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:
15 March 2006 - 04 April 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
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:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests
Version / remarks:
1995
Qualifier:
according to
Guideline:
other: CH Requirements for Registration of Pharmaceuticals for Human Use, Genotoxicity: a Standard Battery for Genotoxicity Testing of Pharmaceuticals (Step 4, recommended for adoption 16 July 1997).
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recommended species according to Guideline.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 223 - 239 g
- Assigned to test groups randomly: yes, under following basis: according to a random !ist which had been provided by the Rando 96 program developed and used at Merck KGaA, Darmstadt
- Housing: individually in Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm) on softwood chippings
- Diet: ad libitum (Standard diet from Provimi Kliba SA)
- Water: ad libitum (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Miglyol 812 neutral oil
- Concentration of test material in vehicle: 20 mL/kg bw
Details on exposure:
To achieve maximal sensitivity of the test system, the test material was administered once intraperitoneally in this investigation. This procedure is in compliant with the OECD Guideline (dated on 1997) and EU Method B.12 (dated 2000).
Duration of treatment / exposure:
24 and 48 hours after administration of the test material preparation of bone marrow smears took place for the high dose group. Bone marrow smears were prepared 24 hours after administration for the low and mid dose group.
Frequency of treatment:
once
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
633 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): With this dose a statistically significant increase in the number of polychromatic erythrocytes with micronuclei, as compared to the negative control, was to be expected.
- Route of administration: oral
- Doses / concentrations: 16.5 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose (2000 mg/kg bw) was selected to produce signs of toxicity but no mortality. The mid (633 mg/kg bw) and low dose (200 mg/kg bw) is obtained by dilution in half-log ranges, i.e. 3.16.

TREATMENT AND SAMPLING TIMES: The test material was administered once. Preparation of bone marrow smears took place 24 and 48 hours after administration.

DETAILS OF SLIDE PREPARATION: The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum 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 in 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).

METHOD OF ANALYSIS: A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics. Round particles with about 1120 - 115 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic. For determination of the quotient of nonnochromatic 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. Nonnochromatic erythrocytes with micronuclei, however, observed during scoring were registered also. Thus, based on this value and on the quotient, the number of micronucleated nonnochromatic erythrocytes per 1000 could be extrapolated.
Evaluation criteria:
The primary parameter for evaluation of the results of this test system is the number of micronucleated, polychromatic erythrocytes (MN-PCE). This number is increased by treatment of cells with mutagenic test materials as compared to the respective negative controls.

- A positive effect in this test system is defined by the occurrence of mean MNPCE 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 (cf. 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 bw has been applicated, 3 different test material doses have to be administered in the supplementary experiment. The criteria mentioned above for a negative or positive test result apply for this experimental design likewise.



Statistics:
Descriptive statistics
For all groups, mean values were calculated ofthe following parameters:
NCE/PCE - number ofnonnochromatic erythrocytes (NCE)/ number of polychromatic erythrocytes (PCE)/ animal
MN-NCE - number of micronuclei-containing cells/ 1000 NCE/ animal
MN-PCE number ofmicronuclei-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.

Statistical tests
For further statistical analysis, the numbers of micronuclei-containing polychromatic erythrocytes (MN-PCE), nonnochromatic erythrocytes (MNNCE) and the quotient of NCE/PCE per animal were used.

Pairwise comparison
Bach 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.

Software
The numerical calculation was performed using the computer-aided micronucleus test pro gram (Version 2.3c) and for the exact Mann Whitney test and Dunnett's t-test the SPSS-System (Version 9.0), running under Windows NT was used.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in the highest dose group
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: The test material was diluted in Miglyol 812 neutral oil.
- Clinical signs of toxicity in test animals: Clear toxic effects (ptosis, abdominal position and a loss in body weight) but no mortality were observed in the high dose group (2000 mg/kg bw).
- Rationale for exposure: Dose was selected to produce signs of toxicity but no mortality.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): No treatment-related variation was observed.
- Appropriateness of dose levels and route: The route chosen is in compliant with the OECD Guideline (dated on 1997) and EU Method B.12 (dated 2000). Appropriate dose levels were selected as the highest dose group induced some clinical signs but no mortality. No clinical signs were observed in the mid and low dose group, however, a decrease in body weight was observed.

Applicant's summary and conclusion

Conclusions:
The test material did not cause a relevant increase in the number of polychromatic erythrocytes with micronuclei and did not increase the number of normochromatic cells with micronuclei. Therefore, the test item is not considered to be mutagenic in the micronucleus test in rats under conditions wher the positive control exerted potent mutagenic effects.
Executive summary:

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

In the current experiment the test material was given once intraperitoneally to male rats. Rats of the negative control group received the solvent for the test material alone, i.e. an intraperitoneal dose of 20 mL/kg body weight Miglyol 812 neutral oil. The animals of the positive control group were treated with an oral dose of 16.5 mg /kg body weight cyclophosphamide.

Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the high dose group (2000 mg/kg bw), preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the low and mid dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. A total of 30 animals (5 male rats per dose group) were used.

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 micronucleated 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 some clinical signs of toxicity and, in addition, a weak decrease in body weight. No relevant treatment-related variation was observed for the quotient of 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 biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed.

The number of normochromatic cells with micronuclei was not increased.

In conclusion, the test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.