Acetamide, 2-chloro-N-methyl-N-(4-nitrophenyl)-

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:

19 March - 20 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in GLP compliance and in accordance with an OECD guideline (see below).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

four instead of five test animals were used due to comet test restrictions

Principles of method if other than guideline:

REFERENCE comet assay:
Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P,
Collins A, Smith A, Speit G, Thybaud V and Tice RR. Recommendations for
conducting the in vivo alkaline Comet assay. Mutagenesis 2003; 18:45-51

GLP compliance:

yes

Type of assay:

other: combined micronucleus and comet assay

Test material

Test material information

Constituent 1

Reference substance name:

CDBE 0055 XX

IUPAC Name:

CDBE 0055 XX

Test material form:

other: powder

Details on test material:

Test substance: BIBF 1120 ES / CDBE 55 XX
Batch number: 7
Physical description: grey solid powder
Certificate of analysis: 6 February 2007
To be re-tested: 31 December 2007
Purity: 100% (HPLC)
Storage conditions: At room temperature, in the dark (ambient humidity)
Sponsor: Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach (Germany)
Manufacturer: Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim (Germany)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Species/Sex: 17 male rats
Strain: Crl:WI(Han)
Breeder: Charles River Laboratories, Research Models and
Services, Deutschland GmbH, Sulzfeld (Germany)
Body weight: 255 – 293 g (at first administration)
Acclimatization time: 4 days
Food/water: Tap water and food ad libitum

Housing: The animals were housed in groups of 2-4 animals in Noryl cages, type V.
Temperature: 22 ± 2°C
Relative humidity: 45 to 75%
Light/darkness cycle: 12/12 hours
Illumination period: 6.00 a.m. - 6.00 p.m.
Average illumination: Ca 60 lx (dependent on the cage position). During the experimental and observational periods, the light intensity was increased up to 450 lx.
Air circulation: Ca 10 air changes per hour

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% hydroxyethylcellulose (Natrosol)

Details on exposure:

STUDY DESIGN:
CDBE 55 XX was suspended in 0.5% hydroxyethylcellulose (Natrosol) and given orally
(gavage) three times to 4 male rats/group of the strain Crl:WI(Han) at an interval of 24 hours.
The animals were treated with 0, 100 and 500 mg/kg CDBE 55 XX 52, 28 and 4 hours before
necropsy (positive control 28 and 4 hours before necropsy). The time points were based on
the protocols for the micronucleus and comet assays. The sampling time points are 24 and 48
h for the micronucleus assay (OECD guideline) and 2-6 h post administration (p.a.) for the
comet assay depending on the expected Cmax which was presumed to be at 4 h p.a..
Only male animals were tested for reasons of animal welfare. A negative vehicle
control (0.5% hydroxyethylcellulose) and a positive control group (110 mg/kg methyl
methanesulfonate/MMS, 2 animals, two administrations) were treated concurrently.

Duration of treatment / exposure:

4 hrs, 28 and 52 before necropsy (positive control 28 and 4 hours before necropsy)

Frequency of treatment:

3 times in 24 hrs

Post exposure period:

The sampling time points are 24 and 48h for the micronucleus assay (OECD guideline) and 2-6 h post administration (p.a.) for the
comet assay depending on the expected Cmax which was presumed to be at 4 h p.a.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 and 500 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 males 100 mg/kg
5 males 500 mg/kg
includes 4 males/dose for Comet assay test


5 males 100 mg/kg
5 males 500 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

A positive control group (110 mg/kg methyl methanesulfonate/MMS, 2 animals, two applications) was treated concurrently.

Examinations

Tissues and cell types examined:

micronucleus assay: Anucleated erythrocytic cells from bone marrow of one femur

comet assay: liver (right medial lobe), jejunum and urinary
bladder

Details of tissue and slide preparation:

Micronucleus assay:
Four hours after the third (positive control after the second) treatment the animals were anaesthetized (i.p.) with Ketamin/Xylazin and exsanguinated by puncture of the abdominal aorta. Bone marrow of one femur was flushed and cells were suspended in fetal bovine serum (FBS). 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. After centrifugation of the eluate smears were prepared from the sediment 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.

Comet assay:
At necropsy samples from the target organs liver (right medial lobe), jejunum and urinary bladder were immersed in chilled tissue buffer. A small piece was cut up with scissors in 200 µL of tissue buffer. 20 µL of the cell suspension were mixed with 100 µL low melting agarose and spread by coverslips on pre-coated slides (2 per organ). The slides were placed on ice to prevent DNA repair and to aid gel setting. After solidification the coverslips were removed, the slides immersed in ice-cold lysis buffer and stored in the refrigerator until the next day.
Electrophoresis For unwinding and separation of the DNA in single strands, the slides were incubated in icecold electrophoresis buffer (pH>13) for 20 min. Then electrophoresis was run 20 min at 25 V/300 mA. After neutralization (15 min) and rinsing in 100% ethanol the slides were airdried.

Evaluation criteria:

The assays are considered valid when a sufficiently high number of animals is available foranalysis and the negative and positive control groups show the expected response.Micronucleus assay: The criterion for a positive result is a statistically significant, dosedependentincrease 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

Comet assay: The dose-dependent and statistical significant increase of the OTM in thetarget organs compared to the negative control is indicative for a primary DNA damage.

Statistics:

Micronucleus assay: The statistical analysis of the data was performed using the Fisher- Pitman permutation test (R97-0699). This method takes into account the characteristics of the micronucleus assay (the animal is the experimental unit, small absolute incidences with nonnormal distributions). All comparisons were made to the vehicle control. Mean and p-values are given in the summary table. A probability of p<0.05 was considered statistically significant (*).

Comet assay: The standard deviation from the single and mean values was calculated by the computerized system. There was an increase in mean values for the jejunum in the high-dose group higher than 1.5-fold above the negative control. Therefore, an additional statistical evaluation was done using a Jonckheere-Terpstra and a Wilcoxon test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Clinical observations:

Mortality

No treatment-related deaths had to be noted during the study.

Clinical Signs

There were no clinical signs observed in the course of this study. At necropsy, all rats given 500 mg/kg showed an excess of fluid in the stomach and intestines.

Micronucleus Assay:

Myelotoxicity

CDBE 55 XX did not induce myelotoxicity since there was no treatment-related change in the mean percentage of polychromatic erythrocytes of 18.2 and 17.3 in rats given 100 and 500 mg/kg, respectively compared with the concurrent vehicle control (19.3). The historical control range in this rat strain is 18.3-43.5%. MMS showed a treatment-specific decrease of PCE (7.8%).

Micronucleus Induction

CDBE 55 XX did not induce a treatment-related increase in the percentage of micronucleated polychromatic erythrocytes (0.10%) compared with the concurrent negative control (0.10%). All values were within the historical control range (0.07 - 0.20%) obtained in this laboratory with this rat strain. As expected, MMS caused an increase of micronucleated cells (0.88%).

Comet Assay:

There was no increase in the mean OTM values in liver and jejunum cells from rats treated three times with 100 and 500 mg/kg CDBE 55 XX p.o. The respective values in the liver were 0.20, 0.19 and 0.25 for negative control, 100 and 500 mg/kg CDBE 55 XX. The mean OTM values in jejunum cells were 0.24, 0.28 and 0.37, respectively. In contrast, the mean OTM value in urinary bladder cells showed an increase at the highest dose. The respective mean values were 0.26, 0.31 and 1.27. The increase was due to a single rat (No. 303). Since a single value was observed in a previous study and the repeat experiment showed OTM values within the range of the negative control, this single high value was considered to be an artifact due to the processing of the bladder tissue. This was supported by the fact, that only this rat showed high percentage for hedge hog-like figures (80%) indicating cellular toxicity (necrosis and apoptosis). Histopathological evaluation showed that proteinaceous material in bladder lumen was found in this rat while all other urinary bladders were inconspicuous.

As expected, the positive control MMS showed a clear increase of comet positive cells with OTM values of 1.77, 3.93 and 1.86 in liver, jejunum and urinary bladder cells, respectively. The %DNA in tail values showed the same tendencies as the OTM values. There was no increase in the mean %DNA values in liver and jejunum cells. The respective values in the liver were 2.53, 2.35 and 2.94 for negative control, 100 and 500 mg/kg CDBE 55 XX. The mean %DNA in tail values in jejunum cells were 3.09, 3.44 and 3.52, respectively. In contrast, the %DNA value in urinary bladder cells showed an increase at the highest dose. The respective mean values were 2.56, 3.16 and 6.31. The increase was again due to one rat (No. 303) with a value of 15.44. This value was higher than in the positive control cells (9.33).

The percentage of hedge hogs indicating cellular toxicity (necrosis and apoptosis) was estimated. There was no treatment-related increase of hedge hogs in liver, jejunum and urinary bladder compared to the negative control with exception of an increase in the urinary bladder of animal 303 (80%) while all other animals were within the range of the negative control (3-20%). The estimated percentages in control and CDBE 55 XX treated groups were 1-2% (liver), 2-10% (jejunum) and 3-80% (urinary bladder).

The positive control substance MMS induced also a clear increase of hedge hog cells in the liver (15-20%), jejunum (30-40%) and urinary bladder (8-10%). However, there was no corresponding histopathological damage present.

Hispatology:

At necropsy, all rats given 500 mg/kg showed an excess of fluid in the stomach and intestines. In addition Animal No. 303 showed an increase of the OTM in the urinary bladder. For this reason, the jejunum and urinary bladders from all rats were examined histopathologically. All rats given 500 mg/kg CDBE 55 XX had focal hyperemia, an increase in mitoses of crypt epithelial cells and acute degeneration and necrosis of the villi in the small intestines. In contrast, the positive control rats had a slight increase in apoptoses of crypt epithelial cells. The urinary bladders were inconspicuous except for animal No. 303 (500 mg CDBE 55XX) which had proteinaceous material in the bladder lumen.

Applicant's summary and conclusion

Conclusions:

CDBE 55 XX did not induce micronuclei in the bone marrow of rats treated orally with 100 and 500 mg/kg CDBE 55 XX. Furthermore, it did not induce damage (single strand breaks and alkaline-labile sites) in the liver, jejunum and urinary bladder as assessed by the comet assay.
In terms of risk assessment, CDBE 55 XX did not show clastogenicity and DNA reactivity under biologically relevant conditions up to toxic doses. Therefore, CDBE 55 XX is an in vitro genotoxin only. Based on the weight of evidence from all the genotoxicity tests with CDBE 55 XX, the two negative in vivo mutagenic endpoints allow the conclusion that workers handling CDBE 55 XX are not exposed to an undue genotoxic hazard.