where the positive control exerted potent mutagenic effects.