1-Piperazineacetamide, N-(4-aminophenyl)-N,4-dimethyl-

Genetic toxicity: in vivo

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:

6 february 2003 - 11 march 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to an OECD guideline and in compliance with GLP. Therefore, the result is considered to be reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: combined micronucleus and comet assay

Test material

Test material information

Constituent 1

Reference substance name:

BIBF 1120/CDBB 213 BS

IUPAC Name:

BIBF 1120/CDBB 213 BS

Details on test material:

- Name of test material (as cited in study report): BIBF 1120/CDBB 213 BS
- Physical state: weak yellow-white solid substance
- Analytical purity: 99.2 % (HPLC)
- Purity test date: 23 january 2003
- Lot/batch No.: 8260050
- Expiration date of the lot/batch: 31 december 2003
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld (Germany)
- Weight at study initiation: males: 222 g; females: 157 g (means)
- Housing: animals were housed in groups of 5 animals in Noryl cages type V
- Diet (e.g. ad libitum): Food Kliba No. 3438.0.25 (Provimi Kliba AG, Kaiseraust, Switzerland) and municipal tap water were provided ad libitum
- Water (e.g. ad libitum): municipal tape water ad libitum
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+-3°C
- Humidity (%): 45 to 75 %
- Photoperiod (hrs dark / hrs light): 12/12 hours light -dark cycle
- Ilumination period: 6:00 a.m.- 6.00 p.m.
- Average ilumination: Ca. 60 lx (dependent on the cage position); during the experimental and observational periods, the light intensity was increased up to 100 lx.
- ca. 12 air changes per hour

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% aqueous hydroxyethylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was suspended in 0.5% hydroxyethylcellulose and administered daily by gavage (10 mL/kg body weight). A concurrent negative vehicle control (0.5% hydroxyethylcellulose) was included.

Duration of treatment / exposure:

2-weeks, 7 days per week

Frequency of treatment:

daily

Post exposure period:

Micronucleus assay: The animals were killed and bone marrow and liver prepared 24-30 hours after the last administration.

Comet assay: The samples were assessed after continous treatment (recommendation: for the in vivo Comet assay is to assess the organs at 3-6 hr and 22 - 26 hr after a single acute treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males/5 females per group at a dose of 1, 3, 10 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

No extra positive control group was included due to the large experience with this assay and for reasons of animal welfare.

Examinations

Tissues and cell types examined:

micronucleus assay: polychromatic erythrocytes from bone marrow per animal

comet assay: liver

Details of tissue and slide preparation:

Micronucleus assay:

DETAILS OF SLIDE PREPARATION: Animals were anaesthetized with sodium pentobarbital (80 mg/kg, intraperitoneal) and exsanguinated by puncture of the abdominal aorta 24-30 hours after the last treatment. The bone marrow of one tibia was flushed and cells suspended in fetal calf serum (FCS). Anucleated erythrocytic cells were separated from other myeloic cells using cellulose column fractionation. This purification step enables the preparation of slides containing only polychromatic (PCE) and normochromatic erythrocytes (NCE) without any nucleated cells or mast cell granules which can occur particularly in rats (Romagna and Staniforth, 1989). Smears were prepared from the sediment after centrifugation of the eluate on clean and grease-free slides. One slide per animal was stained with May-Grünwald/Giemsa. The slides were mounted with Entellan (Merck, Darmstadt), coded and scored.

METHOD OF ANALYSIS: Five animals per group and sex were analyzed. A 1000 x magnification with oil immersion was used for scoring. A total of 2000 polychromatic erythrocytes (PCE) per animal were scored "blindly" and randomly for micronucleated cells (MNE). By definition, micronuclei
are darkly stained and generally round nuclear bodies between 1/10 to 1/5 the size of PCE. The unit of scoring is the micronucleated cell, not the micronucleus. Standard forms were used to record the data. The frequency of polychromatic erythrocytes with micronuclei was determined for each animal and the mean number for the respective dose-group was calculated as percentage (MNE %). Data and results for individual animals and dose groups were summarized in tables. As a measure of myelotoxicity the ratio of bluish-staining polychromatic (PCE) and red normochromatic erythrocytes (NCE) was determined per animal by counting 200 normocytes on each slide. Standard forms were used to record the data. Then, the mean number for the respective dose-group was calculated and expressed as percentage of polychromatic erythrocytes (PCE%) to the total erythrocytes.

Comet assay:
A small piece of the liver, from the same five male rats per group that were investigated in the micronucleus assay, was immediately stored on ice in 10% Hanks balanced Salt Solution (HBSS) without Calcium and Magnesium, 10% DMSO and 25 mmol//L EDTA, pH 7.5. A single cell suspension was prepared by cutting the tissue manually and resuspending it with a pipette. The suspended cells were embedded into low-melting point agarose on slides coated with agarose. Then, the cells were lysed overnight at 4°C in 1% Triton X-100, 10% DMSO in 2.5 mmol/L NaCl, 0.1 mmol/L EDTA and 0.01 mmol/L Tris, pH 10. The slides were put into ice-cooled electrophoresis buffer containing about 300 mmol/L NaOH and 1 mmol/L EDTA, pH 13, first for 20 minutes to allow unwinding of the DNA and then for about 15 - 20 min under an electric field (18-25 V/300 mA) to migrate single-stranded fragmented DNA out of the nucleus (head) forming a tail. After neutralization with 400 mmol/L Tris-HCl, pH 7.5, 3 times each 5 minutes , the slides were stained with ethidium bromide and the intensity of the head and tail of 2x50 single cells was visualized in a fluorescence microscope and quantified using the software Komet 5.0 from Kinetic Imaging and calculated as Olive tail moment (=(Tail.mean - Head.mean)* Tail%DNA / 100). The study was conducted under non-GLP conditions because the present software version is not GLP-compatible.

Evaluation criteria:

The criterion for a positive result is a statistically significant, dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared with the negative vehicle control. Additionally, historical control frequencies obtained in similar experiments using this rat strain are taken into consideration.

Statistics:

The statistical analysis of the data was performed using the Fisher-Pitman permutation test. This method takes into account the characteristics of the
micronucleus assay (the animal is the experimental unit, small absolute incidences with non-normal distributions). All comparisons were made to the vehicle control. Mean and P-values are given in the summary table. A probability of P≤5% was considered statistically significant.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Any other information on results incl. tables

Micronucleus Assay

Myelotoxicity

The mean percentage of polychromatic erythrocytes did not differ substantially between the BIBF 1120/CDBB 213 BS treated groups (M: 31.7-36.1%, F: 24.6-37.3%) and the negative vehicle control animals (M: 30.2%, F: 26.2%). The statistically significant increase in the percentage in low and high dose females (36.0 and 37.3%, respectively) is without any biological relevance since it lacks of a clear dose response. All values were within the historical control range (see page 17) experienced in this laboratory.

Micronucleus Analysis

BIBF 1120/CDBB 213 BS treated groups showed comparable mean numbers of polychromatic erythrocytes containing micronuclei in bone marrow (M: 0.10-0.18%, F: 0.10-0.13%) as those of the negative vehicle control (M: 0.07%, F: 0.16%). The slight

increase of micronucleated cells in mid dose males (0.18%) is without biological relevance since it lacks of a dose response and the extreme low control value (0.07%; historical control range: M: 0.09-0.19%, F: 0.10-0.35% and historical mean value: 0.15% in male

and female animals based on 15 (M)/8 (F) studies). Based on these results it was concluded that BIBF 1120/CDBB 213 BS did not induce micronuclei in rat bone marrow under the conditions of the study. However, no final conclusions can be drawn whether BIBF 1120/CDBB 213 BS would induce micronuclei at the maximum tolerated dose since there were no signs of toxicity or myelotoxicity

observed in this study as required by the relevant guidelines. Although substantial plasma levels were achieved, the absence of consensus about safety factors for genotoxic impurities based on plasma levels renders the results of this study as preliminary only.

Comet Assay

There was no treatment-related increase in the intensities of the tail versus the head values in liver cells isolated from animals from the repeated oral dosing study with BIBF 1120/CDBB 213 BS. The respective Olive tail moments given in mean and standard deviation from five male animals were 0.57 ± 0.70, 0.68 ± 0.73 and 0.74 ± 0.95 for the low-, mid- and high-dose group as compared to the negative control of 0.63 ± 0.71, respectively (Table on page 19). Therefore, there was no indication of the presence of DNA damage in this potential target organ. However, the samples were assessed after continuous treatment while the recommendation for the in vivo Comet assay is to assess the organs at 3 -6 hr and 22 - 26 hr after a single acute treatment. Therefore, this deviation

in addition to the lack of toxicity render the data obtained as preliminary.

Applicant's summary and conclusion

Conclusions:

BIBF 1120/CDBB 213 BS did not induce micronuclei in bone marrow cells of rats after 2 week oral treatment with daily doses up to 10 mg/kg and substantial plasma levels up to Cmax of 1690 ng/mL and (AUC0-24h) of 5440 ng·h/mL. However, no final conclusions can be drawn whether BIBF 1120/CDBB 213 BS would induce micronuclei at the maximum tolerated dose due to the absence of dose-limiting toxicities. The same is true for the negative results obtained in a non-GLP Comet assay in which DNA damage in liver cells was assessed.