1H-Indole-6-carboxylic acid, 1-(2-chloroacetyl)-2,3-dihydro-2-oxo-, methyl ester

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:

23 March - 22 April 2009

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:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: combined micronucleus and comet assay

Test material

Test material information

Constituent 1

Reference substance name:

PD282 (intermediate in the synthesis of BEBF 1120)

IUPAC Name:

PD282 (intermediate in the synthesis of BEBF 1120)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): BIBF 1120 ES , PD 282 XX
- Molecular weight (if other than submission substance): 267.67
- Physical state: powder
-Appearance: Brownish solid
- Analytical purity: 100 %
- Impurities (identity and concentrations):-
- Purity test date: 2009-03-02
- Lot/batch No.: 10
- Expiration date of the lot/batch: 2010-02-28
-Sponsor: Boehringer Ingelheim Pharma GmbH & Co. KG Birkendorfer Str. 65 88397 Biberach an der Riss Germany

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/Sex: 30 male rats
- Strain: Han Wistar HsdRccHan
- Acclimatization time: 6 days
- Source: Harlan UK Ltd., Oxon, England
- Age at study initiation: 8 days
- Weight at study initiation: 220 – 254 g
- Assigned to test groups randomly: yes
- Housing: The animals were housed in groups of up to six, in cages that conformed with the
'Code of practice for the housing and care of animals used in scientific procedures'
(Home Office, London, 1989).
- Diet (e.g. ad libitum): the animals had access ad libitum to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham). Each batch
of diet was analysed for specific constituents and contaminants
- Water (e.g. ad libitum): Mains water was provided ad libitum via water bottles. The water was periodically
analysed for specific contaminants

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 40-70 %
- Air changes (per hr): 15 air changes / hour
- Photoperiod (hrs dark / hrs light): 12 hours light (6:00 to 18:00 Fluorescent lighting)/ 12 hours dark
- Housing: The animals were housed in groups up to 6 animals in cages, in line with Code of practice for housing and care of animals used in scientific procedures.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% hydroxyethyl cellulose (Natrosol) 250 HX in purified water
- physical state: white powder
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 50 mg/L, 100 mg/L, 200 mg/L
- Amount of vehicle (if gavage or dermal):
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): 60474
- Sponsor: Boehringer Ingelheim Pharma GmbH & Co. KG Birkendorfer Str. 65 88397 Biberach an der Riss Germany

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
PD282 was suspended in 0.5% hydroxyethylcellulose .
All treatments were given via oral gavage to 6 male rats/group in the Micronucleus /Comet experiment. The dose levels were 500, 1000 and 2000 mg/kg/day PD282. The high dose was considered to be the maximal tolerated dose based on the results of range finder experiment.
Animals were not fasted prior to dose administration. The test article was given as three administrations at time points of approximately 0, 24 and 45 hours apart and animals were sampled 3 hours after the final administration (48 hours). This dosing and sampling regimen enables examination of cells exposed to the test article over a period of 3 to 24 hours prior to sampling for the Comet assay, and 24 hours prior to sampling for the Micronucleus assay.

Duration of treatment / exposure:

45 hours

Frequency of treatment:

three administrations at time points of approximately 0, 24 and 45 hours apart and animals were sampled 3 hours after the final administration.

Post exposure period:

The sampling time points are 24 h after administration for the micronucleus assay and 3 to 24 hours post administration for the
comet assay.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS) from Sigma-Aldrich Chemical Co, Poole, UK. EMS was freshly prepared in purified water and
administered using the same regimen as the Test Article.

- Doses / concentrations: 250 mg/kg/day.

Examinations

Tissues and cell types examined:

micronucleus assay: erythrocytic cells from bone marrow of one femur

comet assay: liver , stomach, kidney
bladder

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

DETAILS OF SLIDE PREPARATION:
Bone marrow sampling and slide preparation:
3 hours after the final dose administration; test article, EMS and vehicle-treated rats were killed by an overdose of sodium pentabarbitone, given via intraperitoneal injection and exsanguinated by puncture of the abdominal aorta/vena cava. Bone marrows of one femur were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge. 60%-70% of the nucleated cell fraction were removed througha cellulose filtration system. An additional 4 mL of serum was added to each bone marrow sample prior to adding to pre-prepared cellulose filtration columns. Once filtered, the bone marrow cells were centrifuged at 200 g for 5 minutes at room temperature. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. A second centrifugation step at 200g for approximately five minutes followed with the serum aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two slides. A smear was made from the drop by
drawing the end of a clean slide along the labelled slide.
Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. Slides were stained
for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4.

Micronucleus slide analysis:
Scoring was carried out using fluorescence microscopy. Initially the relative proportions of PCE, seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Then at least 2000 PCE/animal were examined for the presence of micronuclei (MN).#

Comet Slide preparation:
Single cell suspensions were produced from the target organs from each control (negative and positive) and each test article-treated animal in Merchants solution.
Four slides were prepared per single cell suspension per tissue. An aliquot of each single cell suspension was added to 0.7% low melting point agarose (LMA) held at approximately 37 ± 1°C. 100 μL of cell suspension/agarose mix was subsequently placed onto a slide that had been previously coated in normal melting point agarose (NMA). Once gelled the coverslips were removed and slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH,1% Triton X-100, 10% DMSO). Following lysis three slides per animal were transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) and the DNA allowed to unwind for 30 minutes (liver and kidney) and 20 minutes (stomach). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 40 minutes (liver and kidney) and 20 minutes (stomach). At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation, the slides were dried and stored at room temperature prior to scoring.

Evaluation criteria:

Micronucleus evaluation criteria
For valid data, the test article was considered to induce clastogenic / aneugenic damage if: 1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels. 2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data 3. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed). The test article was considered as positive in this assay if all of the above criteria were met. The test article was considered as negative in this assay if none of the above criteria were met.
Comet scoring was performed using Perceptive Instruments 'Comet Assay III' image analysis system. Where possible, measurements of tail moment and tail intensity (% DNA in tail) were obtained from 100 cells/animal/tissue. The number of 'clouds' (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) out of 100 cells were scored for each slide
Comet evaluation criteria
For valid data, the test article was considered to induce DNA damage if:
1. A dose related change in tail moment or tail intensity in any tissue, or
2. A statistically significant change in tail moment or tail intensity in any tissue
between the vehicle and at least a single dose group.
The test article was considered as positive in this assay if all of the above criteria
were met.
The test article was considered as negative in this assay if none of the above
criteria were met.
Results which only partially satisfied the evaluation criteria above were dealt with
on a case-by-case basis.

Statistics:

Treatment of micronucleus data:
After completion of microscopic analysis and decoding of the data the following were calculated:
1. % PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated
animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN/2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (± standard deviation).
The numbers of MN PCE in vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable.
For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square test. The numbers of micronucleated PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p ≤ 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Comet data: 1. Values were added together from replicate slides. 2. The median was calculated. 3. The mean of the medians and standard error of the mean was calculated for each group.
The tail moment and tail intensity values from each treated group were compared with the respective values in the vehicle control groups using one-way analysis of variance (ANOVA) for each tissue. This was performed along with Dunnett’s test for pairwise comparisons of each treated group with the vehicle control. Levene’s test for equality of variances between the groups was performed, where this showed evidence of heterogeneity (p≤0.01), the data was analysed using the same methods after applying a rank-transformation. The vehicle control group was compared to both the treated groups and the positive control group, using a two-sample t-test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Micronucleus data

Groups of rats treated with PD282 exhibited % PCE values that were similar to vehicle controls and which fell within acceptable ranges. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in % PCE values compared to the vehicle control group or dose dependant decrease). At all doses, individual frequencies of MN PCE were similar to the values for the

concurrent vehicle control group and also fell within the laboratory’s historical control distribution data. Group mean MN PCE data were within the laboratory’s historical 95% confidence interval. There were no instances of statistically significant increase in MN PCE frequency for any of the groups receiving the test article.

Clinical signs

Clinical signs of toxicity, including piloerection, ataxia and tremors were noted on Day 3 only in the 2000 mg/kg/day PD282 group. No clinical signs were observed in animals dosed with the vehicle control or PD282 at 500 or 1000 mg/kg/day. A reduction in body weight was observed from Day 1 to Day 3 in both the 1000 (-4.2%) and 2000 mg/kg/day (-5.9%) groups compared to the vehicle control (+4.6%).

 

Stomach and Liver Data

Comet analysis of stomach and liver provided tail moment and tail intensity values that were considered consistent with the concurrent vehicle control group. There were no instances of statistically significant increases in either tail moment or tail intensity frequency for any of the groups receiving the test article. The low magnitude of increases/decreases observed in test article treated groups, together with the inter-animal variation in Comet parameters within each group, were not sufficient to be considered indicative of any cross-linking or DNA damage effects.

 

Kidney Data

Group mean tail moment and tail intensity values showed evidence of a small but non-statistically significant dose related increase in both parameters, particularly in the high dose group (~2.3 fold increase in tail intensity over vehicle control). Analysis of individual median tail intensity and tail moment values from this group revealed one animal (352) with values of 8.59 and 2.01 respectively (with maximal values of 2.32 and 0.47 respectively in the vehicle control group).

Histogram analysis (Appendix 6) of the two replicate slides scored for animal 352 were compared to the individual replicate slides scored from all animals in the vehicle control group. Analysis of the individual replicate slides show that the data for this animal were not replicated between slides, with two distinct populations of cells scored for each replicate slide. Furthermore, animals 355 and 336, from the same dose group exhibited both median tail moment and tail intensity values which marginally exceeded individual animal values in the concurrent vehicle control.

Whilst a marginal increase in DNA damage was observed at the high dose group, the biological relevance (in particular animals 355 and 336) were not considered conclusive evidence of increased DNA damage over that of the concurrent vehicle control. Furthermore, no statistically significant dose related increase in DNA damage was observed. Therefore, whilst caution should be taken when interpreting these results from this tissue, the overall conclusion from the data in the kidney is considered negative.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that PD282 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow, or DNA damage in the stomach, liver and kidney of male rats treated up to 2000 mg/kg/day (the maximum recommended dose in accordance with current regulatory guidance), under the conditions of the study.