# PMN Submission For TS-EV07J3 SanitizedCopy BookII

# ofII

# Attachment #19

## STUDYTITLE REPORT IN VITRO CHROMOSOME ABERRATION ASSAY WITH 

# D

IN V79 CELLS DATA REQUIREMENT OECD No. 473 EC 

## 2000/32, B.10 EPAlOPPTS 870.5375 AUTHORS

STUDY COMPLETED ON 20 Nov 2006 PERFORMING LABORATORY

LABORATORY PROJECT IDENTIFICATION 

## Project No.:**~--------------~** 

## SPONSOR

This document contains manufacturing and trade secrets ofthe Sponsor(s). It is the property ofthe Sponsor(s) and may be used

a encies actin only for that purposeforwhich it was intended by the Sponsor(s). Every other or additional use. exploitation, reproduction, publication or submission to otherparties require the written permission ofthe Sponsor(s), with the exception of regulatory within the limits oftheir administrative authorit

.

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## Page 2 of 99

## GLPCOMPLIANCESTATEMENT

This studywasconducted in accordancewith the OECD Principles ofGood Laboratory Practice and the GLP Principles ofthe German "Chemikaliengesetz" (ChemicalsAct)which meetthe United States Environmental Protection Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part792 (TSCA)], with the exception that recognized differencesexist between the GLP Principles/Standards ofOECD and the Principles/Standards ofFIFRA and TSCA.

To be filled for USA EPAsubmission only:



## Sponsor Date



## Submitter Date

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## 3 of99

## Statementfor US EPA submissions only:



## FLAGGING CRITERIA The flagging criteria of40 CFR 158.34are notapplicable forthis studytype

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## 99

## SIGNATURE PAGE

Study Director:

Management:

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## Page 50f99

## CONTRIBUTORSTOTHE STUDY

## ISUPERVISORY LABORATORY PERSONNEL

Head of Experimental Toxicology and Ecology: Study Director:
Data processing:
Statistics: Analytical Chemistry: QualityAssurance Unit (QAU): Genotoxicity:

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# -

STATEMENTOFTHE QUALITYASSURANCE UNIT


## The QualityAssurance Unit (QAU) inspected the study and reported any inspection results to the Study Director and to Management. The final report reflects the raw data.

Phase ofstudy Date of inspection Reported to Study Director and to Management (mm-dd-yyyy) (mm-dd-yyyy) Study Plan: 07-06-2006 07-06-2006 Conduct ofstudy: 07-14-2006 07-14-2006 Report: 11-09-2006 11-09-2006

# ~~~~~---.--

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## Page 70f99

## GLP·CERTIFICATE (FROM THE COMPETENT AUTHORITY)

Gute Laborpraxls I

### Good Laboratory Practice 

## GLP·Bescheinigung 

## IStatementofGLP Compliance 

# •

(gem./according to § 

### 19Abs. 

### 1 Chemikaliengesetz)

Eine GLP-Inspektlon zur Oberwachung und Assessment of conformity with GLP acoording der Einhaltung der GLP-Grundsllize gemaB 

### to Chemikallengeselz and Directive 88/3201 Chemikaliengeselz bzw. Richtlinie 88/320/EG EECat:: wurde durchgefilhrt in:
prilfejndchh 100 I

### Testfacility

PrQfung nach Kategorien/ Areas ofExpertise (gem.Iaccording ChernVwV-GlPNr. 5.3/OECD guidance) 

### 1,2,3,4,5,8,9 Kat. 9 - Biochemische und pathologische Untersuchungen zu Wirkmechanismen/ Biochemicaland pathological examinationsconceming modeofaction Datumder Inspektion/ Date of Inspection (Tag.MonatJahr1day.monthyear) 

### 08.06.2005 und

### 25. bls 

### 27.07.2005
Die genannte PrOfeinrichlung befindet slch im The above mentioned testfacility is included in nalionalen GLP-Oberwachungsverfahren und the national GLP Compliance Programme and wlrd regelmllBig auf Einhaltung der GLP­ is inspected on a regular basis. Grundsatze uberwacht. Auf der Grundlage des InspekliQnsberichtes Based on the Inspection report it can be con­ wird hiermn bestatigt, dass in dieser PrUf­ firmed. that the test facUlty Is able to conduct einrichtung die oben genannten PrOfungen the aforementioned studies in compliance with unter Einhaltung der GLP-Grundsatze durch­ the Principles ofGLP. gefUhrt werden kOnnen.



### 55116 Mainz**~1llilw;;1:dU:!a1LIIlL1..SlQllll1lure.** Date

landesamtfur Umwelt.Wasserwirtschaft und Gewerbeaufsicht Kaiser-Friedrich-SIraBe

### 7

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## CONTENTS


## TITLE PAGE BLANK PAGE GLP COMPLIANCE STATEMENT FLAGGING CRITERIA SIGNATURE PAGE CONTRIBUTORS TO THE STUDY 

## I SUPERVISORY LABORATORY PERSONNEL STATEMENT OF THE QUALITYASSURANCE UNIT GLP-CERTIFICATE (FROM THE COMPETENT AUTHORITY) CONTENTS 

# 1. SUMMARY 2. INTRODUCTION 2.1. OBJECTIVES 2.2. SELECTION OF DOSES/CONCENTRATIONS 2.3. TEST GUIDELINES 2.4. STUDY DATES 2.5. RETENTION OF RECORDS 

# 3. MATERIALAND METHODS 3.1. TEST SUBSTANCE 3.2. BIOLOGICAL TEST SYSTEM 3.2.1. Cell line, storage 3.2.2. Culture media 3.2.3. Cell culture 3.3. EXOGENOUS METABOLIC ACTIVATION 3.3.1. S-9 fraction 3.3.2. S-9 mix 3.4. SELECTION OF VEHICLE, TEST GROUPS AND DOSES 3.4.1. Choice ofthe vehicle 3.4.2. Pretests for dose selection 3.4.3. Test groups and doses
- 1stexperiment 3.4.4. Test groups and doses
- 2nd experiment

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# 3.5. 3.6. 3.7. 3.7.1. 3.7.2. 3.7.3. 3.7.4. 

# 3.7.5. 3.7.6. 3.8. 3.8.1. 3.8.2. 3.8.3. 3.8.4. 

# 3.8.5. 3.8.6. 3.8.7. 3.8.8. 

# 4. 4.1. 4.1.1. 4.1.2. 4.2. 4.2.1. 4.2.2. 4.2.3. 4.3. 4.4. 

## 4.5. 4.6. 4.7. 

# 5. 6. 7. Test Substance Preparation ANALYSIS OFTEST SUBSTANCE PREPARATION EXPERIMENTAL PROCEDURE Cell cycle time Sampling times Control articles Preparation oftest cultures Treatment oftest cultures Preparation ofmetaphase spreads EVALUATION Mitotic index Cell counts Cell morphology Treatment conditions Chromosome analysis Acceptance criteria Assessment criteria Statistics 

# RESULTS CHROMOSOMEANALYSIS - 1ST EXPERIMENT Assay without S-9 mix; 4-hour exposure. 18-hoursampling time Assay with S-9 mix; 4-hour exposure, 18-hoursampling time CHROMOSOMEANALYSIS

- 2ND EXPERIMENT Assay without S-9 mix; 18-hourexposure. 18-hoursampling time Assay without S-9 mix; 18-hourexposure, 28-hour sampling time Assaywith S-9 mix; 4-hour exposure. 28-hoursampling time MITOTIC INDEX CELL COUNTS CELL MORPHOLOGY ASSESSMENT OFTHE SLIDES TREATMENT CONDITIONS 

# DISCUSSION CONCLUSION REFERENCES

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## APPENDIX 1 1ST EXPERIMENT (TABLES 1 - 12) 

## APPENDIX 2 2ND EXPERIMENT (TABLES 

## 13
- 28) APPENDIX 3 MITOTIC INDEX, CELL COUNT APPENDIX 4 CELL MORPHOLOGY, ASSESSMENT, TREATMENT CONDITIONS APPENDIX 5 HISTORICAL NEGATIVE CONTROL DATA - CLASTOGENICITY APPENDIX 6 HISTORICAL NEGATIVE CONTROL DATA - ANEUGENICITY APPENDIX 7 HISTORICAL POSITIVE CONTROL DATA

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# 1. SUMMARY The substancel

# ~as assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the numberofchromosomes (aneugenic activity) in V79 cells in vitro both in the presence and in the absence ofa metabolizing system. According to an initial range-finding cytotoxicitytest forthe determination ofthe highest experimental doses, the test substance did notexhibit any pronounced toxicity up to the highest recommended doses, i.e. 

## 1500 IJg/mL 

## (-10 mM), the following doses were tested and the test groups in bold type were evaluated:



## • 1stexperiment 4-hourexposure, 18-hoursampling time, without5-9 mix 

## 0; 187.5; 375; 750; 1 

## 500 IJg/mL 

## 4-hourexposure, 18-hoursampling time, with 5-9 mix 

## 0; 187.5; 375; 750; 1 

## 500 IJg/mL



## • 2nd experiment For confirmation ofthe results ofthe 1

## st experiment including continuous exposure and a second sampling time, the following doses were tested and the test groups in bold type were evaluated:


## 18-hourexposure, 18-hoursampling time, without 5-9 mix 

## 0; 187.5; 375; 750; 1 

## 500 IJg/mL 

## 18-hourexposure, 28-hoursampling time, without 5-9 mix 

## 0; 375; 750; 1 

## 500 IJg/mL 

## 4-hourexposure, 28-hoursampling time, with 5-9 mix 

## 0; 187.5; 375; 750; 1 

## 500 IJg/mL

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## About 2 - 3 hours priorto harvesting the cells, Colcemid was added to arrest cells at a metaphase-like stage of mitosis (c-metaphases). After preparation ofthe chromosomes and staining with Giemsa, 100 metaphases foreach culture in the case ofthe test substance, and vehicle controls, or 50 cells for each culture in the case ofthe concurrent positive controls, were analyzed forchromosomal aberrations. The negative controls (vehicle controls) gavefrequencies ofaberrations within the range expected forthe V79 cell line. Both ofthe positive control chemicals, i.e. EMS and cyclophosphamide, led tothe expected increase in the numberof cells containing structural chromosomal aberrations. On the basis ofthe results ofthe presentstudy, the test SUbstance did not cause any increase in the numberofstructurally aberrant metaphases inc!. and excl. gaps at both sampling times eitherwithout S-9 mix and/or after adding a metabolizing system in two experiments performed independently ofeach other. No increase in the frequency ofcells containing numerical aberrations was demonstrated either. Thus, underthe experimental conditions ofthis assay, is considered notto be a chromosome-damaging (clastogenic) agent under in vitro conditions in V79 cells.

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# 2. INTRODUCTION

2.1. OBJECTIVES The aim ofthe present study was to assess the potential ofthe test substancd lor its metabolite(s) to induce structural chromosomal aberrations as a consequence ofclastogenic activity and/or polyploidy including endoreduplicated cells due to an aneugenic mode of action. Forthis purpose, an in vitro cytogenetic assaywas carried out in V79 cells (1, 2, 3). Most substances are not mutagenic orcarcinogenic themselves, but only after metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems ofthe liver. Therefore, the tests are carried out notonly directly, but also in the presence ofan exogenous metabolic activation system. The most commonly used system is a cofactorsupplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an enzyme-inducing agent, e.g. Aroclor 1254 (4).

2.2. SELECTION OF DOSES/CONCENTRATIONS The test doses/concentrationswere selected onthe basis ofthe non-GLP range finding part ofthe study (see also section 3.4.). The raw data ofthis part are archived togetherwith the rawdata ofthis study.

2.3. TEST GUIDELINES The study was conducted according to the following test guidelines:

• OECD No. 473 (21 Jul 1997) • EC Directive 2000/32, 

## B.10 (19 May 2000) • EPA/OPPTS 870.5375 (August 1998)

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## 2.4. STUDY DATES Test dates: Study initiation date: 06 Jul2006 Experimental starting date (seeding ofcells, 1st experiment): 12Jul2006 Experimental completion date (draft report to QAU): 19 Oct 2006



## 2.5. RETENTION OF RECORDS GLP-relevant records and materials are stored at

# I 

# J for at least the period oftime specified in the GLP principles. Details concerning responsi ilities or locations ofarchiving can be seen from the respective SOPs and from the raw data.

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# 3. MATERIALAND METHODS

3.1. TEST SUBSTANCE 

## The analyses ofthe test substancewill be 

## Iwere carried out atthe

# I 

# I I**~--~** Name oftest substance:
Test substance No.:
Batch identification:
CAS No.: Purity: 

## 99.9 g/100 9 (see test substance characterization; Project No.1

# ~ Homogeneity: The homogeneityofthe testsubstancewas guaranteed on account ofthe high purity and was ensured by mixing before preparation ofthe test substance preparations. Storage stability: The stabilityofthe test substance under storage conditions throughoutthe study period will be verified by reanalysis on completion ofthe experimental phase of the studyorwill be substantiated otherwise. The results will be reported in an amendmenttothis report.

ADDITIONALTEST SUBSTAI\JCE INFORMATION

Date ofproduction:
Molecularweight: Physical state, appearance: Storage conditions: 

## 1995
148.19 glmol Solid, beige
Room temperature

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## 16 of

## 99

## 3.2. BIOLOGICAL TEST SYSTEM

3.2.1. Cell line, storage The 

## V79 cell line (1, 2) derived from the Chinese hamster has a

• high proliferation rate (doubling time ofabout 12
- 16 hours) • high plating efficiency 90%) • stable karyotype (modal numberof22 chromosomes).

Stocks ofthe 

## V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used forthe cytogenetic experiments was checked for

• mycoplasma contamination • karyotype stability • plating efficiency 

## (= colonyforming ability) incl. vital staining.



## 3.2.2. Culture media MEM (with Earle's salt) MEM medium with glutamine supplemented with 10% (v/v) fetal calf serum (FCS) 1 % (v/v) penicillin/streptomycin (10 000 I U / 10 000 IJg/mL) 1 % (v/v) amphotericin B (250 IJg/mL) During exposure to the test substance (4-hourtreatment), MEM medium was used without FCS supplementation.



## 3.2.3. Cell culture Deep-frozen cell suspensionswere thawed at

# 3rC in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 

## plastic flasks containing about 5.0 mL MEM (minimal essential medium incl. glutamine), supplemented with 10% FCS (fetal calfserum) and antibiotics (peniCillin/streptomycin and amphotericin). Cells were grown with 5%**C~** 

## at 

# 3rC and**~** 

## 90% humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium afterdispersion with 0.25% trypsin solution (about 1.0 mL).

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## 3.3. EXOGENOUS METABOLIC ACTIVATION



## 3.3.1. S-9fraction 

## The S-9 fraction was prepared according to AMES etal. (4). At least 5 male Sprague-Dawley rats (200

- 300 g, Charles River Laboratories, Germany GmbH) received a single intraperitoneal injection of500 mg Aroclor 1254 (as a 200 mg/mL solution in corn oil) per kg bodyweight 5 days before sacrifice. During this time, the animalswere housed in Makrolon cages and were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of20
- 24"C and a relative humidity of30
- 70%. The day/night rhythm was 12 hours (12 hours light from 6.00 

## -1B.00 hours and 12 hours darknessfrom 

## 1B.00
- 6.00 hours). Standardized pelleted feed and tap waterfrom bottles were available ad libitum. 5 days afteradministration, the rats were sacrificed and the livers were prepared (all preparation stepsfor obtaining the liver microsome enzymes were carried out using sterile solvents and glassware atatemperature of+4"C). The livers were weighed and washed in a weight-equivalentvolume ofa 150 mM KCI solution, then cut into small pieces and homogenized in three volumes of KCI solution. Aftercentrifugation ofthe homogenate at 9 000 x g for 10 minutes at +4"C, 5-mL portions ofthe supernatant (so-called S-9 fraction) were stored at -70"C to 

## -BO"C.



## 3.3.2. S-9 mix 

## The S-9 mixwas prepared freshly priorto each experiment (4). Forthis purpose, a sufficient amount ofS-9 fraction was thawed at room temperature, and 1volume of S-9 fraction was mixed with 9 volumes ofS-9 supplement (cofactors). This preparation, the so-called S-9 mix, was kept on ice until used. The concentrations ofthe cofactors in the S-9 mixwere: MgCI2 

## B mM KCI 33 mM Glucose-6-phosphate 5 mM NADP 

## 4 mM Phosphate buffer (pH 7.4) 15 mM The phosphate buffer (5) was prepared by mixing an NB2HP04 

## solution with an NaH2

## P04 

## solution at a ratio ofabout4 : 1.

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## 3.4. SELECTION OF VEHICLE, TEST GROUPS AND 

## DOSES

3.4.1. 

## Choice ofthe vehicle Due to the limited solubilityofthe test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and forwhich historical control data are available.

3.4.2. 

## Pretestsfor dose selection The doses for the 1stexperiment (18-hour sampling time) were determined from appropriate range-finding cytotoxicitytests with cultures exposed forthe duration of4 hours to awide dose range ofthe test article, i.e. 1 IJg/mL - 1 500 IJg/mL culture medium both without S-9 mix and afteradding a metabolizing system. In these pretests, various parameters were checked for all orat least some selected doses; the results were given in the following tables on pages 20
- 21

.As a rule, the highest dose 

## I concentration for nontoxictest substances should not exceed a limit of5 mg/mL or 10 mM, as recommended bythe EC, OECD and by an ICPEMC Task Group (6). According to the results ofthe pretests, the test substance is nontoxic after an exposure time of4 hours up tothe highest recommended dose of 10 mM (- 1500 IJg/mL). On the basis ofthe findings from the pretests, thetop dose in the 1st experimentwas selected afteran exposure period of4 hours with and without S-9 mixtaking into account the current guidelines (see item 1.2.) or recommended criteria (6). The results ofthe pretests after continuous treatment applied in the 2nd experiment are given in thetable on page22. Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.

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## Table 1: 

## 4-hour exposure, 18-hoursamplingtime, without

## 8-9 mix
Test groups pH Osmolarity Solubility Cell Cell Mitotic Assesscount attach- index mentof ment the slides Veh Culture Oh 3-4h Doses mM mOsm Ma Ma Ma 

# tv!.[ ReI. % ReI. % Vehicle control 

# - 7.4 409



### - 

# - 

# - 100.0 1 100.0 E1 DMSO 

### 1**~g/mL** 

### 0.007


# - - S



### - 

# - 90.6


# - - 5**~g/mL** 

### 0.034


# - - S


# - 

# - 97.2


# - - - 

### 10**~g/mL** 

### 0.067


# - 

# - S 

# - - 

# - 102.2



# - - 

# - 50**~g/mL** 

### 0.337


# - - S


# - - 106.4


# - - - 

### 100**~g/mL** 

### 0.675


# - - S



### - - 108.6



# - - - 500**~g/mL** 

### 3.374 

# - S 

### S S S 

### 101.4 1


# - E1 

### 1000**~g/mL** 

### 6.748 7.5 405 S 

### S S S 

### 95.3 1



### - E1 

### 1500**~g/mL** 

### 10.122 7.6 399 S S S S 

### 89.7 1 109.1 E1


## For keyto abbreviations and explanations see page 23.

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## Table 2: 4-hourexposure, 18-hoursampling time, with 5-9 mix

: Test groups

Doses mM pH Osmolarity

mOsm Solubility Cell Cell Mitotic count attach- index ment

ReI. 

### % ReI. 

### % Assess- 

# I ment of the slides Veh Ma Culture Oh 3-4h Ma Ma Mi Vehicle control 

# - 7.3 418


# - - 

# - 100.0 1 100.0 E1 DMSO 

### 1 IJg/mL 

### 0.007 

# - - S



### - 

# - 

# - 98.3 

# - - 

### 5 IJg/mL 

### 0.034 

# - S 

# - - - 105.3



### - 

# - 10 IJg/mL 

### 0.067


# - S



### - - 98.6



### - 

# - 50 IJg/mL 

### 0.337


# - - S 

# - - 108.6


# - - 100 fJg/mL 

### 0.675


# - - S


# - - 101.9


# - - 

### 500 fJg/mL 

### 3.374


# - 

# - S S S S 103.9 1


# - E1 

### 1000 IJg/mL 

### 6.748 7.4 378 S S S S 100.3 1



# - E1 

### 1 500 fJg/mL 

### 10.122 7.4 379 S S S S 99.2 

### I 1 119,4 E1


## For keyto abbreviations and explanations see page 23.

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## Table 3: 

## 18-hourexposure, 18-hoursamplingtime, without5-9 mix
Test groups pH Osmolarity Solubility Cell Cell Mitotic Assesscount attach- index mentof ment the slides I Veh Culture Oh 

### 16-18h_ Doses mM mOsm Ma Ma Ma Mi Ret % ReI. % Vehicle control 

## DMSO 

# - 7.6 427


# - - - 

# - 100.0 

### 1 100.0 E1


### 1 1J9/mL 

### 0.007


# - - S 

# - - - 103.4



### - - 

### 5 1J9/mL 

### 0.034


# - 

# - S


# - 

# - 99.3 

# - - 

# - 10 1J9/mL 

### 0.067



### - 

# - S 

# - - - 98.8


# - - 50 1J9/mL 

### 0.337



### - 

### - S 

# - 102.7



### - - 

# - 100 1J9/mL 

### 0.675


# - - S


# - - 

# - 98.3


# - - - 500 1J9/mL 

### 3.374



### - 

# - S S S S 

### 102.0 1


# - E1 

### 1000 1J9/mL 

### 6.748 7.7 419 S S S S 

### 96.6 1 

# - E1 

### 1500 1J9/mL i 

### 10.122 7.7 408 S S S 

# s 

# I 77.1 1 102.3 E1


## For key to abbreviations and explanations see page 23.

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## Key to abbreviations and explanations:
Veh 

# = vehicle S 

# = solution Su 

# = suspension 

### E 

# = emulsion 

### p 

# = precipitation Ma 

# = macroscopically Mi 

# = microscopically mM 

# = millimolar mOsm 

# = milliosmolar


### E 

# = evaluation possible



## =sufficient metaphases ofgood quality 2 = reduced number ofanalyzable metaphases 3 

### = reduced number ofanalyzable metaphases, some ofpoor quality
R 

### = evaluation possible but with reservations



### = considerably reduced number ofanalyzable metaphases 2 

## =considerably reduced number ofanalyzable metaphases, some ofpoor quality 3 = evaluation of metaphases influenced by precipitation ofthe test substance


### N = evaluation not possible



### = no or only a few metaphases for evaluation 2 

### = no or only a few metaphases for evaluation and some of poor quality 3 

# =complete cell death (lysed cells) 4 = evaluation of metaphases strongly influenced by precipitation ofthe test substance

+ = complete attachment, i.e. fibroblast-like cells ++ 

### = slightly reduced attachment, i.e. few cells rounded +++ reduced attachment, i.e. most cells rounded ++++ = complete detachment, i.e. all cells rounded

= not determined

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## Page 23 of99

## 3.4.3. Test groups and doses - 1stexperiment The numberoftest groups selected on the basis ofthe pretests and evaluated in the 1st main cytogenetic experiment can be seen from the following table. Duplicate cultures were used forall experimental groups.
Test groups Metaphases analyzed Time (hours) S-9 mix Exposure Sampling 

## 4 18



# - Vehicle control (DMSO) 200

** 

## 4 18 187.5 fJg/mL


# - 4 18



# - 200 375.0 fJg/mL 

## 4 18



# - 750.0 fJg/mL 200 

## 4 18



# - 1 500.0 fJg/mL 200

100· 

## 4 18 

# - 500.0 fJ9/mL EMS


## 4 + Vehicle control (DMSO) 18 200


## 4 + 18 187.5 fJ9/mL ** 

## + 

## 4 18 375.0 fJ9/mL 200 

## 4 + 18 750.0 fJ9/mL 200 

## + 18 

## 4 1 500.0 fJ9/mL 200



## + 

## 4 18 0.5 fJg/mL CPP 100* * 

### Due to a clear increase in chromosomally damaged cells, the numberof metaphases analyzed was reduced from the planned 200 mitosesto 100. ** No evaluation

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## 3.4.4. Test groups and doses
- 2nd experiment The number of dose groups evaluated in the 2nd cytogenetic experiment can be seen from the following table. Again, duplicate cultures were used for all experimental groups. Time (hours) Exposure Sampling 18 18 S-9 mix



# - Test groups

Vehicle control (DMSO) Metaphases analyzed

200
18 18 18 18 18 18 18 18 

# -



# -

-

- 187.5**~g/mL** 

## 375.0**~g/mL** 

## 750.0**~g/mL** 

## 1 500.0**~g/mL** 

## ** 200 200 200
18 18



# - 500.0**~g/mL** 

## EMS 100*
18 28



# - Vehicle control (DMSO) 200
18 18 18 28 28 28 

# -

-

- 375.0 750.0 

## 1 500.0**~g/mL ~g/mL** 

### ~g/mL 

## ** ** 200
18 28



# - 500.0**~g/mL** 

## EMS 100*


## 4 28 + Vehicle control (DMSO) 200


## 4 4 4 4 28 28 28 28 + + + + 187.5 375.0 750.0 

## 1 500.0**~g/mL ~g/mL** **~g/mL ~g/mL**  

## ** 200 200 200


## 4 28 + 0.5**~g/mL** 

## CPP 100* I * 

### Due to a clear increase in chromosomally damaged cells, the number of metaphases analyzed was reduced from the planned 200 mitoses to 100. ** 

### No evaluation

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# 3.5. TEST SUBSTANCE PREPARATION The substancewas dissolved in DMSO. To achieve a solution ofthe test substance inthe vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.



# 3.6. ANALYSIS OFTEST SUBSTANCE PREPARATION The stabilityofthe test substance at roomtemperature in the vehicle DMSO and in water each overa period of4 hours was verified analytically.

More detailed information may be requested from the sponso**~------------------~**

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## Page 26 of 99

# 3.7. EXPERIMENTAL PROCEDURE


# 3.7.1. Cell cycle time 

## The cell cycle ofthe untreated V79 cells lasted for about 13
- 14 hours [last measurement based on the BrdU method ofSPEIT, G. et al. (12): July 2006] underthe selected culture conditions. Thus, the selected 1st sampling time of 18 hours (see item3.7.2.) was within the 1
- 1.5x the normal cell cycle time, as recommended bythe OECD Guideline No. 473 "In Vitro Mammalian Chromosome Aberration Test". The latersampling time of28 hours was chosen to covera possible cell cycle delay.



# 3.7.2. Sampling times 

## Chromosomal aberrations were generally analyzed in the first metaphase afterthey had formed to avoid loss during mitoses orconversion ofthe initial aberrations into more complex derivatives during subsequent cell cycles. Since aberrations are induced by the majority ofchemical clastogens during DNA replication, the sampling time must allow cellsto progress through the S-phase aftertreatment so that initial DNAdamage is converted to chromosome alterationsvisible at mitosis. Since V79 cells are asynchronous and different chemicals may affect different stages ofthe cell cycle, more than one sampling time is necessary. Furthermore, mitotic delay may result from clastogen exposure and thus considerably delaythe first post-treatment mitosis. Therefore, samples were taken at 18 hours and 28 hours covering the intervals atwhich maximum aberration frequencies were expected.

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## Page 27 of99

### --~--------------------------------------------------------------~ 

## 3.7.3. Control articles

• Vehicle control The vehicle controls with and without S-9 mix only contained the vehicle forthe test substance atthe same concentration and volume used in the test culture.



## • Positive controls The following positive control substances forclastogenicitywere used to demonstrate the sensitivity ofthe test method and the activity ofthe S-9 mix:

- Without metabolic activation, 500 IJg ethyl methanesulfonate (SIGMA, M-0880) (EMS)/mL culture medium added in a volume of 1.0 mL

- With metabolic activation (S-9 mix), 0.5 IJg cyclophosphamide (Baxter Oncology GmbH, Reg. Nr. E 432-1)/mL culture medium added in a volume of 1.0 mL

The stability of EMS and CPP iswell-defined underthe selected culture conditions, since both positive control articles are well-established reference clastogens.



## 3.7.4. Preparation oftest cultures

• Logarithmica"y growing cultures (each afterthe 2nd passages in the 1stand 2nd experiments) which were more than 50% confluent were trypsinized (0.25% trypsin solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Priorto trypsin treatment, the cellswere rinsed once with 5 mL Ca-Mg-free HBSS.

• This process was stopped by adding MEM supplemented with 10% FCS.

• A single cell suspension was prepared, and about 5 mL MEM supplemented with 10% FCS and containing about 30 000
- 80 000 cells were seeded in each chamberof Quadriperm dishes. Two chambers ofa Quadriperm dish were used foronetest culture.

• The Quadriperm dishes were incubated with 5% CO2 at 3rC and**~** 

## 90% humidity.

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## Page 28 of99

# -

3.7.5. Treatment oftest cultures About 24
- 30 hours afterseeding and incubating the cells, the medium was replaced by 4 mL (with S-9 mix) or 5 mL (without S-9 mix) fresh medium. The test article, dissolved in 50 IJL DMSO, was added to the culture medium (final volume: 5 mL) with orwithout 1 mL S-9 mix. Concurrent negative and positive controls (see item3.7.3.) were tested. After incubation (5% CO2, 

## 3rC and**~** 

## 90% humidity) for 4 hours, the serum-free medium was replaced by MEM supplemented with 10% FCS after being rinsed twice with HBSS. Subsequently, the Quadriperm dishes were incubated again for another 14 hours or24 hours until the cells were harvested. After continuous treatment, i.e. 18 hours without S-9 mix, cells were treated in culture medium supplemented with 10% FCS and in the case ofa sampling time of28 hours incubated again for another 10 hours.



## 3.7.6. Preparation of metaphase spreads

• Cell harvest and preparation ofslides The cells were prepared based on the methods described by SCHMID, W. (7) and SPEIT, G. and HAUPTER, S. (8). 2 - 3 hours prior to harvesting the cells, 0.2 IJg Colcemid/mL culture medium (= 1 IJg Colcemid dissolved in 0.1 mL PBS/culture) was added to each chamber in order to arrest mitosis in the metaphase. After incubation at 

## 3rC, the culture medium was completely removed. For hypotonictreatment, 5 mL ofa 0.4% KCI solution which was at 

## 3rCwas added for about 20 minutes. Subsequently, 5 mL offixative (methanol: glacial acetic acid/3 : 1) which was added at 4°C and kept for at least 15 minutesand then replaced. After about another 10 minutes, the fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation. The slides were taken out ofthe Quadriperm chambers, briefly allowed to drip and then rapidly passed through a Bunsen burner flame.

• Staining The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

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# 3.8. EVALUATION


# 3.8.1. Mitotic index 

## A mitotic index based on 1 000 cells/culture was determined forall evaluated test groups in both experiments.



# 3.8.2. Cell counts 

## Forthe determination ofcytotoxicity, additional cell cultures (using 25 cm2 

## plasticflasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the numberofcells in the dose groups in comparison with the concurrent vehicle control at the end ofthe culture period using a counter (easy 1).



# 3.8.3. Cell morphology 

## About 3 hours and 16
- 18 hours aftertest substance treatment, cultures ofall test groups were checked for cell morphology, which is an indication ofattachment ofthe cells to the slides.



# 3.8.4. Treatment conditions 

## pH values and osmolaritywere measured. The solubility ofthetest substance in the vehicle used and in the aqueous culture medium at the beginning and at the end oftreatmentwas checked to ensure proper culturing and to avoid extreme treatment conditions (6).

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## 3.S.S. Chromosome analysis As a rule, the first 100 consecutive well-spread metaphases ofeach culture were counted for all test groups, and ifcells had 

## 20

- 22 chromosomes, theywere analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. The chromosome analysiS was carried out according to the following definitions (9, 10, 11):

• Structural chromosome aberrations

- G' and Gil Chromatid gapand chromosome gap (isochromatid gap) Unstained regions (so-called achromatic lesions.::::width ofone chromatid) without dislocation ofthe segment which appears to be separated

- S' and S" Chromatid breakand chromosome break Visible discontinuity in chromatid or chromosome structure with lateral or longitudinal dislocation ofthe fragment

- F' and F" Chromatid fragment and chromosome fragment Acentric chromosome segments which occur singly or in pairs

- 0' and Oil Chromatid deletion and chromosome deletion Loss ofa segment at the level ofchromatids or chromosomes

- m.A. Multiple aberrations Metaphaseswith 5 or more aberrations excl. gaps

- Disintegration ofchromosomal structure (pulverization 

## = P) The chromosomes are present as irregular particles and a chromosomal structure can no longer be detected.

- Exchanges 

## (translocations) These exchange aberrations (Ex) are divided into intrachanges and interchanges:

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## Int' and Int" Intrachanges at the level ofchromatids and chromosomes Thejoining ofbroken ends capable of reuniting two or several chromatid regions within a chromosome, e.g. centric ring chromosomes, acentric ring chromosomes, pericentric inversions

- I' and I" Interchanges at the level ofchromatids and chromosomes Thejoining of broken ends capable of reuniting two orseveral chromosomes. Theyare classified as:

- symmetric interchanges, e.g. reciprocal translocations between nonhomologous chromosomes, centric fusions, quadriradial structures

- asymmetric interchanges, e.g. dicentric and polycentric chromosomes, triradial and quadriradial structures

• Numerical chromosome aberrations (so-called heteroploidies) Aneuploidy Metaphaseswith absent (hypoploid) oradditional (hyperploid) chromosomes Only hyperploid metaphases are registered

- Polyploidy Changes in the numberofchromosomes bywhole chromosome sets Endopolyploidy Tetraploid metaphases with diplo-chromosomes (products ofendomitotic chromosome reduplication)

Slides were coded before microscopic analysis. Ifonlya few cells were found or ifthe metaphaseswere of low quality, a chromosome analysiswas not carried out. Ifthere is a clear increase in chromosomally damaged cells, the numberof metaphases to be analyzed is reduced from the planned 200 mitoses/test group.

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## 3.8.6. Acceptance criteria The V79 in vitro cytogeneticaSiay is considered valid ifthe following criteria are met:

• The quality ofthe slides must allowthe identification and evaluation of a sufficient number ofanalyzable metaphases.

• The numbers ofcells with structural! numerical aberrations in the negative control (vehicle control) has to be within the normal range ofthe historical negative control data (see Appendix 5 and 6).

• The positive control chemicals both with and without 8-9 mix haveto induce a significant increase in the numberofcells with structural aberrations (see Appendix 7).



## 3.8.7. Assessment criteria The test chemical will be assessed as "positive" in this assay ifthe following criteria are met:

• A dose-related and reproducible significant increase in the numberofcells with structural! numerical chromosomal aberrations.

• The numberofaberrant cells exceeds both theconcurrent negative control range and the highest value ofthe negative historical control range.

A test substance generallywill be considered as "negative" ifthefollowing criteria are met:

• The numberofcells with structural! numerical aberrations in the dose groups is not significantly above the concurrent negative control and iswithin the historical control data.



## 3.8.8. Statistics 

# the statistirl evaluation ofthe data was carried out using the MUCHAN program system

The proportion of metaphases with aberrations was calculated for each group. A comparison ofeach dose group with the vehicle control group was carried out using Fisher's exact test forthe hypothesis ofequal proportions. Thistestwas Bonferroni-Holm corrected versus the dose groups separately foreach time and was performed one-sided. If the results ofthis test are significant, labels (* p 

## <0.05, ** 

# P**~** 

## 0.01) are printed in the tables.

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## Page 33 of99

# 4. RESULTS

4.1. CHROMOSOME ANALYSIS

- 

# 1ST EXPERIMENT 

## (Tables 1 - 12; Appendix 1)

• Clastogenic mode ofaction 

## After a treatmenttime of4 hours no increase in the numberofchromosomally damaged cells was observed eitherwithout 8-9 mix orafterthe addition ofa metabolizing system. The types and frequencies ofaberrations observed are given on pages35.
- 36.

• Aneugenic mode ofaction 

## No increase in the number of cells with changes in the numberofchromosomes was demonstrated eitherwithout 8-9 mix orafterthe addition ofa metabolizing system.

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## 4.1.1. Assaywithout 8-9 mix; 4-hourexposure, 18-hoursamplingtime

Vehicle control: 8 (4.0%) metaphases inc!. gaps 3 (1.5%) metaphases excl. gaps, i.e. 1 x Bit; 2 x Ex

375 J-Ig/mL:
12 (6.0%) metaphases incl. gaps

2 (1.0%) metaphases excl. gaps, i.e. 2 x Ex

750 J-Ig/mL: 10 (5.0%) metaphases incl. gaps

4 (2.0%) metaphases excl. gaps, i.e. 2 x Ex; 1 x 2Ex; 1 x B'Ex

1 500 J-Ig/mL: 12 (6.0%) metaphases incl. gaps

3 (1.5%) metaphases excl. gaps, i.e. 1 x B'; 2 x Ex

500 J-Ig EM8/mL: The positive control chemical led to 17.0% aberrant cells both incl. and exel. gaps including 12 cells with exchanges.

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# 4.1.2. Assaywith 5-9 mix; 4-hourexposure, 18-hoursampling time Vehicle control: 

## 11 (5.5%) metaphases inc!. gaps

6 (3.0%) metaphases excl. gaps, Le. 1 x B'; 2 X 

## B"; 1 x F"; 1 x Ex; 1 x 2Ex

375 IJg/mL:



## 9 (4.5%) metaphases inc!. gaps 5 (2.5%) metaphases excl. gaps, i.e. 1 x B"; 1 x D"; 2 x Ex; 1 x B'Ex

750 IJg/mL: 

## 22 (11.0%) metaphases incl. gaps

4 (2.0%) metaphases excl. gaps, i.e. 1 x D"; 2 x Ex; 1 x2Ex

1 

## 500 IJg/mL: 

## 13 (6.5%) metaphases inc!. gaps

8 (4.0%) metaphases excl. gaps, i.e. 1 x B'; 1 x F"; 5 x Ex; 1 x m. A. 

## incl. Ex



# 0.5 IJg CPP/mL: 

## The positive control chemical led to 22.0% aberrant celis incl. gaps and 19.0% aberrant metaphases excl. gaps including 15 cells with exchanges.

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# 4.2. CHROMOSOME ANALYSIS - 2ND EXPERIMENT 

## (Tables 13
- 28; Appendix 2)

• Clastogenic mode ofaction 

## Both with and without 

## 8-9 mix, no increase in the numberofstructurallydamaged metaphases was observed either after a treatment time of4 hours or aftera continuous treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours. Thetypes and frequencies ofaberrations are given on pages38
- 40.

• Aneugenic mode ofaction 

## No increase in the numberofcells with changes in the number ofchromosomes was demonstrated eitherwithout 

## 8-9 mixorafterthe addition ofa metabolizing system.

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## Page 37 of

## 99

## 4.2.1. Assaywithout S-9 mix; 18-hourexposure, 18-hoursampling time Vehicle control: 

## 19 (9.5%) metaphases incl. gaps



## 7 (3.5%) metaphases excl. gaps, i.e. 

## 3 x 8'; 

## 4 x Ex
375lJg/mL: 

## 12 (6.0%) metaphases incl. gaps



## 3 (1.5%) metaphases excl. gaps, i.e. 

## 1x 8"; 

## 1 X 

## 0"; 

## 1x Ex

750 IJg/mL: 

## 17 (8.5%) metaphases incl. gaps



## 2 (1.0%) metaphases excl. gaps, Le. 

## 2 x Ex

1 500 lJg/mL: 

## 17 (8.5%) metaphases inc!. gaps



## 8 (4.0%) metaphases excl. gaps, i.e. 

## 1x 8'; 

## 1x 0"; 

## 1 x F"; 

## 5 x Ex

500 lJg EMS/mL: The positive control chemical led to 

## 26.0% aberrant cells incl. gaps and 

## 22.0% aberrant mitoses excl. gaps including 

## 17 cells with exchanges.

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## Page 

## 38 of

## 99

## 4.2.2. Assaywithout S-9 mix; 18-hourexposure, 28-hour sampling time Vehicle control:


## 7 (3.5%) metaphases incl. gaps 2 (1.0%) metaphases excl. gaps, i.e. 1 x 8'; 1 x Ex

1 500 IJg/mL:



## 21 (10.5%) metaphases inc!. gaps

7 (3.5%) metaphases excl. gaps, i.e. 2 x B'; 2 X

## B"; 1X

## F"; 2 x Ex

500 IJg EMS/mL: 

## The positive control chemical led to 20% aberrant cells both incl. and excl. gaps including 16 cells with exchanges.

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## 4.2.3. Assaywith 

## 5-9 mix; 4-hourexposure, 28-hoursamplingtime Vehicle control:
4 (2.0%) metaphases incl. gaps 2 (1.0%) metaphases excl. gaps, i.e. 1 x 8'; 1 x 8n


## 37Sl-lg/mL:

5 (2.5%) metaphases inc!. gaps 4 (2.0%) metaphases excl. gaps, Le. 1 x 8 f 

## 1 x 8 n ; 

## 2 x 2Ex ;


## 7S0 J.lg/mL:

4 (2.0%) metaphases incl. gaps 2 (1.0%) metaphases excl. gaps, i.e. 1 x 

## F'; 1 x Ex

1 SOO J.lg/mL:
5 (2.5%) metaphases incl. gaps 3 (1.5%) metaphases excl. gaps, i.e. 1 x 8'; 2 X 

## 8"

O.S J.lg CPP/mL: The positive control chemical led to 13.0%aberrant cells both incl. and excl. gaps including 6 cells with exchanges.

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# 4.3. MITOTIC INDEX The mitotic index is based on 1 000 cells per culture for the differenttest groups without and with metabolic activation. The numberof mitotic cells in the samples scored are given in Appendix 3. According tothe results ofthe determination ofthe mitotic index, no suppression ofthe mitotic activitywas observed under all experimental conditions.



# 4.4. CELL COUNTS The results ofthe cell count are given in Appendix 3. According to the results ofthe cell count, no growth inhibition was observed under all experimental conditions.**~5.** 

# CELL MORPHOLOGY Observations on cell morphology (attachmentto slides) are given in Appendix4. Cell attachmentwas not influenced at anydose evaluated for structural chromosomal aberrations.



## 4.6. ASSESSMENT OF THE SLIDES Details on the qualityof metaphases are given in Appendix 4.



# 4.7. TREATMENT CONDITIONS The osmolarityand pH values and the observations on the solubilityofthe test substance are given in Appendix 4. According to this, osmolarity and pH valueswere not influenced bytest substance treatment.

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# 5. DISCUSSION According to the results ofthe present in vitro cytogenetic study, the test substancell did not lead to an increase in the numberofstructural chromosomal aberrations

# inc~ excl. gaps eitherwithout S-9 mix orafterthe addition ofa metabolizing system in two experiments performed independently ofeach other selecting different exposure times (4 hours treatment and continuous treatment) and sampling times (18 and 28 hours); the types and frequencies ofaberrations were closeto the range ofthatofthe concurrent negative control values at both sampling times and in the range ofthe historical control data (see Appendix 5). No increase in the numberofcells containing numerical chromosomal aberrations was demonstrated. The increase in the frequencies ofchromosomal aberrations induced bythe positive control agents EMS and CPP clearly demonstrated the sensitivity ofthe test method and ofthe metabolic activity ofthe S-9 mix employed.



# 6. CONCLUSION Thus, underthe experimental conditions chosen here, the conclusion is drawn thatDis nota chromosome-damaging (clastogenic) agent under in vitro conditions using V79 cells.

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# 7. REFERENCES

1. BRADLEY, M.O.; BHUYAN, B.; FRANCIS, M.C.; LANGENBACH, R; PETERSON, A.; HUBERMAN,E.: Mutagenesis by chemical agents in V79 Chinese hamstercells: A review and analysis in literature. A report ofthe Gene-Tox Program. Mut. Res., 87, 81
- 142 (1981) 2. PRESTON, RJ.; AU, W.; BENDER, M.A.; BREWEN, J.G.; CARRANO, A.V.; HEDDLE, J.A.; McFEE, RF.; WOLFF, S.; WASSOM. J.S.: Mammalian in vivo and in vitro cytogenetic assays: A report ofthe U.S. EPA's Gene-Tox Program. Mut. Res., 87, 143
- 188 (1981) 3. AARDEMA, M.J.; ALBERTINI. S.; ARNI, P.: HENDERSON, L.M.; KIRSCH-VOLDERS, M.; MACKAY

...I.M.; SARRIF. A.M.; STRINGER, D.A.; TAALMAN, RD.F.: Aneuploidy: a report ofan ECETOC taskforce. Mut. Res

.. 410, 3 - 79 (1998) 4. AMES, B.N.; McCANN, J.; YAMASAKI, E.:

. Methods for detecting carcinogens and mutagens with the Salmonella/mammalianmicrosome mutagenicitytest. Mut. Res., 

# 11, 347
- 364 (1975) 5. DeMARINI; D.M., DALLAS, M.M

.. LEWTAS, J.: Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay. Ter. Canc.**Mut..~,** 

## 287
- 295 (1989) 6. SCOTT, D.; GALLOWAY, S.M.; MARSHALL, RR; ISHIDATE Jr., M.; BRUSICK, D.; ASHBY, J.; MYHR, B.C.: Genotoxicity under extreme culture conditions. A reportfrom ICPEMC Task Group 9. Mut. Res.• 257. 147
- 204 (1991) 7. SCHMID, W.: A technique for in situ karyotyping of primary amnioticfluid cell cultures. Humangenetik, 30, 325
- 330 (1975) 8. SPEIT, G.; HAUPTER, S.: Cytogenetic effects of penicillamine. Mut. Res., 190,197
- 203 (1987)

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## Page 43 of 99

## 9. EVANS, H.J.; O'RIORDAN, M.L.: Human peripheral blood lymphocytesforthe analysis ofchromosome aberrations in mutagen tests. Mut. Res., 31, 135
- 148 (1975) 10. SAVAGE, J.R.K.: Classification and relationships of induced chromosomal structural changes. J. Med. Genet., 12, 103
- 122 (1975) 11. Standard-Protokoll zur cytogenetischen Auswertung von Mitose- und Meiosechromosomen bei der Routineuntersuchung; ausgearbeitetvon der "Arbeitsgruppe der Industrie, Cytogenetik", 1987 12.SPEIT, G.; HAUPTER, S.; VOGEL, W.: Characterization of mitosis with sisterchromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells. Hum. Genet., 71,358
- 360 (1985)

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## Page 44 of99

## Table 1:

Table 2:

Tables 3 -7:

Tables 8 - 12: APPENDIX 1 1ST EXPERIMENT

(TABLES 1 -12)

Summarytable without S-9 mix: 4-hourexposure, 18-hoursampling time Summarytable with S-9 mix: 4-hour exposure, 18-hoursampling time Individual cultures without S-9 mix: 4-hourexposure, 18-hoursampling time Individual cultures with S-9 mix: 4-hour exposure, 18-hoursampling time

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## Page 45 of99

# I -I ::r iii' a. 0 n <:: 3 <Tl 3. iii' 

## s: <Tl 

# " a 

# "**~** '< 

# a s: <Tl U> 

## "8 ::J**~** iil (j) 

## -g ""0

# ~ tI.l a. 0 

## co :::J ('1) Ol**~** 

# " (0 0') <Tl 0 ::-' c U> 

# - r.D <1l r.D cr '< 

### Sl. ::r**~** 

## 15 ;J. 

### m' U> (il .0 <:: @' 

# i f/)

::J

# ~**~.** 

### o· ::J cr '< 

## s: <Tl U> 

# " 0 ::J**~** UI ANALYSIS OF CHROMOSOMES PROJECT-NO. SUBSTANCE NAME SUMMARY TABLE

S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: 16 h

NO. OF METAPHASES WITH ABERRATIONS METAPHASES Dose Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. Dis. n % n % n % n % n %

Vehicle DMSO 200 8 4.0 3 1.5 2 1.0 

### a 0.0 

## o 0.0

375.0 ug/ml 200 12 6.0 2 1.0 2 1.0 

### a 0.0 

## o 0.0 750.0 ug/ml 200 10 5.0 4 2.0 4 2.0 

### o 0.0 

## o 0.0 1 500.0 ug/ml 200 12 6.0 3 1.5 2 1.0 

## a 0.0 

## o 0.0

EMS 500 ug/ml 100 17 17.0** 17 17.0** 12 12.0** 

## o a a a 0.0

FISHER'S EXACT TEST (ONE-SIDED) WITH BONFERRONI-HOLM CORRECTION: 

### *: p 

### <= 0.05, 

### **: p 

### <= 0.01 A PAIRWISE COMPARISON OF EACH DOSE GROUP WITH THE SOLVENT CONTROL GROUP ,BONFERRONI-HOLM CORRECTED

/ Meupl. n %



## o 0.0



## o 0.0


## a 0.0


## a 0.0



## o 0.0

FOR EACH Polypl. n %

5 2.4

5 2.4 10 4.8 5 2.4



### a 0.0

TIME 1 TABLE

16-0CT-2006

Endopl. n %



## o 0.0



### a 0.0


## a 0.0


## o 0.0



## o 0.0-; ::T iii' a. 0 <: 

# " 3 (!) 

## .a iii' 

## s: (!) "0 

# a "0 (!) ;:l. '< 

### Sl. 

## s: (!) "0 

## '" 0 ;;;l 

## '" 0 iil 

### u;­ "0 (!) Q. ::;, 

### "lJ (!) ID a. 0 

## co :J (1) "0 .j:l.. III <0 -.,J (!) 0 

# -**~** c: (0 

## '" (!) (0 c:r '< $l. ::T 

### !!l "0 III 

## a (!) 

## '" Ol .0 5. Ol 

# "'**~.** ::; (!) => 

### -g**~.** !11. 

# '" 0 :J c:r '< 

## s: (!) "0 

## '" 0 :J (J)

### ~ ANALYSIS OF CHROMOSOMES TABLE 2 PROJECT-NO. SUBSTANCE NJ\ME SUMMARY TABLE

S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h 16-0CT-2006

NO. OF METAPHASES WITH ABERRATIONS METAPHASES Dose Incl.Gaps Excl.Gaps Exchanges Mul.Aber. 

### Chr. Dis. Aneupl. PolypI. Endopl. n % n % n % n % n % n % n % n %

Vehicle DMSO 200 11 5.5 6 3.0 2 1.0 

### o 0.0 

### o 0.0 

### o 0.0 6 2.9 

### o 0.0

375.0 ug/ml 200 9 4.5 5 2.5 3 1.5 

### o 0.0 000 

### o 0.0 5 2.4 2 1.0 750.0 ug/ml 200 22 11.0 4 2.0 3 1.5 

### o 0.0 

### o 0.0 

### o 0.0 7 3.4 

### o 0.0 1 500.0 ug/ml 200 13 6.5 840 6 3.0 1 0.5 

### o 0.0 

### o 0.0 7 3.4 

### o 0.0

CPP 0 5 ug/ml 100 22 22.0** 19 19.0** 15 15.0** 

### o 0.0 000 

### o 0.0 

### o 0.0 

### o 0.0

FISHER'S EXACT TEST (ONE-SIDED) WITH BONFERRONI-HOLM CORRECTION: *: p 

### <= 0.05, **: p 

### <= 0.01 A PAIRWISE COMPARISON OF EACH DOSE GROUP WITH THE SOLVENT CONTROL GROUP ,BONFERRONI-HOLM CORRECTED FOR EACH TIMEANALYSIS OF CHROMOSOMES -I :::T (ii' PROJECT-NO, INDIVIDUAL DATA C. 0 SUBSTANCE NAME

; ,----I -------' n c 3 S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: 18 h CD**~** TABLE 3 

# I

16-0CT-2006 (ii'**~** CULT. NO. OF CD NO. METAPH, -0 

# a Dose -0 CD**~** '< 

### g,**~** Vehicle DMSO CD (f) -0 0 004 0100 ::J (f) 0 Cil 010 0100 

## en -0 CD Q, ::!l 

### "'U CD Q) C. 0 CO ::J CD -0 OJ**~** <0 (XI CD 0 "::-'



## ...... C (0 (f) (0 CD C" '< $1 :::T 

### !!: -0 OJ**~,** CD (f) iil .n c**~.** (f)

### ~, :::: CD ::J -0

# ~ (ii' !!?, 0 ::J C" '<**~** CD (f) -0 0 ::J (f) 0 Cil MITOTIC METAPHASES WITH ABERRATIONS INDEX Inc1.Gaps Exc1.Gaps Exchanges Mu1.Aber. Chr. Dis. Aneup1. Po1yp1. Endop1. % n % n % n % n % n % n % n % n %

11.2 7 7.0 3 3.0 2 2.0 

### o 0.0 

### o 0.0 

### o 0.0 5 4.8 

### o 0.0

13.2 1 1.0 

### o 0.0 

### o 0.0 

### o 0.0 

### o 0.0 

### o 0.0 

### o 0.0 

### o 0.04 .l\.NALYSIS OF CHROMOSOMES -i ::r 

### iir PROJECT-NO. INDIVIDUAL DATA Cl. 0 SUBST.l\.NCE NAME n c 3 <II S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: 18 h**~** 

### or S' <b CULT. NO. OF ""0 NO. METAPH. 

# a ""0 Dose <b ::l. '<**~** 5' <b 375.0 ug/ml
U> ""0 0 014 0100 ::J U> Q (JJ Ul 015 0100 ""0 <b n 

### "'U 5 <b 

### m Cl. (Q CD .j::>. 

## co 0 ::J ""0 III <0 <b 

# o**~** -+. c 

## co co (JJ <b cr '< 

### s;:. ::r**~** "0 III do. <b 

## '"**~** .c c**~.** Ul

### ~.**~** ::J 

### 1£ 

## 3 <ii' Ul 

### o· ::J cr '< 5' <b Ul "0 0 ::J 

### g: Ul MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges**Mul.Abe~. Ch~.**  % n % n % n % n % n TABLE

16-0CT-2006

Dis. Aneupl. Polypl. Endopl. % n % n % n %

9.4 7 7.0 1 1.0 1 1.0 0 0,0 0 0.0 0 0.0 2 2.0 0 0.0

11.0 5 5.0 1 1.0 1 1.0 0 0,0 0 0.0 0 0.0 3 2.9 0 0.05 ANALYSIS OF CHROMOSOMES -; :::T iii' PROJECT-NO, INDIVIDUAL DATA n. SUBSTANCE NAME 0 0 c 3 S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: lS h Cl> 

### ;a iii' S' CULT. NO. OF Cl> NO. METAPH. "0 

# a Dose "0 Cl> ::1­ '< Q. S' 750.0 ug/ml Cl> (J) "0 0 002 0100 ::l (fI 0 iii (j) 006 0100 "0 Cl> Q, 

### -U Cl> 

# "" Q) Cl. <.Cl ::l 0 ('j) "0 OJ 

## c..n <0 0 Cl>

c (fI 

# - 0 

## co 

# -

Cl> 

## co cr '< Sf. :::T 9; 

## al**~** Cl> 

# '" m .a 5, ib (J) :I; ::!, ::: Cl> 

# 1 ::l**~** 0' ::l cr '< S' Cl> (fI 

## -g ::l**~** iil MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n TABLE

16-OCT-2006

Dis. Aneupl. Polypl. Endopl. % n % n % n %

7.6 1 1.0 0 0.0 0 0.0 0 0.0 0 

## o 0 0 0.0 3 2.9 0 0.0

10.7 9 9.0 4 4.0 4 4.0 0 0.0 0 0.0 0 0.0 7 6.5 0 0.06 -I ::r (ii' PROJECT-NO, 0­ SUBSTANCE NAME 0

# ~ C1 c 3**S-~** MIX: WITHOUT (1) 3­ (ji' g. (1) '0 

# a Dose 

## a;**~** 

# s. g. 1 500,0 ug/ml (1) '0 

# '" 0 :J 

# '"**~** (ij" 

## a; 12, 

### '"0 ::!1 Q)**~** 0 

## co :J (t) '0 Q) 01 r.o


# ..... (1) 

# a ':­ II> 

## c:.o c (1) 

## c:.o 0' '< 0 g.**~** 

### -g 3: (1) II> 

### til .0 !;, 

### <i! II>

# ~ (1) ::l 

## a; 3 (ii' 

# 6' :J 0' '< 5' (1) '0 

## '" 0 :J 

## '" 0 til ANALYSIS OF CHROMOSOMES INDIVIDUAL DATA

L..--I -----l EXPOSURE TIME: 4 h SAMPLING TIME: 18 h

CULT. NO. OF NO. METAPH.

OlB 0100

024 0100 MITOTIC METAPHASES WITH ABERRATIONS 

### INDEX Incl.Gaps Excl.Gaps ExChanges Mul.Aber. Chr. Dis. % n t n % n % n t n %

10.2 4 4.0 

## o 0.0 

## o 0.0 

## o 0.0 

### o 0,0

11.5 B 

### B.O 3 3.0 2 2.0 

### o 0,0 

### o 0.0 TABLE

16-0CT-2006



### Aneupl. Polypl. Endopl. n % n % n %



### o 0.0 3 2.9 

### o 0.0



### o 0,0 2 2,0 

## o 0.07 -i :r iii' PROJECT-NO. Q. 0 SUBSTANCE NAME n

# ~ c: 3 S-9 MIX: WITHOUT <1>**~** ANALYSIS OF CHROMOSOMES INDIVIDUAL DATA 

# 1'-------1

EXPOSURE TIME: 4 h SAMPLING TIME: 18 h iir 5' CULT. NO. OF <1> NO. METAPH. -0 

## a Dose -0 <1>**~** '< 

### S. 5' EMS 500 ug/ml <1> Ul -0 0 007 0050 ::J 0 

# '" til 008 0050 (i) <1> 

# " Q. 

### '"C <1> 

# "" Q. lD 0 <0 ::l (l) ID 0'1 

# " (Q 

### tv <1>



# - 0 -::- c: 

## c.o 

# '" c.o <1> c:r '< 0 5' 

### !!t
ID 

# "**~** roO Ul 

## ro .D c:**~.** Ul 

# " 

# ***~** ::l <1> 

## 3 or Ul 0" ::l c:r '< 5' <1> 

# '" 0 

# " ::l (II 0 til MITOTIC METAPHASES WITH ABERRATIONS INDEX Inel.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. Dis. % n % n % n % n % n %

6.9 8 16.0 8 16.0 6 12.0 0 0.0 0 0.0

8.7 9 18.0 9 18.0 6 12.0 0 

## o 0 0 0.0 TABLE

16-QCT-2006

Aneup1. PolypI. Endopl. n % n % n %

0 0.0 0 0.0 0 0.0

0 0.0 0 0.0 0 0.08 ANALYSIS OF CHROMOSOMES --I ::r 

### or PROJECT-NO. INDIVIDUAL DATA co. SUBSTANCE NAME 0 (") c: 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h 

### 3­ <1l <ii' 

## s: <1l "0 

# a Dose "0 <Il

### ~ 

## a 

## s: Vehicle DMSO ro (Jl "0 0 ::l 0 

## '" Ul 

## 16 

# '" Q. :::!I 

### \J 

## l!l. 

## co 0 ::l 

## m (1) "'0 

### OlJj 

### W <1l 

### 0 ':;-" 

# -.. c: <.0 (Jl <.0 ro 0­ '< !2. ::r**~** 

### -g 

## a ro 

## '" til .t:> S. 

### a; 

## '" =: ;:" [if ::l 

## 16**~,** (Jl !!!. 0 ::l 0­ '< 

## s: ro Ul "0 0 ::l**~** Ul CULT. NO. OF MITOTIC METAPHASES WITH ABERRATIONS NO. METAPH. INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n TABLE

16-0CT-2006

Dis. Aneupl. polypl. Endopl. % n % n % n %

005 0100 12.7 3 3.0 2 2.0 1 1.0 0 

### o 0 0 0.0 0 0.0 3 2.9 0 0.0

019 0100 11.6 8 8.0 4 4.0 1 1.0 0 

### o 0 0 0.0 0 0.0 3 2.9 0 0.09 ANALYSIS OF CHROMOSOMES -i :T 00" !?ROJECT-NO. INDIVIDUAL DATA 0. 0 SUBSTANCE NAME () c: 3 S-9 MIX: WITH EXPOSURE TIME: 4 b SAMPLING TIME: 18 b <D ;l 00" 

## s: CULT. NO. OF MITOTIC METAPHASES WITH ABERRATIONS NO. METAPH. INDEX <D 

# " a Dose Incl.Gaps Exc1.Gaps Exchanges MUl.Aber. Cbr. 

## lil % n % n % n % n % n ;:l. '< Q. 

## s: 375.0 ug/ml <D (Jl 

# "" TABLE

16-0CT-2006

Dis. Aneupl. Polypl. Endopl. % n % n % n %

0 012 0100 16.4 2 2.0 1 1.0 1 1.0 0 

## o 0 0 0.0 0 0.0 4 3.8 2 1.9 ::> (Jl

### ~ 016 0100 14.0 7 7.0 4 4.0 2 2.0 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0 ti> <D Q" 

# " '1J t\)

## ~ 0 

### c.o CD ::> 

### -g CJ1 <0**~** <D


# ... 9, c: <0**~** <0 cr '< 

### 8. :T 

### !!l 

### -g 

## a C1> (Jl 

### til .0 5. 

### til (Jl**~**

# ~ ::> 

## lil 

## 3 1ii" !!!" 0 ::> cr '< 

## s: C1> 

## '" 

# "" g (Jl

### ~ANALYSIS OF CHROMOSOMES TABLE 10 

### ;l iir PROJECT-NO. INDIVIDUAL DATA Cl. 0 SUBSTANCE NAME (') c: 3 (I) S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h 16-0CT-2006 

# a iir 5' (I) "0 CULT. NO. NO. OF METAPH. MITOTIC INDEX METAPHASES WITH ABERRATIONS 

# a "0 Dose Inel.Gaps Exel.Gaps Exchanges Mul.Aber. Chr. Dis. Aneupl. PolllP1

. Endopl. (I) ;:J. '< % n % n % n % n % n % n % n % n % 

# a 5' (I) 750.0 ug/ml 

## '" 

## -g ::l 

## '" 003 0100 12.7 8 8.0 2 2.0 2 2,0 

### a a 0 0 

## o 0 0 0.0 2 2.0 0 0.0

### ~ Vi 017 0100 11.7 14 14.0 2 2.0 1 1.0 0 0.0 0 0.0 

### a 0.0 5 4 8 0 0.0 "0 

### lJ D.l (I) 0, :::!l (I) Cl. 

## co CD 0 ::l "0 01 01 III (£l (I) 

# o -;­

-

# ~ c: 

# '" (I) c:r '<

## ~**~** 

## 5l a, (!) VI CD ..0 c:**~.** 

# '"**~,** 

## 15' ::l "0 (I) 

## 3 iii' 

## '" 0' ::l c:r '< 5' (!) en "0 0 ::l en**~** VJ

### I ANALYSIS OF**CHROMOSO~~S** -; ::T (ii' PROJECT-NO, INDIVIDUAL DATA a. SUBSTANCE NAME 0 c: 3 

# " S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h <t>**~** 

# .. s:

' CULT. NO. OF MITOTIC METAPHASES WITH ABERRATIONS <t> '0 NO. METAPH. INDEX (3 Dose Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. 

## -g n % n % n % n % n ::l. '< 

# " TABLE 11

16-0CT-2006

Dis. Aneupl. polypI. Endop1. % n % n % n % Q. 

## s: 1 500.0 ug/ml <t> en '0 0 001 0100 9.9 8 B.O 7 7.0 5 5.0 1 1.0 0 0.0 0 0,0 5 4.8 0 0.0 :> en 0 til til 023 0100 11.0 5 5.0 1 1.0 1 1.0 0 0.0 0 0.0 0 0,0 2 2.0 0 0.0 '0 <t> Q. :::!! 

### ""0 <t> Q) a. (Q 0 :> CD '0 (JJ (Q OJ 0'> <t> 0 ".., c: 

# - (0 en (0 <t> tJ '< 

### sa. ::T 9; '0 OJ ::l. (ii' en 

### til .0 c:**~.** en**~ ~**  <t> :>**~** 

## 3 (ii' !!? 0 :> tJ '< 

## s: <t> en 

## "8 :>**~** iiiANALYSIS OF CHROMOSOMES --! ::T iii· PROJECT-NO. INDIVIDUAL DATA a. SUBSTANCE NAME 0

# ~,----I ­ 0 c 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h 

### ;a. <1> iii· :T <1> "0 CULT. NO. NO. OF METAPH. 

# a "0 Dose <1>

### ~ 

### 8­ :T <1> CPP 0.5 ug/ml
(J) "0 0 011 0050 

# '" (J) 0 iil (j) 022 0050 "0 <1> 0 

### "'U**~** <1> Q) a. 

## co CD 0 

# '" "0 01 

## ....., 

# '" (0 <1> 0 

# - c:-' c 

### c.o c.o (J) <1> 0­ '<

### ~**~** "0 

# '" ;:::\. roO (J) <il .D. C**~.** (J)**~** 

## a <1> 

# '" "0**~** 3 iii· (J) 

### o· 

# '" 0­ '< :T <1> (J) "0 0 

# '" (J) 0 iil MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n Dis. %

8.4 11 22.0 8 16.0 5 10.0 0 0.0 0 0.0

9.3 11 22.0 11 22.0 10 20.0 0 0.0 0 0.0 TABLE 12

16-0CT-2006

Aneupl. Polypl. Endopl. n % n % n %

0 0.0 0 0.0 0 0.0

0 0.0 0 0.0 0 0.0

## Table 13:

Table 14:

Table 15:

Tables 16
- 20:

Tables 21
- 23:

Tables 24
- 28: APPENDIX 2 2ND EXPERIMENT

(TABLES 13 - 28)



## Summarytable without S-9 mix: 18-hourexposure, 18-hoursampling time Summarytable without S-9 mix: 18-hourexposure, 28-hoursampling time Summarytable with S-9 mix: 4-hourexposure, 28-hoursampling time Individual cultures without S-9 mix: 18-hourexposure, 18-hoursampling time Individual cultures without S-9 mix: 18-hourexposure, 28-hoursampling time Individual cultures with S-9 mix: 4-hour exposure, 28-hour sampling time

This document is the property ofthe sponsors (specified on page 1), use by otherparties requires written pemnission bythe sponsors 

## Page 58 of99-1 ::r (ii' 

## g. (") c 3 <I>**~** (ii' 5' <I> 't:l 

# a 't:l <I>

### ~ 

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### en (j) 't:l <I> 0, "'0 

### 15' 0­ III 0 

## c.c ::l CO 't:l 0> 01 <0 <0 <1> 0**~**



# .....**~** c <0 (J) <I> <0 0' '< 

### 9­ ::r (!; 

## 1il d, <1> (J) CD .0 C**~.** 

# i '"

::l 

## 'lil 

## 3 (ii' 

# '" 6' ::l**~** :T (\l 't:l 

## '" 9 13 Cil ANALYSIS OF CHROMOSOMES**~**

# I

..... PROJECT-NO. StJMl.!ARY TABLE SUBSTANCE NAME 8-9 MIX: WITHOUT EXPOSURE TIME: 18 h SAMPLING TIME: 18 h

NO. OF METAPHASES WITH ABERRATIONS METAPHASES 

### Dose InCl,GapS Excl.Gaps Exchange", Mul.Aher. 

### Chr. Dis. n % n % n % n % n "
Vehicle DMSO 200 19 9.5 7 3.5 4 2.0 

### o 0.0 

### o 0.0

375.0 ug/ml 200 12 6.0 3 1.5 1 0.5 

### o 0.0 

### o 0.0 750.0 ug/ml 200 17 8.5 2 1.0 2 1.0 

### o 0.0 

### o 0.0 1 500.0 ug/ml 200 17 8.5 8 4.0 5 2.5 

### o 0.0 

### o 0.0

EMS 500 ug/ml 100 26 26.0** 22 22.0** 17 17.0** 

### o 0,0 

### o 0.0

FISHER'S EXACT TEST (ONE-SIDED) WITH BONFERRONI-HOLM CORRECTION: 

### *: p <= 0.05, **: p <= 0.01 A PAIRWISE COMPARISON OF EACH DOSE GROUP WITH THE SOLVENT CONTROL GROUP ,BONFERRONI-HOLM CORRECTED Aneupl. n %



### o 0.0



### o 0.0


### o 0.0


### o 0.0



### o 0.0

FOR EACH Polypl. n %

4 2.0

3 1.5
7 3.4 2 1.0



### o 0.0

TIME TABLE 13

11i-OCT-2006

Endopl. n %



### o 0.0



### o 0.0


### o 0.0


### o 0.0



### o 0.0-; :::r 0;' 0. 0 (") c 3 (!) 

### ;a 0;' 5' (\l -0

## ~ (\l**~** 

### 9" 5' (\l -0 

# '" 0 ::> 0 

# '" <i! 0;­ -0 (\l Q. => 

### 1J (\l Q) 0. (Q 0 CO ::> -0 (j) to 0> 0 (\l


# - 0 ::­ c <D (f) (\l <D CT '< 0 5'**~** -0 0> ;:l. (ii' (f) 

### a; .0 C**~.** UI**~**

### ~ ::> -0 (\l 

## 3 0;' UI 

### o· ::>**~** 5' (\l (f) -0 0 ::> UI

### ~ ANALYSIS OF CHROMOSOMES TABLE 14 PROJECT-NO. S1JI3STANCE NAME SUMMARY TABLE

S-9 MIX: WITHOUT EXPOSURE TIME: 16 h SAMPLING TIME: 26 h 16-OCT-2006

NO. OF METAPHASES WITH ABERRATIONS METAPHASES Dose Incl.Gaps Excl.Gaps Exchanges MUl.Aber. Chr. Dis. Aneupl. Polypl. Endopl. n % n % n % n % n % n % n % n %

Vehicle DMSO 200 7 3.5 2 1.0 1 0.5 

### a 0.0 

### a 0.0 

### a 0.0 4 2.0 

### a 0.0

1 500.0 ug/ml 200 21 10.5** 7 3.5 2 1.0 

### a 0.0 

## o 0.0 

## o 0.0 6 2.9 

## o 0.0

EMS 500 ug/ml 100 20 20.0** 20 20.0** 16 16.0** 

### a 0.0 

### a 0.0 

### o 0.0 

### a 0.0 

### a 0.0

FISHER'S EXACT TEST (ONE-SIDED) WITH BONFERRONI-HOLM CORRECTION: 

### *: p 

### <= 0.05, 

### **: p 

### <= 0.01 A PAIRWISE COMPARISON OF EACH DOSE GROUP WITH THE SOLVENT CONTROL GROUP ,BONFERRONI-HOLM CORRECTED FOR EACH TIME-i :T iii" 0. 0 (') c: 3 <1>**~** 

### or :;. <1>

## ~ <1> 

# " ;l. '< Q, 

### :;. <1> (II 0 

# " g: 

# '" ii! (jj' <1> 

# " () 

### "'0 <1> Q) c

### to o CD "'0 

# '" 0'> OJ <0 

# ..... <1> 

# o .,.­

- c: <D (II <D <1> !J '< )1 :T**~** 

## 11 

# it· '" iO ,t:J c:**~.** :Ii 

# a <1> :J 

## 16 3 ur 

## '" o· 

# '" !J '< 

### :;. til 

# '"**~** 

# '" '"**~** 

# ] ANALYSIS OF CHROMOSOMES PROJECT-NO.

- SUMMARY TABLE SUBSTANCE NAME S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h

NO. OF METAPHASES WITH ABERRATIONS METAPHASES Dose Incl.Gaps Excl.Gaps Exchanges Mul.Aher. Chr. Dis. n % 

### n % 

### n % n % n %

Vehicle DMSO 200 4 2.0 2 1.0 

### o 0.0 

## o 0.0 

### o 0.0

375.0 ug/ml 200 5 2.5 4 2.0 2 1.0 

### o 0.0 

### o 0.0 750.0 ug/ml 200 4 2.0 2 1.0 1 0.5 

### o 0.0 

## o 0.0
1 500.0 ug/ml 200 5 2.5 3 1.5 

## o 0.0 

### o 0.0 

### o 0.0

CPP 0.5 ug/ml 100 13 13.0** 13 13.0** 6 6.0** 

## o 0.0 

### o 0.0

FISHER'S EXACT TEST (ONE-SIDED) WITH BONFERRONI-HOLM CORRECTION: *: p 

### <= 0.05, 

### **: p 

### <= 0.01 A PAIRWISE COMPARISON OF EACH DOSE GROUP WITH THE SOLVENT CONTROL GROUP ,BONFERRONI-HOLM CORRECTED Aneupl. n %



### o 0.0

1 0.5
1 0.5


### o 0.0



### o 0.0

FOR EACH PolypI. n %

4 2.0

3 1.5
4 2.0
2 1.0



## o 0.0

TIME TABLE 15

16-0CT-2006

Endopl. n %



### o 0.0



### o 0.0 

## o 0.0


### o 0.0



### o 0.0ANALYSIS OF CHROMOSOMES -i :r 00' PROJECT-NO. INDIVIDUAL DATA e. 

# [ SUBSTANCE NAME 0 (") c: 3 S-9 MIX: WITHOUT EXPOSURE TIME: 16 h SAMPLING TIME: 16 h 

## '" :::. TABLE 16

17-0CT-2006
oj" 5' CULT. NO. OF 

# '" NO. METAPH. 

### <3 "0 Dose "0 

# '"**~** 

## sa. S"­ Vehicle DMSO 

# '" "0 

# "' 0 034 0100 :;, (I) 0 til 037 0100 (i) "0 Q. 

# '" '"0 ::!1 Q) 

# '" e. 0 CO ::J Cl) 

## m-g

# "'~ ..... 

# a c: <0 <0 

## '" 

# "' 0­ '< 0 5' 9( "0 Q)

# goo 

## m .0 c: @" rn

## ~ m ::J "0 

# '" 3 00' 

### o· 

## '" ::J**~** 5' 

# '" -l3 0 ::J rn 0 til MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Exc1.Gaps Exchanges Mul.Aber. Chr. Dis. Aneupl. Polypl. Endopl. % n % n % n % n % n % n % n % n %

9.3 9 9.0 3 3.0 1 1.0 0 0.0 0 0.0 0 0.0 2 2.0 0 0.0

9.4 10 10.0 4 4.0 3 3.0 0 0.0 0 0.0 0 0.0 2 2.0 0 0.0-i 2: 

# '" 0­ 0 (") c 3 <l> 

## a iii' 5' <l> -0 

# a**~** ::l. '< 

# a 5' <l> 

## -g 

# '"
::J 0 

# '" iii Oi' -0

### ~. 

### ""0 

# '"' Q)**~** (0 0 ::J CD -0**m~** (.oJ <l> 

## o ::-­

.... c (0 <l> 

## '" (0 0­ '< 0 5' 

### !!1 -0 Ol 

## g '" in .J::J C 

### @' (J)**~** 

### a <l> ::J <l> 

# "**~. ~.**  

# '" 0 ::J 0­ '< 

# :r <l> -0 

## '" 0 ::J**~** 

### U! ANALYSIS OF CHROMOSOMES PROJECT-NO. SUBSTANCE NAME

# J INDIVIDUAL DATA

S-9 MIX: WITHOUT EXPOSURE TIME: 16 h SAMPLING TIME: 16 h

Dose CULT. NO. NO. OF METAPH.

375.0 ug/ml 035 0100

047 0100 MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n Dis. %

8.5 6 6.0 1 1.0 

### a 0.0 0 0.0 

### a 0.0

7.9 6 S.O 2 2.0 1 1.0 0 0.0 

### a 0.0 TABLE 17

17-0CT-200S

Aneupl. Polypl. Endapl. n % n % n %

0 0.0 2 2.0 0 0.0

0 0.0 1 1.0 0 0.0-l :;;r iii' 0. 0 () c:: 3 <l> 

### ;:; iii' 5' <l> "0 

# a "0 <l> ::I. '< Q. 5' <l> <II "0 0 :J 1ft 0 Ul 

### u;­ "0 <l> Q. 

### 1J <l> 0,) 0. 0 

### co (J) :J "0 0) Ql <0 .j':l. <l> -,.­ 0 

# - c:: <II <0 (1) <0 c:r '< 0 5' 

### !l1 "0 Ql

# gIft iil .0 C ;jj' <II 

# "**~** :J "0 

### !l1 3 iii' 1ft 0' :J**~** 5' (1) 1ft "0 0 :J 

## '" 0 Ul ANALYSIS OF CHROMOSOMES PROJECT-NO. SUBSTANCE NAME

# :~- 

# :LL

_______....J INDIVIDUAL DATA

S-9 MIX: WITHOUT EXPOSURE TIME: 18 h SAMPLING TIME: 18 h

CULT. NO. OF NO. METAPH. Dose

750.0ug/ml 030 0100

043 0100 MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. Dis. % n % n % n % n % n %

7.0 6 6.0 0 0.0 0 0.0 0 0.0 0 0.0

9.4 11 11.0 2 2.0 2 2.0 0 0.0 0 0.0 TABLE 18

17-0CT-2006

Aneupl. Polypi. Endopl. n % n % n %

0 0.0 4 3.8 0 0.0

0 0.0 3 2.9 0 0.0ANALYSIS OF CHROMOSOMES -l

### ~ PROJECT-NO. INDIVIDUAL DATA a. SUBSTANCE NAME 0 n c: 3 S-9 MIX: WITHOUT EXPOSURE TIME: 18 h SAMPLING TIME: 18 h en**~** (ii' SO CULT. NO. OF en NO. METAPH. "0 

# a Dose "0 en ::l. '< 8, SO 1 500.0 uq/ml en "0 

# '" 0 029 0100 

# " '"**~** (j) 056 0100 

## 1il !c!. 

### ""0 en 

# "" Q.l a. CO 0 CD "0 

# " OJ 

## m co 01 m 

## o ::­ -+> c: <.0 (Jl <.0 m <:r '< 

### 2­ :::r**~ ~**  <l-. en 

## '" iil .n c: 

# '"**~if** :IE :::!. :+ en "0 

# " m 

## 3 Cii**~.** 

# " 0 0­ '< SO m "0 

# '" 0 

# " 0 

# '" iil MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n TABLE 19

17-0CT-2006

Dis. Aneupl. Polypl. Endcpl. % n % n % n %

e.1 10 10.0 5 5.0 3 3.0 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0

6.3 7 7.0 3 3.0 2 2.0 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0-I :::r (ir 0­ 0 (") c 3 a> ::2­ (ii' g: a> 

# " a 

# " $ 8. g: a> VI 

## 15 ::J 

### :g til Cil 

# "**~.** -0 ::II Q) 

## '" 0­ 0 CC ::J CD OJ (j) 

# " (Cl (j) <I) 

# sa. .,.­ c;; 

### to 

## '" to 

## '" £! 2­ :::r**~** 

### dl 

# a <I) (II aJ .0 c**~.** <II ::E ::l, 

## iB' ::J a> 

# " 3 (ii' (II 15' ::J c:r '< g: (I) (II 0 

# " ::J 

## '" 0 til ANALYSIS OF CHROMOSOMES PROJECT-NO. SUBSTANCE NAME

# ;[ INDIVIDUAL DATA

S-9 MIX: WITHOUT EXPOSURE TIME: 18 h SAMPLING TIME: 18 h

Dose CULT. NO. NO. OF METAPH.

EMS 500 ug/ml 048 0050

050 0050 MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. Dis. % n % n % n % n % n %

7.7 14 28.0 11 22.0 8 16.0 0 0.0 0 0.0

8.4 12 24.0 11 22.0 9 18,0 0 0.0 0 0.0 TABLE 20

17-0CT-2006

Aneupl. Polypl. Endopl. n % n % n %

0 0.0 0 0.0 0 0.0

0 0,0 0 0.0 0 0.0-i ::r 

### en'Co 0 

# '" 3 (1) 

# :a en'::f

### ro 

# "'" a 

# "'" ro ::l. '< 

### 8­::f

### ro 

## '" 

## "8 :> 

## '"**~** 'iii" 

## i 0 

### 1J (1) c. <.0 0 :> 

### CD "'" OJ 

## m.2l 0 

# '"**~** c 

# - to 

## '" ro 

### to cr '< 0 ::f­**~** 

### lil 

## a, ro 

## '" iil .Q C 

### @' fII**~ ~.**  :J 

## i 

## '3 en' 0' 

## '" :J cr '<::f

### ro 

## '" 0 

# "'" :J 

## :3 01 ANALYSIS OF CHROMOSOMES PROJECT-NO. SUBSTANCE NAME 

# ;I INDIVIDUAL DATA

S-9 MIX: WITHOUT EXPOSURE TIME: 18 h SAMPLING TIME: 28 h

Dose

Veh.icle DMSO CULT. NO. OF NO. METAPH.

036 0100

051 0100 MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges MuI.Aber. Chr. Dis. % n % n % n % n % n % TABLE 21

lI5-OCT-2006

Aneupl. PolypI. Endopl. n % n % n %

13.8 5 5.0 1 1.0 0 0.0 0 0.0 0 0.0 0 0.0 4 3.8 0 0.0

11.1 2 2.0 1 1.0 1 1.0 0 0.0 0 

## o 0 0 0.0 0 0.0 0 0.0ANALYSIS OF CHROMOSOMES -f ::T iir 

## g. (") PROJECT-NO. SUBSTANCE NAME

# ~I**.~** INDIVIDUAL DATA

c 3 

## '" S-9 MIX: WITHOUT EXPOSURE TIME: 1B h SAMPLING TIME: 2B h ::l iir SC (I) CULT. NO. OF MITOTIC METAPHASES WITH ABERRATIONS "0 NO. METAPH. INDEX 

# a "0 Dose Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. D:l.s. (I) % n % n % n % n % n % TABLE 22

16-OCT-2006

Aneupl. Polypl. Endopl. n % n % n %

### ~ 

# s. sc 1 500.0 ug/ml (I) "0 

# '" 0 033 0100 13.0 15 15.0 3 3.0 

## a 0.0 

## a 0.0 0 0.0 0 0.0 4 3.B 

## a 0.0 :l 0 

## '" iil (jj' 040 0100 15.B 6 6.0 4 4.0 2 2.0 

## a 0.0 0 0.0 

## a 0.0 2 2.0 0 0.0 "0 (") 

## '" :5 

### "U (l) 0. OJ 0 

## co :l ('I) 

## O')-g c;o'g 0**~** -+. c ttl <:0 (I) <:0 cr '< 

### ::l. ::; i1; "0 0>::l0)' 

## '" 

# ! c @' ttl**~ ~.**  (l) :l 

## 1il 

## 3 iii' ttl 

## o· :l cr '< SC (I) "0 

# '" 0 ::l 0 

# '" iilANALYSIS OF CHROMOSOMES -i :::r (ii' PROJECT-NO. INDIVIDUAL DATA a. SUBSTANCE NAME 0 <> c: 3 S-9 MIX: WITHOUT EXPOSURE TIME: 19 h SAMPLING TIME: 28 h (J) 

### ;::. (ii' 5' CULT. NO. OF NO. METAPH. (J) 

# " (3 Dose**~** :l. '< 

# a 5' EMS 500 ug/ml (J) 

## -g 

# '" 026 0050 ::l 0 

# '" (jl 

# '" 042 0050 1;l (J) Q. 

### ""0 

## iii' a. OJ 0 <0 ::l (0 "@ (j) <0 (!) CD 0**~** c: 

# - CD 

# '" (!) CD cr '<

### ~ !Ii
Ql 

# " ;:), (!) 

# '" <il .0 c: 

# af <II**~**

# ~ ::l**~** 

## 3 iii' 

## '" c)" ::l**~** 5' (1) 

## -g 

# '"
::l 0 

# '" iii MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges MUI.Aber. Chr. Dis. 

# , , , n 'Is n 'Is n n n % TABLE 23

16-0CT-2006

Aneupl. PolypI. Enc\opl. n % n % n %

9 5 10 20.0 10 20 0 7 14.0 0 0.0 0 0.0 0 0.0 0 

## o 0 0 0.0

11 1 10 20.0 10 20.0 9 18.0 0 0.0 0 0.0 0 

## o 0 0 0.0 0 0.0ANALYSIS OF CHROMOSOMES TABLE 24 -I :::r iii" PROJECT-NO. INDIVIDUAL DATA 0.. SUBSTANCE NAME 

# [ g c: 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h 16-0CT-2006 m::liii" S' m CULT. NO. OF ""0 NO. METAPH. 

# a ""0 Dose m 

## q g, S' m Vehicle DMSO Ifj ""0 0 039 0100 ::I Ifj 0 (il Ii) ""0 m 054 0100
C') 

### "'"0 

# = m Q) 0.. (.Q 

## co ""-I 0 0 ::I ""0 w <0 m 

# - Q, c: CD CD 

## '" m 0" '< 0 S'**~** ""0 W::l0;" Ifj til .0 c:**~.** 

# '" 

# i

::I ""0 m 

## 3 iii" 

# '" 0" => 0" '< S' m en ""0 0 ::I 

# '" 0 (il MITOTIC METAPBASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. 01$. Aneupl. Polypl. Endopl. 'II n 'II n 'II n 'II n % n 'II n % n 'II n %

15.4 3 3.0 1 1.0 0 0.0 0 0.0 0 0.0 0 0.0 1 1.0 0 0.0

13.3 1 1.0 1 1.0 0 0.0 0 0.0 0 0.0 0 0.0 3 2.9 0 0.0ANALYSIS OF CHROMOSOMES -i =r 

### iir PROJECT-NO" :

# 1-- 

# l

INDIVIDUAL DATA a. SUBSTANCE NAME :

# I 0 (") c: 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h :::::. (!) iii' 5' (!) CULT. NO. OF 

# " NO. METAPH. 

# a " Dose (!) ::l. '< 

### So 5' (!) 375.0 ug/ml (J) -a 0 ::l 028 0100 <J) 0 iil 

## en 045 0100 -a ro Q, 

### '"0 tl) 

# ""**~** (0 ('I) 0 ::l -0 -....I



# ..... OJ co ro 0 

### .,.... 

# - c: CD CD (/) ro c:r '< 0 5' 

### !l: -0 OJ ::l. (ji' 

## '" (il .0 c:**~.** <J)

# ~ (!) ::l -0 ro 

## '3 Ui" <J) 0" ::l c:r '< 5' ro 

## '" 11 ::l 

## '" Q <J) MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n % n % n % n % n Dis. %

15.0 4 4.0 3 3.0 1 1.0 0 

## o 0 0 0.0

16.4 1 1.0 1 1.0 1 1.0 0 0.0 0 0.0 TABLE 25

16-0CT-2006

Ancupl. polypI. Endopl. n % n % n %

1 1.0 1 1.0 0 0.0

0 0.0 2 2.0 0 0.0ANALYSIS OF CHROMOSOMES -I**~** 0;' PROJECT-NO. INDIVIDU1\L DATA a. SUBSTANCE NAME 0 (") c 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h (1)**~** 0;' g: CULT. NO. OF MITOTIC METAPHASES WITH ABERRATIONS (1) u NO. METAPH. INDEX (3 Dose Incl.Gaps Excl.Gaps Exchanges HUl.Aber. Chr. u (1) % n '!r n '!r n % n % n 

### ::. TABLE 26

16-0CT-2006

Dis. Aneupl. Polypl. Endopl. % n '!r n % n % '< Q. g: 750.0 ug/ml (1) en u 0 025 0100 13.5 2 2.0 2 2.0 1 1.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 :J en 0 Ol 

### <n 044 0100 15.8 2 2.0 0 0.0 0 0.0 0 0.0 0 0.0 1 1.0 4 3.8 0 0.0 

## 1il**~** 

### -U 

## or Il> a. 

## co g CD u OJ -..J <0 N (1) 

# a ::­ c (/) 

## co (1) 

## co cr '<

### ~ tQ 

## 51 

## a (1) (/) iil J:I C 

## ar en

# ~ :J U (1)

## ~. (/) !!1. 0 :J cr '< g: (1) (/) u 

## g (/) 0 OlANALYSIS OF CHROMOSOMES -I ::r iii" PROJECT-NO. INDIVIDUAL DATA a. SUBSTANCE NAME 0 C'l 

# '" 3 S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h (1) 

## :a <Ii 

## s: CULT. NO. OF (1) NO. METAPH. {3 '"0 Dose '"0 (1) ::l '< 

### So 

## s: 1 500.0 ug/ml (1) ((j '"0 0 049 0100 ::J 

### lS til 052 0100 C;; '"0 (1) C'l 3i 

### 1J ell 0 CO ::J CD '"0



### ....... J!j 

### W (1) 

# 8. ::­ ((j CD 

# '" CD (1) cr '<

### ~**~** '"0 OJ ::l­ a;' (Jl 

### iii ..Cl**~.** 

# '" '"**~. ~**  ::J '"0 (1) 

## 3 (ii' !e. 0 ::J cr '< 

## s: (1) (J) '"0 0 ::J 0 

# '" til MITOTIC METAPHASES WITH ABERRATIONS INDEX Incl.Gaps Excl.Gaps Exchanges Mul.Aber. Chr. % n ,. n % n % n ,. n TABLE 27

16-0CT-2006

Dis. Aneupl. Polypl. End.opl. % n % n % n ,.

14.3 2 2.0 2 2.0 

### o 0.0 

## o 0.0 

## o 0.0 

### o 0.0 2 2 0 

## o 0.0

14.6 3 3.0 1 1.0 

### o 0.0 

### o o 0 

## o 0.0 

### o 0.0 

### o 0.0 

## o 0.0ANALYSIS OF CHROMOSOMES -I ::r 0;' PROJECT-NO. INDIVIDUAL DATA Q. 0 SUBSTANCE NAME c: 3 

# " S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 26 h <t> 

# a 0;' 

### g. CULT. NO. OF <t> "0 NO. METAPH. 

# a Dose "0 <t>

### ~ Q, 

### :;: CPP 0.5 ug/ml <t> (/) "0 0 055 0050 :J UJ 0 iil (j) 058 0050 "0 <t> 3i 

# " "'0 <t> Q) Q. 0 

## co :::l (1) "0 ID <0 <t>

# ~ 

# a
. c: <0 (/) <0 <t>**~** Q. ::r !!; "0 ID 

### a. (1) UJ 

### til .0 c:**~}** (J)**~** 

## a (1) ::J "0 (1) 

## 3 (ii' Ul 

### o· ::J cr '< 

### :;: <t> Ul "0 0 ::J <II 0 iil MITOTIC METAPHASES INDEX Incl.Gaps Excl.Gaps Exchanges % n % n % n %

13.7 7 14.0 7 14.0 3 6.0

16.0 6 12.0 6 12.0 3 6.0 TABLE 26

16-0CT-2006

WITH ABERRATIONS

WUl.Aber. Chr. Dis. Aneupl. polllPI. Endcpl. n % n % n % n % n %

0 0.0 0 0.0 0 0.0 0 0.0 0 0.0

0 0.0 0 0.0 0 0.0 0 0.0 0 0.0

## APPENDIX 3 MITOTIC INDEX, CELL COUNT

This document is the propertyofthe sponsors (specified on page 1), use by other parties requires written pennission by the sponsors 

## Page 75 of 99

## MITOTIC INDEX

The mitotic index based on 1000 cells perculture forthe differenttest groups without and with metabolic activation can be seen in the following tables. The numbers of mitotic cells in the samples scored are given as "absolute" values. The "relative" figures are related to the corresponding vehicle controlswhich are setat 100%.



## • 1st experiment
Time (hours) Exposure Sampling 4 18 

### Testgroups

Vehicle control DMSO 

### S-9 mix



# - 1stculture
% Abs.

11.2 

### 2nd culture
%Abs.

13.2 

### Mean
%Abs.

12.2 

### % Rei. 100.0

4 4 4 4 18 18 18 18 187.5 375.0 750.0 1 5000 1J9/mL 1J9/mL 1J9/mL 1J9/mL 

# -



# -

-

- 

# - 9.4 7.6 10.2 11.0 10.7 11.5 

# - 10.2 9.2 10.9 

# - 83.6 75.0 88.9

4 18 500.0 1J9/mL EMS 

# - 6.9 8.7 7.8 63.9

4 18 Vehicle control 

### + 12.7 11.6 12.2 100.0

4 4 4 4 18 18 18 18 187.5 1J9/mL 375.0 1J9/mL 750.0 1J9/mL 1 5000 1J9/mL 

### + + + + 

# - 16.4 12.7

9.9 

# - 14.0 11.7 11.0 

# - 15.2 12.2 10.5 

# - 125.1 100.4 86.0

4 18 0.5 1J9/mL 

### cpp + 8.4 9.3 8.9 72.8 

# -=notdetermined

This document is the propertyofthe sponsors (specified on page 1). use by other parties requires written penmission bythe sponsors 

## Page 

## 76 of99

## • 2nd experiment
Time (hours) Exposure Sampling 

## Time (hours) 5-9 

# 11st culture mix • %Abs. 2nd culture %Abs. Mean
%Abs. % Rei.

18 18 Vehicle control DMSO 

# - 9.3 9.4 9.4 100.0

18 18 18 18 18 18 18 18 187.5 1J9/mL 375.0 1J9/mL 750.0 1J9/mL 15000 1J9/mL 

# -

-

-

- 

# - 8.5 7.0 8.1 

# - 7.9 9.4 6.3 

# - 8.2 8.2 7.2 

# - 87.7 87.7 77.0

18 18 500.0 1J9/mL EMS


# - 7.7 8.4 8.1 86.1

18 28 Vehicle control DMSO 

# - 13.8 17.1 15.5 100.0

18 18 18 28 28 28 375.0 1J9/mL 750.0 1J9/mL 1 5000 1J9/mL 

# -



# -

- 

# -

- 13.0 -



# - 15.8 

# -

- 14.4 

# -

- 93.2

18 28 500.0 1J9/mL EMS


# - 9.5 11.1 10.3 66.7
4 28 Vehicle control DMSO + 15.4 13.3 14.4 100.0

4 4 4 4 28 28 28 28 187.5 1J9/mL 375.0 1J9/mL 750.0 1J9/mL 15000 1J9/mL + + + + 

# - 15.0 13.5 14.3 

# - 16.4 15.8 14.6 

# - 15.7 14.7 14.5 

# - 109.4 102.1 100.7
4 28 0.5 1J9/mL cpp 

### + 13.7 

### I 16.0 14.9 103.5

• 

# - =not determined

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission bythe sponsors 

## Page 77 of99

## CELL COUNT

The results ofthe cytotoxicitytest cell count can be seen in the following tables. The number ofcells counted in the dose groups are expressed as a percentage ofthe concurrentvehicle control value

.



## • 1stexperiment
Time (hours) Testgroups S-9 mix I 

## Cell count No. ofcells 1 x 105/ mL Exposure Sampling Rei 

## % 4 18 

## Vehicle control 3.03 100.0 

# - DMSO

2.94 97.0 4 18



# - 187.5 IJg/mL 

### 3.37 111.2 18 4 375.0 IJg/mL 

# - 3.02 99.7 18 4 750.0 IJg/mL 

# - 3.12 103.0 18 1 500.0 IJg/mL


# - 4

4 18 

## Vehicle control + 3.25 100.0 DMSO

3.58 110.2 18 4 + 187.5 IJg/mL 

### 3.51 108.0 4 18 + 375.0 IJg/mL 

### 3.68 113.2 4 18 + 750.0 IJg/mL 

### 3.20 98.5 4 18 1 500.0 IJg/mL 

### + I 

# I
not determined

This document is the property ofthe sponsors (specified on page 1). use byother parties requires written permission bythe sponsors 

## Page 78 of99

## • 2nd experiment
S-9 mix Time (hours) Test groups Cell count No. ofcells Exposure Sampling 

## 1x 105 

### /mL 

## Rei 

## % 18 18 

## Vehicle control 2.32 100.0 

# - DMSO

3.09 133.2 18 18 187.5 IJg/mL


# - 2.94 126.7 18 18 375.0 IJg/mL


# - 2.70 116.4 18 18 750.0 1J9/mL


# - 2.56 110.3 18 18 1 500.0 1J9/mL


# -
18 

## Vehicle control 28 5.03 100.0 

# - DMSO

4.36 86.7 18 28 375.0 IJg/mL


# - 4.40 87.5 18 28



# - 750.0 IJg/mL 3.81 75.7 18 28 1 500.0 IJg/mL


# -

4 28 

## Vehicle control + 3.97 100.0 

# DMSO

4.15 104.5 4 4 28 + 187.5 IJg/mL 4.04 101.8 28 + 4 375.0 1J9/mL 4.05 102.0 28 + 4 750.0 IJg/mL 3.68 92.7 28 1500.0 1J9/mL +



## - = notdetermined

This document is the property ofthe sponsors (specified on page 1), use byother parties requires written permission bythe sponsors 

## Page 79 of99

## APPENDIX 4 CELL MORPHOLOGY, ASSESSMENT, TREATMENT CONDITIONS

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

## Page 80 of 99

## CELL MORPHOLOGY, ATTACHMENT TO SLIDES, ASSESSMENT OF THE SLIDES

• 1stexperiment
Time (hours)

Exposure Sampling 

### Test groups S-g mix Cell morphology

## I Attachmentto slides Assessment ofthe slides



## 4 18 Vehicle control DMSO 

# - + 

### E1



## 4 4 4 4 18 18 18 18 187.5 

## 1J9/mL 375.0 

## 1J9/mL 750.0 

## 1J9/mL 1500.0 

## 1J9/mL 

# -



# -

-

- + + + + 

### E1 E1 E1 E1



## 4 18 Vehicle control DMSO + + 

### E1



## 4 4 4 4 18 18 18 18 187.5 

## 1J9/mL 375.0 

## 1J9/mL 750.0 

## 1J9/mL 1500.0 

## IJg/mL + + +
+ 

### I + + + + 

# I E1 E1 E1 E1
E

N 

### = evaluation possible 1 

### =sufficient metaphases ofgood quality 2 

### = reduced numberofanalyzable metaphases 3 

# =reduced numberofanalyzable metaphases, some of poor quality evaluation not possible 1 

# =no oronlya few metaphasesfor evaluation 2 

# =nooronlya few metaphases for evaluation and some ofpoor quality 3 

### =complete cell death (lysed cells) 4 

### =evaluation ofmetaphases strongly influenced bythe precipitation ofthe test substance 

### + 

### =complete attachment, Le. fibroblast-like cells ++ 

### = slightly reduced attachment, Le. few cells rounded 

### +++ reduced attachment, Le. most cells rounded ++++ 

### complete detachment, i.e. all cells rounded 

# (-) = notdetermined 

# =assessment not possible due to test substance precipitation

This document is the property ofthe sponsors (specified on page 1 j, use byother parties requires written permission bythe sponsors 

## Page 81 of99

## • 2nd experiment
Time (hours)

Exposure I

### Sampling Test groups 

## 5-9 mix Cell morphology

## I Attachmentto slides Assessment oftheslides



## 18 18 Vehicle control 

# DMSO - + 

## E1

18 18 18 18 18 18 18 18 187.5 tJ9/mL 

## 375.0 tJ9/mL 

## 750.0 tJ9/mL 

## 1500.0 tJ9/mL 

# -

-

-



# - + + + + 

## E1 E1 E1 E1
18 28 Vehicle control 

# DMSO 

# - + 

## E1

18 18 18 28 28 28 375.0 tJ9/mL 

## 750.0 tJ9/mL 

## 1500.0 tJ9/mL 

# -

-

- + + + 

## E1 E1 E1
4 

## 28 Vehicle control 

# DMSO + + 

## E1

4 4 4 4 

## 28 28 28 28 187.5 tJ9/mL 

## 375.0 tJ9/mL 

## 750.0 tJ9/mL 

## 1500.0 tJ9/mL + + + + + + + + 

## E1 E1 E1 E1
E :: evaluation possible 1 :: sufficient metaphases ofgood quality 2 :: reduced number of analyzable metaphases 3 

### =reduced numberofanalyzable metaphases, someof poorquality N 

### =evaluation not possible 1 

## =no or onlya few metaphasesforevaluation 2 :: no or onlya few metaphasesfor evaluation and some ofpoorquality 3 complete cell death (lysed cells) 4 

### =evaluation ofmetaphases strongly influenced bythe precipitation ofthetest SUbstance + 

# =complete attachment, Le. fibroblast-like cells ++ slightly reduced attachment, i.e. few cells rounded +++ 

### = reduced attachment, i.e. most cells rounded ++++ 

### = complete detachment, Le. all cells rounded :: not determined 

# (-) =assessment not possible duetotest substance precipitation

This document is the propertyofthe sponsors (specified on page 1), use by other parties requires written permission bythe sponsors 

## Page 82 of99

## TREATMENT CONDITIONS

The osmolarity and pH valuesand the observations on the solubility ofthe test substance can be seen from the following tables.



## • 1

## stexperiment
Time (hours) Test groups S-9 mix pH Osmolarity Solubility
Vehicle Culture medium

Exposure 4 Sampling 

## 18 Vehicle control DMSO 

# - 7.6 mOsm 

## 417 Ma



# - Oh Ma



# - 3-4h Ma Mi



# - -

4 

## 4 4 4 18 18 18 18 187.5 375.0 750.0 1 500.0 I-lg/mL I-lg/mL I-lg/mL I-lg/mL 

# -

-

-



# - -

- 7.4 7.5 

# -

- 404 405 S S S S S S S S S S S S S S S S



## 4 18 Vehicle control DMSO + 

## 7.3 371 

# - - - 

# -

4 4 4 4 18 18 18 18 I 

## 187.5 I-lg/mL 

## 375.0 I-lg/mL 

## 750.0 I-lg/mL 

## 1 500.0 I-lg/mL + + + + 

# -



# - 7.3

I 

## 7.4 

# -
380 368 S S S S S S S S S S S S S S S S
S 

# = solution Su 

# = suspension E 

# = emulsion p 

# = precipitation Ma 

# = macroscopically Mi 

# = microscopically mOsm 

# = milliosmolar 

# = not determined

This document is the property ofthe sponsors (specified on page 1), use byother parties requires written permission bythe sponsors 

## Page 83 of99

## • 2nd experiment
Time (hours)

Exposure Sampling 

## 18 18 Test groups

Vehicle control DMSO 

### S-9 mix



# - pH



## 7.3 Osmolarity

mOsm



## 425 Vehicle Ma



# - Solubility Culture medium Oh 16

### -18 h Ma Ma Mi



# - 

# - -



## 18 18 18 18 18 18 18 18 187.5 375.0 750.0 1500.0 jJ9/mL jJ9/mL jJ9/mL 1J9/mL 

# -

-

-

- -



# - 7.7 7.7 

# -

- 410 403 S S S S S S S S S S S S S S S S


## 18 28 Vehicle control DMSO 

# - 7.7 426



# - 

# - - -

18 18 18 28 28 28 375.0 1J9/mL 

## 750.0 1J9/mL 

## 1500.0 1J9/mL 

# -

-

- 

# - 7.7 7.8 

# - 428 406 S S S S S S S S S S S S 

### Time (hours) Test groups S-9 pH Osmolarity Solubility mix Vehicle Culture medium Oh 3-4h Exposure Sampling mOsm Ma Ma Ma Mi 

## 4 28 Vehicle control 

### + 

## 7.3 389



# - 

# - - 

# - DMSO 

## 4 28 187.5 1J9/mL 

### + 

# - - S S S S 

## 4 28 375.0 1J9/mL 

### + 

# - S S S S 

## 4 28 750.0 1J9/mL 

### + 

## 7.3 380 S S S S 

## 4 28 1500.0 1J9/mL 

### + 

## 7.4 392 S S S S S 

# = solution Su 

# = suspension 

### E emulsion 

### p 

# = precipitation Ma 

# = macroscopically Mi 

# = microscopically mOsm = milliosmolar 

# = not determined

This document is the property ofthe sponsors (specified on page 1). use by other parties requires written permisSion by the sponsors 

## Page 84 of99

## APPENDIX 5 HISTORICAL NEGATIVE CONTROL DATA- CLASTOGENICITY

This document is the property ofthe sponsors (specified on page 1), use byother parties requires written permission by the sponsors 

## Page 85 of 99

# Historical Negative Control Data Summary Negative Control

- Without5-9 Mix Exposure 

### ISampling 

## 4/18 h 

## 4/28 h
Aberrations 

### [%] Inc!. gaps Excl. gaps Exch. Incl. gaps Excl. -.9.8ps Exch.

Mean 

## 4.1 1.8 0.8 4.8 2.0 0.8
Min 

## 0.5 0.0 0.0 0.5 0.0 0.0
Max 

## 9.5 4.5 4.0 10.5 5.0 3.0 No.ofexp. 

## 180 51
Exposure 

### ISampling 

## 18/18 h 

## 18/28 h
Aberrations 

### [%] Inc!. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 3.7 1.5 0.7 4.4 1.9 0.9
Min 

## 1.0 0.0 0.0 

# I 0.5 0.0 0.0 Max 

## 10.0 3.0 14.5 5.5 3.0 No. ofexpo 

## 51 50 

## Negative Control

- With 5-9 Mix Exposure 

### I Sampling 

## 4/18 h 

## 4/28 h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 4.9 2.1 0.8 4.3 1.9 0.7 Min 

## 0.5 0.0 0.0 0.0 0.0 0.0 Max 

## 10.5 5.5 3.5 13.5 7.5 3.5 No.ofexp. 

## 187 87

This document is the propertyofthe sponsors (specified on page 1), use byother parties requires written permission bythe sponsors 

## Page 86 of99

# Historical Negative Control Data

Untreated Control

- Without S-9 Mix Exposure 

### I Sampling 

## 4/18 h 

## 4/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.

Mean 

## 4.3 1.8 0.8 4.4 1.8 0.7
Min 

## I 1.5 0.0 0.0 1.5 0.5 0.0
Max 

## 9.0 4.0 3.0 7.5 3.0 1.5
No. ofexpo 

## 63 14
Exposure ISampling 

## 18/18 h 

## 18/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 3.1 1.4 0.6 3.7 1.6 0.7
Min 

## 1.0 0.0 0.0 0.5 0.0 

# I 0.0 Max 

## 7.0 3.5 3.0 8.0 5.5 2.5 No. ofexpo 

## 15 16



## Untreated Control

- With S-9 Mix Exposure 

### ISampling 

## 4/18 h 

## 4/28 h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. aaps Excl. aaps Exch.
Mean 

## 4.7 2.0 0.8 4.1 1.7 0.6 Min 

## 1.5 0.0 0.0 1.5 0.5 0.0 Max 

## 10.5 5.0 3.0 9.0 4.5 2.5 No.ofexp. 

## 62 29

This document is the propertyofthe sponsors (specified on page 1), use by other parties requires written permission bythe sponsors 

## Page 87 of99

## --L-

________________________________________________________________**~** 

# Historical Negative Control Data

Vehicle Control DMSO

- Without S-9 Mix Exposure 

### ISampling 

## 4/18 h 

## 4/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 4.0 1.S O.S 5.0 2.0 O.S
Min 

## 0.5 0.0 0.0 0.5 0.0 0.0
Max 

## 9.5 4.5 4.0 10.5 5.0 3.0
No. ofexpo 

## 106 36
Exposure 

### I Sampling 

## 18/18 h 

## 18/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 4.4 1.6 O.S 5.0 2.1 1.0
Min 

## 1.0 0.0 0.0 1.0 0.5 0.0 Max 

## 10.0 4.5 2.5 14.5 4.5 3.0 No. ofexpo 

## 31 29


## Vehicle Control DMSO

- With S-9 Mix Exposure 

### I Sampling 

## 4/18 h 

## 4/28 h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 5.0 2.0 O.S 4.3 1.9 O.S Min 

## 0.5 0.0 0.0 0.0 0.0 0.0 Max 

## 10.5 5.0 3.5 13.5 7.5 3.5 No.ofexp. 

## 112 51

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

## Page 88 of99

# Historical Negative Control Data

Vehicle Control Ethanol- Without 5-9 Mix Exposure 

## 1Sampling 

## 4/18 h 4/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.

Mean 

## 3.0 1.3 0.3 2.5 2.5 2.5
Min 

## 1.5 0.5 0.0 2.5 2.5 2.5
Max 

## 5.0 2.0 0.5 2.5 2.5 2.5
No. ofexpo 

## 4 1
Exposure 

### I Sampling 

## 18/18 h 18/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.

Mean 

## 0.0 0.0 0.0 0.0 0.0 0.0
Min 

## 0.0 0.0 0.0 0.0 0.0 0.0
Max 

## 0.0 0.0 0.0 0.0 0.0 0.0
No. ofexpo 

## 0 0



## Vehicle Control Ethanol
- With 5-9 Mix Exposure 

### I Sampling 

## 4/18 h 4/28 h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 5.0 3.8 1.5 2.0 1.5 0.0
Min 

## 4.0 2.0 0.0 2.0 1.5 0.0 Max 

## 6.0 5.5 3.0 2.0 1.5 0.0 No. of expo 

## 4 1

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

## Page 89 of99

# Historical Negative Control Data

Vehicle Control Acetone
- Without 5-9 Mix Exposure ISampling 

## 4/18 h 

## 4/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch.

# I~~EXCI. ga gaps Exch.
Mean 

## 4.5 I 

## 2.1 0.8 0.0 0.0 0.0
Min 

## 2.0 1.0 0.5 0.0 0.0 0.0
Max 

## 9.0 3.5 2.0 0.0 0.0 0.0
No. ofexpo 

## 7 0
Exposure 

### I Sampling 

## 18/18 h 

## 18/28 h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean



# .

# ­ 1.7 0.9 0.4 2.8 1.5 0.9
Min 

## 1.5 0.5 0.0 1.5 1.0 0.5 Max 

## 2.5 2.0 1.0 6.0 2.0 1.5 No.ofexp. 

## 5 5



## Vehicle Control Acetone
- With 5-9 Mix Exposure ISampling 

## 4/18 h 

## 4/28 h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

## 5.4 1.8 0.7 5.2 2.4 1.0 Min 

## 2.5 0.5 0.0 2.5 1.0 0.0 Max 

## 9.5 3.0 1.0 11.0 5.0 2.5 No.ofexp. 

## 9 6

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## Page 90 of99

## APPENDIX 6 HISTORICAL NEGATIVE CONTROL DATA - ANEUGENICITY

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## Page 91 of 99

# Historical Negative Control Data Summary Negative Control- Without5-9 Mix Exposure

### I Sampling 

## 4/18 h 4/28 h Aberrations 

### [%] Aneu· Poly. Endopoly· Aneu· Poly. ploidy ploidy ploidy ploidy ploidy 

### Mean 

## 0.2 0.6 0.1 0.6 0.7
Min 

## 0.0 0.0 0.0 0.0 0.0 Max 

## 3.3 4.3 2.4 I 

## 2.4 2.0 No. ofexp.* 

## 147 147 63 22 22 Endopoly· ploidy 

## 0.0 0.0 0.0

1 Exposure 

### ISampling 

## 18/18 h 18/28 h Aberrations 

### [%] Aneu· Poly- Endopoly. Aneu- Poly· Endopoly· ploidy ploidy ploidy ploidy ploidy ploidy 

### Mean 

## 0.1 0.4 0.0 0.2 0.5 0.0
Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 2.0 3.8 0.5 4n 

## 3.8 I No. ofexp.* 

## 80 80 I 

## 34 65 65 33 

## Negative Control

- With 5-9 Mix Exposure 

### I Sampling 

## 4/18 h 4/28 h Aneu- Poly. Pol ndopoly· Aneu· Endopoly 

### Aberrations 

### [%] ploidy ploi ploidy ploidy ploidy ploidy 

### Mean 

## 0.2 0.3 0.2 0.0 0.4 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 0.7

Max 

## 5.0 4.2 2.4 1.5 0.5 3.4 No. ofexp.* 

## 182 182 88 34 63 88
* 

### differentiation between polyploidy and endoploidy since January 2000

This document is the propertyofthe sponsors (specified on page 1), use byother parties requires written permission by the sponsors 

## Page 92 of99

# - Historical Negative Control Data

Untreated Control

- Without 5-9 Mix Exposure 

### I Sampling 

### 4/18 

## h 4/28 

## h ] Aneu- Poly- Endopoly- Aneu- Poly- Endopolyploidy ploidy ploidy ploidy ploidy ploidy 

### Mean 

## 0.1 0.5 0.0 0.7 0.6 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 1.5 3.4 0.5 2.4 1.5 0.0 No. ofexp.* 

## 45 45 17 10 10 1 Exposure 

## ISampling 

### 18/18 

## h 18/28 

## h Aberrations 

### [%] Aneu- Poly- Endopoly- Aneu- Poly- Endopolyploidy ploidy ploidy ploidy ploidy ploidy 

### Mean 

## 0.1 0.4 0.0 0.3 0.6 0.0 Mill 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 1.0 3.0 0.5 4.0 3.B 0.0 No.ofexp.* 

## 30 30 11 24 24 12 

## Untreated Control

- With 5-9 Mix Exposure 

### ISampling 

### 4/18 

## h 4/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopolyploidy 

### Mean 

## 0.3 0.7 0.5 0.2 0.5 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 5.0 2.9 1.4 1.5 3.4 0.5 No. ofexp.* 

## 59 59 17 

## 31 31 11

• differentiation between polyploidy and endoploidy since January 2000

This documentisthe property ofthe sponsors (specified on page 1), use byother parties requires written permission by the sponsors 

## Page 93 of 99

# Historical Negative Control Data

Vehicle Control DM50
- Without 5-9 Mix Exposure

## 1Sampling 

### 4/18 

## h 4/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopoly ploidy 

### Mean 

## 0.2 0.7 0.2 0.5 0.7 0.0
Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 3.3 4.3 2.4 1.9 2.0 0.0 No. ofexp.* 

## 93 93 41 11 11 0 Exposure

## 1Sampling 

### 18/18 

## h 18/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopolyploidy 

### Mean 

## 0.2 0.5 0.1 0.1 0.4 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 2.0 3.8 0.5 1.0 2.0 0.5 No. ofexp.* 

## 44 44 20 35 35 18 

## Vehicle Control DM50
- With 5-9 Mix Exposure

## 1Sampling 

### 4/18 

## h 4/28 

## h Aberrations 

### [%] Aneuploidy Poly. ploidy Endopolyploidy Aneuploidy Poly- 

# I**En~:ioIY** ploidy idy 

### Mean 

## 0.2 0.7 0.2 0.2 0.3 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 2.0 4.2 2.4 1.5 1.5 0.0 No. ofexp.* 

## 111 111 41 49 49 20
* 

### differentiation between polyploidy and endoploidy since January 2000

This document is the propertyofthe sponsors (specified on page 1), use byother parties requires written permission bythe sponsors 

## Page 94 of99

# Historical Negative Control Data

Vehicle Control Acetone
- Without5-9 Mix Exposure 

### 1Sampling 

## 4/18 

## h 4/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopoly ploidy 

### Mean 

## 0.0 0.3 0.0 0.0 0.0 0.0
Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 0.0 1.0 

# I 0.0 0.0 0.0 0.0 No. ofexp.· 

### 7 7 

## 5 0 0 0 Exposure 

## I Sampling 

## 18/18 

## h 18/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopolyploidy 

### Mean 

## 0.0 0.1 0.0 0.0 0.1 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 0.0 0.5 0.0 0.0 0.5 0.0 No. ofexp.· 

## 5 5 3 5 5 3


## Vehicle ControlAcetone - With 5-9 Mix Exposure

## I Sampling 

## 4/18 

## h 4/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopolyploidy 

### Mean 

## 0.1 0.5 0.0 0.3 0.3 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0_ 0.0 Max 

## 1.0 

## 2.4 0.0 1.0 1.0 0.0 No. ofexp.· 

### 9 9 

## 5 6 6 

## 3
* 

### differentiation between polyploidy and endoploidy since January2000

This document is the property ofthe sponsors (specified on page 1). use byother parties requires written permission bythe sponsors 

## Page 95 of99

# Historical Negative Control Data

Vehicle Control Ethanol
- Without 5-9 Mix Exposure 

### I Sampling 

### 4/18 

## h 

# §4/28h

Aberrations 

### [%] AneuPoly- EndopolyPoly­

# ~ ploidy ploidy ploidy ploidy y 

### Mean 

## 0.5 0.0 0.0 1.5 0.0 0.0
Min 

## 0.0 0.0 0.0 1.5 0.0 0.0 Max 

## 1.0 0.0 0.0 1.5 0.0 0.0 No. ofexp.* 

## 2 2 0 1 

## 1 0 Exposure 

### I Sampling 

### 18/18 

## h 18/28 

## h Aberrations 

### [%] Aneu- Poly- Endopoly- Aneu- Poly- Endopolyploidy ploidy ploidy ploidy ploidy ploidy 

### Mean 

## 0.0 0.0 0.0 0.0 0.0 0.0
Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 0.0 0.0 0.0 0.0 0.0 0.0 No. ofexp.* 

## 1 1 0 1 1 0 

## Vehicle Control Ethanol
- With 5-9 Mix Exposure 

### I Sampling 

## 4/18 h 4/28 

## h Aberrations 

### [%] Aneuploidy Polyploidy Endopolyploidy Aneuploidy Polyploidy Endopolyploidy 

### Mean 

## 0.3 1.0 0.0 0.3 0.0 0.0 Min 

## 0.0 0.0 0.0 0.0 0.0 0.0 Max 

## 1.0 2.0 0.0 0.5 0.0 0.0 No. ofexp.* 

## 3 3 0 2 2 0
* differentiation between polyploidy and endoploidy since January2000

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## Page 96 of99

## APPENDIX 7 HISTORICAL POSITIVE CONTROL DATA

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## Page 97 of 99

# Historical Positive Control Data

Without**S~9** 

## Mix**~** 

## Ethylmethanesulfonate (EMS) 350 IJg/ml Exposure/ Sampling 

### 4118 

## h 4/28 

## h
Aberrations 

### ["!o] Inc!. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.

Mean 

### 17.6 15.4 10.5 19.0 16.0 6.0 Min 

### 9.5 7.0 2.0 19.0 16.0 6.0 Max 

### 28.0 25.0 20.0 19.0 16.0 6.0 No.ofexp. 

### 141 1
Exposure 

### I Sampling 

### 18/18 

## h 18/28 

## h
Aberrations 

### ["!o] Inc!. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

### 20.7 19.4 13.7 21.4 20.8 15.5 Min 

### 15.0 13.0 7.0 17.0 16.0 12.0 Max 

### 25.0 23.0 27.0 27.0 i 

### 25.0 21.0 No. ofexpo 

### 43 13



## With**S~9** 

## Mix**~** 

## Cyclophosphamide (CPP) 0.5 IJg/ml Exposure 

### I Sampling 

### 4118 

## h 4/28 

## h Aberrations 

### ["!o] Incl. aaDS Excl. aaDS Exch. Inc!. gaps Excl. gaps Exch.
Mean 

### 20.4 18.4 12.0 19.6 17.9 9.9 Min 

### 11.0 10.0 5.0 13.0 12.0 3.0 Max 

### 36.0 30.0 22.0 26.0 23.0 20.0 No. ofexpo 

### 159 50

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

## Page 98 of99

# Historical Positive Control Data

Without S-9 Mix
- Ethylmethanesulfonate (EMS) 500 J,lg/ml Exposure

## ISampling 

### 4118 

## h 4/28 

## h
Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

### 18.9 

### 16.0 9.2 

### 0.0 0.0 0.0 Min 

### 10.0 8.5 

### 5.0 0.0 0.0 0.0 Max 

### I 

### 24.0 21.0 12.0 0.0 0.0 0.0 No.ofexp. 

### 12 0
Exposure 

### I Sampling 

### 18/18 

## h 18/28 

## h Aberrations 

### [%] Incl. gaps Excl. gaps Exch. Incl. gaps Excl. gaps Exch.
Mean 

### 20.8 19.1 

### 12.0 

# I 21.8 

### 20.3 16.1 Min 

### 16.0 15.0 9.0 

# I 17.0 17.0 13.0 Max 

### 25.0 23.0 16.0 26.0 

### 23.0 21.0 No. ofexpo 

### 10 10

This document is the propertyofthe sponsors (specified on page 1). use byother parties requires written permission bythe sponsors 

## Page 99 of99

# Attachment #20

## STUDY TITLE AMENDMENT NO.1 TO THE REPORT

IN VITRO CHROMOSOME ABERRATION ASSAY WITH

IN V79 CELLS AUTHOR OF THE AMENDMENT

AMENDMENT COMPLETED ON 11 Apr 2007 PERFORMING LABORATORY

LABORATORY PROJECT IDENTIFICATION Project No.: SPONSOR

This documentcontains manufacturing and trade secrets ofthe Sponsor(s). It is the property ofthe Sponsor(s) and may be used only forthat purposeforwhich itwas intended by the Sponsor(s). Every otheror additional use, exploitation, reproduction, publication orsubmission to other parties require the written permission ofthe Sponsor(s), with the exception of regulatory a encies actin within the limits oftheir a,....d

... m
... in

... is"'tr

.... a"'tiv

... ec-:a

... ut

... h"'or"'itL·

____________________-1

This document isthe property ofthe sponsors (specified on page 1), use byother parties requires written permission by the sponsors 

## Page 1of 5

## GLP COMPLIANCE STATEMENT

This studywas conducted in compliance with the OECD Principles ofGood Laboratory Practice and the GLP Principles ofthe German "Chemikaliengesetz" (Chemicals Act) which meetthe United States Environmental Protection Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part 792 (TSCA)], with the exception that recognized differences existbetween the GLP Principles/Standards ofOECD and the Principles/Standards of FIFRA and TSCA.

This documentis Ihe propertyofthe sponsors (specified on page 1). usebyotherparties requires written permission bythe sponsors 

## Page 2 of5

## SIGNATURE PAGE



## Study Director:

Thisdocument Is the propertyofthe sponsors (specified on page 1), use byotherparties requires written permission by the sponsors 

## Page 30f5

# -

STATEMENTOFTHE QUALITYASSURANCE UNIT
TheQualityAssurance Unit (QAU) inspected the study and reported any inspection results to the Study Directorand to Management. Thefinal report reflects the raw data.

Phase ofstudy Date ofinspection (mm-dd-yyyy) Reported to Study Director and to Management (mm-dd-yyyy) Study Plan: 07-06-2006 07-06-2006 Conductofstudy: 07-14-2006 07-14-2006 Report: Amendment No.1 to the Report 11-09-2006 03-26-2007 11-09-2006 03-26-2007

mm ......

# /'---------ll

................

This document is the propertyofthe sponsors (specified on page 1

### l. use byother parties requires written pennisslon bythe sponsors 

## Page4 of5

## Test substance analysis:**~~wlt~s....l=o:J..,fthe** 

## reanalysis will be reported in an amendment. dated 26 Jan 2 7 of**th~** 

# I the test substance was

This document is the propertyofthe sponsors (specified on page 1). use byother parties requires written permission bythe sponsors 

## Page 

# 5 of 

# 5

# Attachment #21

## STUDYTITLE 

## Report
Repeated Dose Oral Toxicity study in Wistar rats; Administration by gavage for 4weeks DATA REQUIREMENT OECD407



## 96/54/EC

STUDY COMPLETED ON 

## March 16, 2007 PERFORMING LABORATORY

LABORATORY PROJECT IDENTIFICATION 

# I I SPONSOR

PART I OF III (REPORT SECTION AND SUMMARYTABLES)

This documentcontains manufacturing and trade secrets ofthe Sponsor(s). It is the property ofthe Sponsor(s) and maybe used only forthat purpose for which itwas intended bythe Sponsor(s). Everyotheroradditional use, exploitation, reproduction, publication orsubmission to other parties require the written permission ofthe Sponsor(s), with the exception ofregulatory agencies actin within the limitsoftheiradministrative authorit

.

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission bythe sponsors 

## Page 1of 368

## THIS PAGE IS INTENTIONALLY LEFT BLANK

This document is the property ofthe sponsors (specified on page 1). use byother parties requires written permission by the sponsors 

## Page 2 of 368

## GLP COMPLIANCE STATEMENT

This studywas conducted in accordancewiththe OECD Principles ofGood Laboratory Practice and the GLP Principlesofthe German "Chemikaliengesetz" (ChemicalsAct). which meetthe United States Environmental ProtectionAgencyGood Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part792 (TSCA)]. with the exception that recognized differencesexist betweenthe GLP Principles/Standards ofOECD and the Principles/Standards of FIFRAand TSCA. 

# .l:1..I!!.f

..

# L~ 

# ..J •.

# ?f!J7­ Study Director Date To befilled for USA EPAsubmission only:

Sponsor Date

Submitter Date

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## Page 3 of 368

## THIS PAGE IS INTENTIONALLY LEFT BLANK

This document is the property ofthe sponsors (specified on page 1), use byother parties requires written permission by the sponsors 

## Page 4 of 368

## SIGNATURE PAGE



## Study Director:

Clinical Pathology:

Pathology:

Analytical Chemistry:

Management:

This document is the property ofthe sponsors (specHied onpage 1), usebyotherparties requires written permissionbythesponsors 

## Page 5of 368--L-

__________________________________________________________________**~** 

## CONTRIBUTORS TO THE STUDY 

## ISUPERVISORY LABORATORY PERSONNEL



## Head of Experimental Toxicology and Ecology: Study director:
Clinical Pathology: Pathology:
Data processing:
Statistics: Analytical Chemistry: Quality Assurance Unit (QAU):
Coordination QAU: Laboratory Subchron.-/ Chron. Tox. Rodent: Clinical Pathology/Hematology: Pathology: Histopathology:

This document is the property ofthe sponsors (specified on page 1). use by other parties requires written permisSion by the sponsors 

## Page 6 of 368

### ,-. 

## STATEMENTOFTHEQUALITYASSURANCE UNIT


## The QualityAssurance Unit (QAU) inspected the studyand reported any inspection results tothe Study Directorand to Management. Thefinal report reflects the raw data.

Phase ofstudy Date ofinspection Reported to Study Director and to Management (mm-dd-yyyy) (mm-dd-yyyy) Study Plan: 10-05-2006 10-05-2006 Conduct ofstudy: 10-20-2006 10-20-2006 10-31-2006 10-31-2006 11-03-2006 11-03-2006 Report: 02-01-2007 02-01-2007



# -

This document isthe propertyofthe sponsors (specifiedon page 1). use by otherparties requireswritten permission bythe sponsors 

## Page 7 of 368

## GLP CERTIFICATE (FROM THE COMPETENTAUTHORITY)

Gute Laborpraxls 

### IGood Laboratory Practice 

## GLP-Bescheinlgung 

# IStatementofGLP Compliance (gem./according to 

### § 19Abs. 1Chemikaliengesetz) 

# •

Eine GLP-Inspektion zur Oberwachung und Assessment ofconformity with GLP according der Einhaltung der GLP-Grundsalze**gema~** to Chemikaliengesetz and Directive 8813201 Chemikaliengesetz bzw. Richtlinie 881320IEG EECat:: wurde durchgefOhrt in: 

### PrOfeinrichtung /Testfacility

PrOfung nach Kategorien/Areas ofExpertise (gem.!aa:o<dlng ChernVwII.GLP Nr. 5.3IOECDguidance) 

### 1,2,3,4,5,8,9 Kat. 9 - 

### Biochemische und pathologische Untersuchungen zu Wirkmechanismen/ Biochemical and pathological examinations concerning mode ofaction Datumder Inspektion/ Date of Inspection (Tag.MonatJahr1day.month year) 

### 08.06.2005und25. bls

### 27.07.2005
Die genannte PrOfeinrichtung beflndet sich im The above mentioned testfacility is included in nationalen GLP-Oberwachungsverfahren und the national GLP Compliance Programme and wird**regelma~1g** auf Einhaltung der GLP­ is inspected on a regular basis. Grundslltze Oberwacht. Auf dar Grundlage des Inspektionsberichtes Based on the inspection report it can be con­ wird hiermit besUitigt, dass in dieser PrOf­ firmed, that the test facility is able to conduct einrichtung die oben genannten PrQfungen the aforementioned studies in compliance with unter Einhaltung der GLP-Grundsatze durch­ the Principles ofGLP. gefOhrt werden kOnnen.



### LandesamtfOr Umwelt, Wasserwirtschafl und Gewerbeaufslcht Kaiser-Friedrich-StraBe 7 55116 Malnz**"cN~';;~·;,;;;iAd~d;,;:Gt:p:o;';;,:.;;.;;j;~;;;';;;ho;.d·~·i·** LandesamtfUr (

# J

Name and ad

.....of!he GLP MoniloringAuthority) Umwelt. Wasserwirtschaft undGewerbeaufsicht

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## Page 8 of368

# - CONTENTS OF PART I (REPORTSECTION AND SUMMARYTABLES)
TITLE PAGE THIS PAGE IS INTENTIONALLY LEFT BLANK GLP COMPLIANCE STATEMENT THIS PAGE IS INTENTIONALLY LEFT BLANK SIGNATURE PAGE CONTRIBUTORS TO THE STUDY 

## I SUPERVISORY LABORATORY PERSONNEL STATEMENT OF THE QUALITYASSURANCE UNIT GLP CERTIFICATE (FROM THE COMPETENTAUTHORITY) CONTENTS OF PART I (REPORT SECTION AND SUMMARYTABLES)



## 1. SUMMARY 

## 1.1. METHODS 

## 1.2. OBSERVATIONS 

## 1.3. RESULTS 

## 1.3.1. Analytics 

## 1.3.2. Findings 

## 2. INTRODUCTION 

## 2.1. OB

..IECTIVES 

## 2.2. SELECTION OF DOSES 

## 2.3. TEST GUIDELINES 

## 2.4. STUDY DATES 

## 2.5. RETENTION OF RECORDS 

## 3. MATERIAL AND METHODS 

## 3.1. TEST SUBSTANCE 

## 3.2. TESTANIMALS 

## 3.2.1. Species and strain 

## 3.3. HOUSING AND DIET 

## 3.4. TEST GROUPS AND DOSES

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## Page 9 of368

## 3.5. 3.6. 3.6.1. 3.6.2. 3.6.3. 3.6.4. 3.6.5. 3.7. 3.7.1. 3.7.2. 3.7.3. 3.7.4. 3.7.5. 3.7.6. 3.7.7. 3.7.8. 3.7.9. 3.7.10. 3.8. 3.8.1. 3.8.2. 3.8.3. 3.8.4. 3.9. 3.9.1. 3.9.2. 3.9.3. 3.9.4. 3.9.5. 4. 4.1. 

## 4.1.1. 4.1.2. 4.1.3. 

## 4.1.4. 4.1.5. 

## 4.1.6. 4.2. 

## 4.2.1. 4.2.2. TEST SUBSTANCE PREPARATIONSAND PREPARATION FREQUENCY ANALYSES Analyses ofthe testsubstance preparations Analytical methods Food analyses Drinking wateranalyses Bedding and enrichment analyses EXPERIMENTAL PROCEDURE Mortality Clinical observations Detailed clinical observations (DCO) Food consumption Water consumption Bodyweight data Food efficiency Functional observational battery (FOB) Motoractivity assessment Statistics ofclinical examinations CLINICAL PATHOLOGY Hematology Clinical chemistry Urinalysis Statistics ofclinical pathology PATHOLOGY Necropsy Organ weights Organ 

## ITissue fixation Histotechnical processing 

## I Examination by light microscopy and assessment offindings Statistics ofpathology RESULTS ANALYSES Stability analyses Homogeneity control analyses Concentration control analyses Food analyses Drinking water analyses Bedding and enrichment analyses CLINICAL EXAMINATIONS Mortality Clinical examinations

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## 4.2.3. 4.2.4. 4.2.5. 4.2.6. 4.2.7. 4.2.8. 4.3. 4.3.1. 4.3.2. 4.3.3. 4.3.4. 4.4. 4.4.1. 4.4.2. 4.4.2.1. 4.4.2.2. 4.4.3. 4.4.4. 

## 5. 

# 6. 7. 7.1. 7.2. 7.3. Food consumption Water consumption Body weight data Food efficiency Functional observational battery Motoractivity measurement CLINICAL PATHOLOGY Hematology Clinical chemistry Urinalyses Other deviations PATHOLOGY Mortality Weight parameters Absolute organ weights Relative organ weights Gross lesions Histopathology DISCUSSION CONCLUSION APPENDIX LIST OF ABBREVIATIONS USED IN TABLES IA (CLINICAL EXAMINATIONS) LIST OF ABBREVIATIONS USED IN TABLES IB (CLINICAL PATHOLOGY) LIST OF ABBREVIATIONS USED IN TABLES IC (PATHOLOGY)

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## Part A: CLINICAL EXAMINATIONS (MEAN VALUES AND SUMMARYTABLES)



## Body weight Motoractivity

Observations Report
- Incidence Food consumption Water consumption Body weight Body weight change Food efficiency Functional observational battery Motoractivity

Part B: CLINICAL PATHOLOGY (MEAN VALUES AND SUMMARYTABLES) 

## Hematological examinations Differential blood count Clotting analyses Enzymes Blood chemistry Urinalyses

Part C: PATHOLOGY (MEAN VALUES AND SUMMARYTABLES) 

## Absolute weights
- mean values (male) Absolute weights
- mean values (female) Relative weights
- mean values (male) Relative weights
- mean values (female) Incidence ofgross lesions Incidence ofall microscopicfindings Figures 

## 1 - 2 3-4

Tables IA- 1 - IA-16 IA-17
- IA-20 IA-21
- IA-24 IA-25
- IA-28 IA-29
- IA-32 IA-33
- IA-36 IA-37
- lA-52 lA-53
- lA-56

IB- 1 - IB- 2 IB- 3 - IB- 6 IB- 7 - IB- 8 IB- 9 - IB-10 IB-11
- IB-14 IB-15 -IB-18

IC- 1 IC- 

## 2 IC- 

# 3 IC- 

# 4 IC- 5 IC- 6 - IC- 8

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## Page 12 of368

## CONTENTS OF PART II (TABLES SECTION; INDIVIDUAL VALUES)



## TITLE PAGE TABLE OF CONTENTS LIST OF ABBREVIATIONS USED IN TABLES

Part

## A: CLINICAL EXAMINATIONS (INDIVIDUAL VALUES)



## Observations Report
- Summary Food consumption Water consumption Bodyweight Functional observational battery Motor activity

Part 

## B: CLINICAL PATHOLOGY (INDIVIDUALVALUES) 

## Hematological examinations Differential blood count Red blood cell morphology Clotting analyses Enzymes Blood chemistry Urinalyses

Part 

## C: PATHOLOGY (INDIVIDUAL VALUES) 

## Absolute weights
- individual values Relative weights
- individual values Single animal sheet Tables IIA- 1 - IIA-16 IIA-17
- IIA-20 IIA-21
- IIA-24 IIA-25
- IIA-28 IIA-29
- IIA-54 IIA-55
- IIA-62

IIB- 1 - IIB- 2 IIB- 3 - IIB- 6 IIB- 7 - IIB- 8 IIB- 9 - IIB-10 IIB-11 -IIB-12 IIB-13
- IIB-16 IIB-17
- IIB-20

IIC- 1 -IIC-16 IIC-17 -IIC-32 IIC-33 -IIC-55

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## CONTENTS OF PART III (SUPPLEMENT)



## TITLE PAGE TABLE OF CONTENTS Final Report Chemical and spectroscopic characterization

# of~or the notification according tothe German

# Chemi~ 

# I I

Final Report Characterization ofi 

# Iaftertermination of toxicological studies'-------' 

# I ANALYTICAL REPORT STABILITYANALYSIS OF

Homogeneity and Concentration Control Analysis

# o~,- __...... in Drinking waterwith 0.5% Carboxymethylcellulose Functional Observational Battery (FOB): Detailed description ofexaminations, ranking and documentation procedures HISTORICAL CONTROL DATA OF CLINICAL PATHOLOGYTESTING

THIS REPORT CONSISTS OF PART I, II AND III.

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## Page 14 of368

# 1. SUMMARY

1.1. METHODS I Iwas administered to groups of5 male and 5 female Wistar rats at dose levels of0 (vehicle control; group 0), 10 (group 1), 30 (group 2), 60 (group 3; from day 0till day 7) and 45 (group 3; from day 8 till day28) mg/kg bodyweight/day over a period of4 weeks. The animals ofgroup 3 received doses of60mg/kg bw/dayfrom day 0 till day 7 and 45 mg/kg bw/dayfrom day 8 till the day28, becauseofsevere toxic effects at 60 mg/kg bw/day.



# 1.2. OBSERVATIONS Food consumption, water consumption and bodyweightwere determined weekly. The animals were examined for signsoftoxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted priorto the startofthe administration period and weekly thereafter. In addition, all animalswere checked daily before and about 1 houras well as 3 - 4 hours after administration for any clinical signs. A functional observational battery (FOB) and measurement ofmotor activitywas carried outafter26 days oftreatment in males and 27 daysoftreatment in females. Clinicochemical, hematological examinations and urinalyses were performed towards the end ofthe administration period. All animals were assessed by gross pathology, followed by histopathological examinations.

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# 1.3. RESULTS


# 1.3.1. Analytics 

## The various analyses:

• Demonstrated the stability ofthe test substance preparationsover a period upto 96 hours at room temperature • Showed the homogeneous distribution ofthe test article in the vehicle • Showed the correctness ofthe prepared concentrations



# 1.3.2. Findings 

## The following test substance-related adverse findings were noted:

60 mg/kg bodyweight/day (from day 0 until day 7)



## • 1 male and 1female were found dead on day 6 and on day 7, respectively • Convulsions, clonicwere observed moderate in 4 females, severe in 2 females, epileptoid slight in 1 male, moderate in 1female, severe in 2 males and 1female, tonic slight in 1 female and severe in 1 male and 2 females • Hyperesthesia was observed slight in 2 males and 5females, moderate in 4 females and severe in 1 male and 2 females • Hyperexcitabilitywas observed slight in 2 males and 5females, moderate in 4 females and severe in 1 male and 2 females • Nose, discharge, red was observed moderate in 1female • Piloerection was observed in 2 females • Salivation was observed slight in 2 females, moderate in 1 male and 4 females and severe in 1 male and female • Skin, paleness was observed moderate in 2females • Twitching was observed slight in 3 malesand 5 females, moderate in 2 males and 3 females and severe in 1 male and 3females • Food consumption in both sexes was statisticallysignificantly reduced of-29.4% in males and -28.2% in females on day 7 • Waterconsumption in both sexes was increased of+13.9% in males and statistically significantly increased of+36.9% in females on day 7 • Body weight was statistically significantly decreased of-13.9% in males and not statistically Significantly decreased of-6.5% in females on day 7 • Bodyweight change in both sexeswas statistically significantly decreased of-60.8% in males and -50.9% in females on day 7 • Food efficiency in both sexes was statistically significantly decreased on day 7

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## Page 16 of368

# - 45 mg/kg bodyweight/day (from day 8 untilthe end ofthe administration period)

• 1female was prematurely sacrificed moribund on day 11 • Anogenital region, smeared with urine was observed severe in 1female • Convulsions, tonic were observed slight, moderate and severe in each 1female • Hyperactivity was observed in 1female • Hyperesthesia was observed slight in 2 males and 1female • Hyperexcitability was observed slight in 4 males and 3 females, moderate in 4 males and females and severe in 1 male and 2 females • Lateral position was observed in 1female • Nose, discharge, red was observed slight in 1female and severe in 2 females • Piloerection was observed in 2 males and 2 females • Reduced general condition was observed slight in 1female and severe in 2 females • Salivation was observed slight in 1female, moderate in 2 females and severe in 1female • Twitching was observed slight in 4 males and 3 females, moderate in 1 male and 4 females and severe in 2 females • Food consumption in both sexes was reduced during the administration period, with a maximum of-11.9% in males and statistically significantly of-15.3% in females on day 14 • Water consumption in both sexes was increased during the administration period, with the exception ofday 14 (-8.5%) in males and amaximum of+8.1% in males on day 28 and +24.7% in females on day 14 • Body weight in both sexes was decreased during the administration period, with a maximum of-8.6% in males and -4.4% in females on day 14 • Body weight change in both sexes was decreased during the administration period, with a maximum of-24.4% in males and -20.5% in females on day 14 • Food efficiency in males was statistically significantly increased on day 14 • Twitching was observed during FOB (home cage observation) in 3/4 males on day 26 and 3/3 females on day 27 • Twitching was observed during FOB (sensorimotor tests/reflexes) in 2/4 males on day 26 and 2/3 females on day 27 • Rearing during FOB (quantitative parameters) was increased of+48.0% in males and +63.7% in females • Grip strength forelimbs and grip strength hindlimbs during FOB (quantitative parameters) was reduced of-9.2% and -16.8% in males and -9.9% and -5.9% in females • Motor activity was statistically Significantly increased in both sexes

• Decreased total protein and globulin levels in both sexes • Decreased albumin levels in the females

30 mg/kg body weight/day (during the entire administration period)

• Convulsions, tonic were observed moderate in 1 male • Hyperactivitywas observed in 1female • Hyperesthesia was observed slight in 1 male and 5 females

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## Page 17 of368

## • Hyperexcitabilitywas observed sUght in 5 males and 5females, moderate in 3 males and 4 females and severe in 1female • Piloerection was observed in 1 male • Twitching was observed slight in 5 males andfemales, moderate in 4 females and severe in 1female • Food consumption in both sexes was reduced during the administration period, with a maximum of-9.8% in males and statistically significantly of-18.0% in females on day7 • Waterconsumption in both sexes was increased during the administration period, with the exception ofday 14 (-13.6%) in males and a maximum of +6.3% in males on day21. a maximum of+26.4% in females on day 14 and s1atistically significantly of+24.6% in females on day28 • Bodyweight in both sexes was decreasedduring the administration period, with a maximum of-7.7% in males on days 7 and 21 and -6.3% in females on day 7 • Bodyweight change in both sexes was decreased during the administration period, with a statistically significantly maximum of-37.7% in males and -48.7% in females on day 7 • Food efficiency in both sexes was statistically significantly decreased on day7 • Rearing during FOB (quantitative parameters) was increased of+26.3% in males and +21.4% in females • Grip strength hindlimbs during FOB (quantitative parameters) was reduced of-24.4% in males and in forelimbs and hindlimbsof-39.9% and -26.2% in females • Motoractivitywas statistically significantly increased in both sexes

• Decreased total protein level in the males



## 10 mg/kg body weight/day (during the entire administration period)

• Food consumption in both sexes was reduced during the administration period, with a maximum of-6.1% in males and statistically significantly of-12.3% in females on day 14

. • Water consumption in both sexes was increased during the administration period, with a maximum of+14.0% in males on day21 and +27.3% in females on day 14 • Bodyweight in both sexes was decreasedduring the administration period. with a maximum of-4.8% in males on days 7 and 28 and -4.3% in females on day 7 • Bodyweight change in both sexes was decreased during the administration period, with a maximum of-20.9% in males and statistically significantly of-35.3% in females on day 7 • Food efficiency in females was statistically significantly decreased on day 7 • Rearing during FOB (quantitative parameters) was increased of+10.5% in males and +14.3% in females • Grip strength hindlimbs during FOB (quantitative parameters) was reduced of-17.9% in males and in forelimbs of-8.8% in females • Motor activity was statistically Significantly increased in both sexes

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## Page 18 of368

## In conclusion, the oral administration

# o~ Iby gavage overa period of4 weeks caused severe, substance-related as well as adverse dinical effects throughout all dose levels in both sexes. Whereas the treatmentwith the test article did not lead to any pathomorphological changes in respectto the tissue and organs examined during necropsy, organ weight determinations and light microscopy. Therefore, the no observed adverse effect level (NOAEL) underthe conditions ofthis study was 

## below 10 

## mg/kg bodyweight/dayfor male as well as female Wistar rats.

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## Page 19 of368

# 2. INTRODUCTION

2.1. OBJECTIVES The aim ofthe studywas to determine the toxicolq;Jical profile ofthe test substance including the target organs and the "no observed adverse effect level" (NOAEL) after4 weeks oforal administration by gavage.


# 2.2. SELECTION OF DOSES By request ofthe sponsor, thefollowing dose levels were selected forthe present study:
60 mg/kg bodyweight/day: as high dose with expected toxic effects 30 mg/kg bodyweight/day: as mid dose 10 mg/kg bodyweight/day: as low dose
Due to severe clinical signs oftoxicity, the high dose level was reduced to 45 mg/kg body weight/day from study day 8 onwards. The oral route was selected since this has been proven to be suitableforthe detection ofa toxicological hazard.


# 2.3. TEST GUIDELINES The study was conducted according 

## tolbased on the following test guidelines:

• OECD Guidelines forTesting ofChemicals; Method No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents; adopted July 27, 1995

• EC Commission directive 

## 96/54/EEC ofSeptember 30, 1996; Part B: Methods forthe determination ofToxicity; Toxicity after 28-dayadministration (oral); Official Journal ofthe European Communities No. L 248, pp. 213-217, 1996

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## Page 20 of368

## --L-

__________________________________________________________________**~**


## 2.4. STUDY DATES In the following table, the relevant intervals for certain study phases are given:

i 

### i

### Males Females 

## September 26,2006

October 04,2006 October04,2006 October 05, 2006 October06, 2006 October30,2006 October 31 ,2006 November01, 2006 November 02, 2006 November 03, 2006 November December 2006 January25, 2007 Phase ofstudy 

## I Examination 

## Experimental starting date: Arrival ofthe animals and beginning ofthe adaptation period Randomization ofthe animals Sampling foranalyses Startofthe administration period Study initiation date Urinalysis FOB1; M1 FOB2; M2
Last weighing Blood sampling and necrops/) Organ fixation, processing and evaluation Experimental completion date; Draft report to QAU Study day 

## -9
-1 -1 0 1 25 26 27
28 29

1) 

## Fasting period (withdrawal offood) ofabout 16to 20 hours before necropsy FOB = Functional observational battery M = Measurementofmotoractivity 

## 1...2 = Identification numberofthe specific examination



## 2.5. RETENTION OF RECORDS GLP-relevant records and materials are stored a or at leastthe period oftime specified in the GLP principles. Detal s concerning responsi llities or locations ofarchiving can be seen from the respective SOPs and from the raw data.

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## Page 21 of368

# 3. MATERIALAND METHODS

3.1. TEST SUBSTANCE The analyses ofthe test substance have been carried out atthel 

# I

Name oftest substance: Testsubstance No.: Batch identification: Purity: Homogeneity: Stability: 

# I**~----~**

99.9 g/1OOgl'-

_____--1 

## given stable until January at room temperature
ADDITIONALTEST SUBSTANCE INFORMATION
Date ofproduction: 

## 1995 Physical state/Appearance: Storage conditions: room temperature

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## Page 22 of368

## 3.2. TESTANIMALS


## 3.2.1. Species and strain
Test species and strain: Rat; Crl:WI(Han) Reason for species selection: Ratswere selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats. Age when supplied; sex: 33 

## +/- 1 day; males/females Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH, SandhoferWeg 7, 97633 Sulzfeld Arrival in the testing facility: September26,2006 Identification: The rats were identified clearly by eartattoo



## 3.3. HOUSING AND DIET The rats were housed singly in type OK III stainless steel wire mesh cages supplied by Becker

# &Co., Castrop-Rauxel (floor area about 800 crrf). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest). Motoractivity measurements were conducted in Polycarbonate cageswith wire covers from Ehret, Emmendingen (floorarea about 800 crrf) and small amounts ofabsorbent material (see above). For enrichmentwooden gnaw;ng blocks (Typ NGM E-022), supplied by Abedd ® 

## Lab. and Vet. Service GmbH, Vienna, Austria, were added. The animals were housed in a fully air-conditioned room. Central air-conditioning guaranteed a range of20
­ 24°Cfor temperature and of30 -70% for relative humidity. The day/night cycle was 12 hours (12 hours lightfrom 06.00 a.m. - 06.00 p.m., 12 hours darkfrom 06.00 p.m. - 06.00 a.m.). Deviationsfrom these ranges did not occur. The animal roomwas completely disinfected priorto the study using a disinfector ("AUTEX",fully automatic, formalin-ammonia-based terminal disinfector). The floor and the wallswere cleaned once a week. The cleansing liquid used was water containing about 0.1% Incidin (supplied by Henkel, DUsseldorf). The food used was ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA. Kaiseraugst. Switzerland. Food and drinking water (from water bottles) were available ad libitum.

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## Page 23 of368

# 3.4. TEST GROUPS AND DOSES Males
Dose group (colorofthe cage cards) Dose mg/kg body

weight1} 

## Concentration 

## g/100 ml No. of animals Animal No.



# 0 (gray) 

# 02) 

# - 5 1 - 5
1 (blue) 

# 10 0.1 5 6 -10
2 (yellow) 

# 30 0.3 5 11

- 15
3 (red) 

## 60/45 0.6/0.45 

# 5 16

- 20

Females 

# I Dose group

! 

## (colorofthe cage cards) Dose mg/kg body

weight1) 

## Concentration 

## g/100 ml No. of animals Animal No.



# 0 (gray) 

# 02)


# - 5 21

- 25 I 

# 1 (blue) 

# 10 0.1 5 26

- 30 I 

# 2 (yellow) 

# 30 0.3 5 31

- 35
3 (red) 

## 60/45 0.6/0.45 

# 5 36

-40

1)

## The dose refers tothe body weight determined most recently. 2)

## Vehicle: drinking waterwith

# 0.5% carboxymethylcellulose

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## Page

# 24 of

# 368

# 3.5. TEST SUBSTANCE PREPARATIONS AND PREPARATION FREQUENCY The test substance was applied as a suspension.To prepare the suspension, the appropriate amount oftest substance was weighed out depending on the desired concentration. Then the vehicle (0.5% carboxymethylcellulose in drinking water) wasfilled up to the desired volume, subsequently mixed using a magnetic stirrer. During application the test substance preparations were kept homogeneous using a magnetic stirrer. Thetest substance preparations were prepared twice aweek.

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## Page 25of368

# 3.6. ANALYSES


# 3.6.1. Analyses ofthe testsubstance preparations The anal ses ofthetest substance preparations were carried outatth

The stability ofthe test substance in the vehicle overa period of up to 96 hours was proven before the start ofthe studyl 

# I
Before the beginning ofthe administration period, 3 samples were taken from the lowest and highest concentration for homogeneity analysis; these samples were used as a concentration control at the same time. Only one samplewas taken from the intermediate dose group for concentratlC'**tIl.!D.DIIW1...1:Uft5.fl:DlfLSiillllJ.J..e....Ql..ealGIl..saJIlrl.lfLlIlli:llS...If:ll[lml.fill......ullL~llillf:l5..JIjfjilli_....,** 

## senttothe



# 3.6.2. Analytical methods The methods used for analytical investigationsofthe test substance preparations can be found in PART III (Supplement).



# 3.6.3. Food analyses The supplier assayed the food used in the study for chemical and microbiological contaminants.



# 3.6.4. Drinking wateranalyses



# 3.6.5. Bedding and enrichment analyses The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

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## Page 26 of368

## 3.7. EXPERIMENTAL PROCEDURE On the dayofarrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad lit)tum. Priorto the first detailed clinical observation, the animals were distributed according toweight among the individual test groups, separated bysex. The weight variation ofthe animals used did not exceed±20 percentofthe mean weight ofeach sex. The list of randomization instructions was compiled with a computer. Atthe start ofthe administration period (day 0) the rats were 42±1 days old. The test substance was administered dailybygavage for about 28 days. Control animals received the vehicle only. The volume to be administered was 10 ml/kg body weight/day (related tothe body weight determined most recently in each case). Atthe end ofthe administration period the animalswere sacrificed after a fasting period (withdrawal offood) for at least 16
- 20 hours.



## 3.7.1. Mortality A checkfor moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. Ifanimals were in a moribund state, they were sacrificed and necropsied.



## 3.7.2. Clinical observations All animals were checked daily before, about 1 hour afterand about 3 - 4 hours after application for any clinically abnormal signs. Abnormalities and changes were documented for each animal.



## 3.7.3. Detailed clinical observations (DCO) Detailed clinical observations were performed in all animals priortothe administration period and thereafter atweekly intervals. The findings were ranked according to the degree of severity, ifapplicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of25 cm high). The following parameterswere examined: 1. abnormal behaviorduring "handling" 2. fur 3. skin 4. posture 5. salivation 6. respiration 7. activity/arousal level 8. tremors 9. convulsions 10. abnormal movements

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## Page 27 of368

## 11. impairment ofgait 12. lacrimation 13. palpebral closure 14. exophthalmus 15. feces (appearance/consistency) 16. urine 17. pupil size



## 3.7.4. Food consumption Individual food consumption was determined weekly overa period of7 days and calculated as mean food consumption grams per animal and day.



## 3.7.5. Waterconsumption Individual water consumption was determined weekly over a period of4 daysand calculated as mean water consumption in grams peranimal and day.



## 3.7.6. Bodyweight data Body weightwas determined before the startofthe administration period in orderto randomize the animals. During the administration period the body weightwas determined on day 0 (start ofthe administration period) and thereafter atweekly intervals. The difference between the body weight on the respective day ofweighing and the bodyweight on day 0 was calculated as body weight change.



## 3.7.7. Food efficiency Food efficiency (group means) was calculated based upon individual values for bodyweight and food consumption:

x 

### 100

# = Food efficiency fordayx FCytox 

## BWx 

### = bodyweight on dayx (g) 

## BWy = body weight on day y (lastweighing date before day x) (g) FCytox 

## = mean food consumption from dayy to day x; calculated as mean daily food consumption on dayx, multiplied with the numberofdays from day y to day x (g)

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## Page 28 of368

# - 3.7.8. Functional observational battery (FOB) 

## A functional observational battery was performed in all animals at the end ofthe administration period starting at about 10.00 a.m
.. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorirrotortests as well as reflextests. The findings were ranked according to the degree ofseverity, ifapplicable. The observations were performed at random. A detailed description ofthe methods, the ranking and documentation system can be found in Part III (Supplement). Home cage observations: 

## The animals were observed in theirclosed home cages; anydisturbing activities (touching the cage or rack, noise) were avoided during theseexaminations in ordernotto influence the behaviorofthe animals. Attention was paid to: 1. posture 2. tremor 3. convulsions 4. abnormal movements 5. impairment ofgait 6. otherfindings Open fieldobservations: 

## The animals were transferred to a standard arena (50 x 50 cm with sides of25 cm high) and observed forat least 2 minutes. Following parameters were examined: 1. behaviorwhen removed from cage 2. fur 3. skin 4. salivation 5. nose discharge 6. lacrimation 7. eyes/pupil size 8. posture 9. palpebral closure 10. respiration 11. tremors 12. convulsions 13. abnormal movements 14. impairment ofgait 15. activity/arousal level 16. feces (number offecal pellets/appearance/consistency) within two minutes 17. urine (appearance/quantity) within two minutes 18. numberof rearings within two minutes

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## Page 29 of368

## Sensorimotor Tests/Reflexes: 

## The animals were removed from the open field and subjected tofollowing sensorimotoror reflex tests: 1. approach response 2. touch response 3. vision ("visual placing response") 4. pupillary reflex 5. pinna reflex 6. audition ("startle response") 7. coordination of movements ("righting response") 8. behaviorduring "handling" 9. vocalization 10. pain perception ("tail pinch") 11. grip strength offorelimbs 12. grip strength ofhindlimbs 13. landing foot-splay test 14. otherfindings



## 3.7.9. Motor

## activityassessment Motor activity was measured on the same dayas FOB was performed. The measurement was performed in the dark usingthe Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material.The animalswere put into the cages in a randomized order. The measurements started atabout 2.00 p.m. The numberofbeam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instantfor all cages; the period ofassessmentfor each animal started when the first beam was interrupted by pushing the cage intothe rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

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## Page 30 of368

## 3.7.10. Statistics ofclinical examinations Means and standard deviations ofeach test group were calculated for several parameters (see tables). Further statistical analyses were performed according to following tables:
Parameters Statisticaltest Markers inthe References tables food consumption, A comparison ofeach * 

### for p**~**

### 0.05 DUNNETI, 

### C.w. (1955): A multiple water consumption, bodyweight, body groupwith the control group was performed ** 

### for p 

### 0.01 comparison procedurefor comparing several treatments with a control. JASA, weightchange, food using DUNNETT's test Vol. 

### 50, 1096-1121 i 

### efficiency (two-sided) forthe hypothesis ofequal DUNNETT, 

### c.w. (1964). Newtables for means multiple comparisons with a control. Biometrics, Vol. 

### 20, 482

- 491 feces, rearing, grip Non-parametricone­ • for p 

### 0.05 SIEGEL, S. 

### (1956): strength forelimbs, way analYSis using

..for p**~**

### 0.01 Non-parametricstatistics forthe grip strength KRUSKAL-WALLIS test behavioural sciences. McGraw-Hili New hindlimbs, foot­ (two-sided).lfthe York splaytest, motor resulting p-value was activity equal orlessthan 

### 0.05, a pairwise comparison ofeach dose groupwith the control group was performed using

.Wilcoxon-test (twosided)forthe equal medians


### Due to technical reasons, on some tables the following markers are given:
* 

# = 0.050 = 

## * for p < 

### 0.05 ** 

# = 0.010 

## = ** 

### for p 0.01

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## Page 31 of368

# 3.8. CLINICAL PATHOLOGY In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequentanalysis ofthe blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal offood and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. The assays ofblood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results ofthe clinical pathology examinations are expressed in units ofthe International System (SI). The following examinations were carried out in all surviving animals pertest group and sex at the end ofthe application period.


# 3.8.1. Hematology The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore differential blood smears were prepared and stained according to Wrightwithout being evaluated.

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## Parameters and methods:
Parameter Unit Method References Leukocyte count gigall cytochemistry coupled with (WBC) Erythrocyte count teral! flow cytometry 

# I**:~;cytometric** laserlight (RBC) attering i 

### Hemoglobin mmolll cyanmethemoglobin 

### I (HGB)­ method; according to ICSH • Hematocrit III calculation: I 

### (HCT) MCVx erythrocytes Mean corpuscular 

### fI RBC/PLT method; mean of volume RBC volume distribution Operator's Guide for Advia 120 System •(MCV) curve (histogram)

Mean corpuscular fmol calculation: hemoglobin (MCH) hemoglobin erythrocytes Mean corpuscular mmolll calculation: hemoglobin concentration hemoglobin (MCHC) hematocrit i 

### Platelet count giga/l flow cytometric laserlight (PLT) scattering Differential blood 

### % and cytochemistry coupled with count qigall flow cytometry ReticuI ocytes 

### % cytochemistry coupled with flow cytometry 

## The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, Germany). Parameter and method:
Parameter Unit Method References Prothrombin time (Hepato Quick's test) (HQT) seconds citrated blood with calcium thromboplastin Fischer, M. and Falkensammer, Ch., Klin. Wschr. 86, 577-583 (1974)

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## Page 33 of368

## 3.8.2. Clinical chemistry An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinico-chemical parameters Parameters and methods: Enzyme (systematic name and system number) Unit Method,wave· length and measuring References
temperature Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.) \Jkat/I kinetic UVtest, 340 nm; 

## 3rC Recommendations ofthe German •Society for Clinical Chemistry: ' "Standardization ofmethods for determining enzyme activities in biological liquids"

. Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.) \Jkat/I kinetic UVtest, 340 nm; 

## 3rC J. Clin. Chem. Clin. Biochem. 

### §"**658~** 

### 660 (1970); 

### J. Clin. Chem. Clin. Biochem.**~,** 

### 464­ 465 (1971); 

### J. Clin. Chem. Clin. Biochem. 10, 182-192 (1972) Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.) \Jkat/I kinetic colortest. 415 nm, 

## 3rC Roche working instructions

,(-Glutamyltransferase (GGT) ('(-glutamyl) peptide: aminoacid-,(­ glutamyl-transferase; EC 2.3.2.2.) nkat/I kinetic colortest, 415 nm. 

## 3rC Szasz. G. et aI., 

### J. Clin. Chem. Clin. Biochem. 12.228 (1974) Roche working instructions

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### Blood 

### Chemistry Parameter Unit Method 

### References

Sodium (NA) mmol/l ion selective electrodes (ISE) Hitachi 917
- working instructions Potassium (K) mmol/l Chloride (Cl) mmol/l Inorganic phosphate (INP) mmol/l molybdate reaction Henry, 

## R.J. in: "Clinical Chemistry", Harper and Row Publishers, NewYork (1974); Roche working instructions Calcium (CA) mmol/l o-cresolphthalein complex without deproteinization Ray Sarkar, B.C. and Chauhan, U.P.S., Anal. Biochem. 20,155 (1967); Roche working instructions Urea (UREA) mmolll enzymatic determinationwith the ureaselglutamate dehydrogenase method Neumann, U. and Ziegenhorn, J.: XVI, Nordiska kongressen for klinisk kemi och klinisk fysiologi 1977, Oulu, Finland; Roche working instructions Creatinine (CREA)**~molll** 

### kinetic Jaffe method without deproteinization Bartels, H. et aI., Clin. Chim. Acta 37, 193 (1972); Roche working instructions Glucose (GlUC) mmol/I hexokinase/glucose-6-phosphate dehydrogenase method Schmidt, F.H., Klin. Wschr. 39,1244-1247 ,(1961); Roche working instructions Total bilirubin (TBll)**~molll** 

### DPD method Wahlefeld, A.w. etaI., Scand. J. Clin. lab. Invest. 29, Suppl. 126 (1972) Abstract 11.12; Roche working instructions Total protein (TPROT) 

### gIl biuret method Weichselbaum, T.E., Amer. J. Clin. Path. 10,40(1946); Roche working instructions Albumin (ALB) g/l bromocresol green method Doumas et aI., Clin. Chim. Acta 31,87 (1971); Roche working instructions Globulins (GLOB) g/l difference between total protein and albumin Triglycerides (TRIG) mmolll enzymatic colortestwith lipase esteraselglycerokinaselglycerol3-phosphate oxidase/4-aminophenazone mod. method by Wahlefeld, A.w., in "Methoden der enzymatischen Analyse" [Methods ofenzymatic analysis] (Bergmeyer, H.U., ed.) Vol. II, 3rd ed., Verlag Chemie Weinheim, GERMANY, pp. 1878-1882 (1974); Roche working instructions Cholesterol (CHOl) mmol/l enzymatic determinationwith cholesterol esterasel cholesterol oxidase/4-amino-phenazone (CHOD-PAP method) Siedel, 

## J. et aI., 

## J. Clin. Chem. Clin. Biochem. 19,838(1981); Roche working instructions Magnesium (MG) mmolll xylidylblue method Mann, CK and Yoe, J.H., Anal. Chem. 28, 202-205 (1956) Bohnon, C., Clin. Chim. Acta 7, 811-817 (1962)

-

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## Page 35 of368--L-

________________________________________________________________________**~**


# 3.8.3. Urinalysis With the exception ofvolume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters and methods:
Parameter Method References pH methyl red and bromothymol blue

Test strip book by Roche, Mannheim, GERMANY (1977) Protein tetrabromophenol-phthaleinethylester (TBPE) Glucose GOD-POD reaction Ketones sodium nitroprusside Urobilinogen p-methoxyaniline-diazonium-salt Bilirubin 2,5-dichloroaniline diazonium salt Blood 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine Specific gravity refractometer Hamilton orAtago operating instructions :Sediment microscopy Hallmann, L., [Clinical Chemistryand Microscopy]

## 12, ed., 233-246, Georg Thieme, Stuttgart, Germany (1966) Color, turbidity by visual evaluation Volume graduated tubes

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## Page 36 of368

# - 3.8.4. Statistics ofclinical pathology Means and standard deviations ofeach test group were calculated for several parameters (see tables). Further statistical analyses were performed according to following tables:
Parameter Statisticaltest Markers in the tables References Clinical pathology parameters, except reticulocytes and differential blood count Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).lf the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) forthe equal medians * 

### for p**~** 

### 0.05 ** for p**~** 

### 0.01 SIEGEL, S. (1956): Non-parametricstatistics forthe behavioural sciences. McGraw-Hili New York



### Parameter Statisticaltest Markers inthe tables References Urinalysis, exceptvolume, color, turbidity and specific gravity Pairwise comparison ofeach dose group with the control group using FISHER's exact testforthe hypothesis ofequal proportions * 

### for p 

### < 0.05 ** 

### for p 0.01 SIEGEL, S. (1956): Non-parametricstatistics forthe behavioural sciences. McGraw-Hili New York

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## 3.9. PATHOLOGY


## 3.9.1. Necropsy 

## The surviving animals were sacrificed by decapitation under Isoba® (Essex GmbH Munich, Germany) anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals that died intercurrently were necropsied as soon as possible aftertheir death and assessed by gross pathology.



## 3.9.2. Organ weights 

## The following weights were determined in all animals sacrificed on schedule: 1. Anesthetized animals 2. Liver 3. Kidneys 4. Adrenals 5. Testes 6. Epididymides 7. Ovaries 8. Uterus 9. Spleen 10. Brain 11. Heart 12. Thymus 13. Thyroid glands



## 3.9.3. Organ 

## ITissue fixation 

## The following organs or tissues were fixed in 4% formaldehyde solution: 1. All gross lesions 2. Brain 3. Pituitary 4. Thyroids (with Parathyroids) 5. Thymus 6. Oesophagus 7. Salivary glands (mandibular and sublingual glands) 8. Trachea 9. Lungs 10. Pharynx 11. Larynx 12. Nose (nasal cavity) 13. Aorta 14. Heart

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## 15. Liver 16. Pancreas 17. Spleen 18. Kidneys 19. Adrenals 20. Testes 21. Ovaries with Oviducts 22. Uterus and Vagina 23. Epididymides, prostate and seminal vesicle 24. Stomach (forestomach and glandularstomach) 25. Duodenum, jejunum and ileum 26. Cecum, colon and rectum 27. Urinary bladder 28. Lymph nodes (mesenteric and axillary lymph nodes) 29. Sciatic nerve 30. Bone marrow (femur) 31. Eyes 32. Extraorbitallacrimal glands 33. Skin 34. Female mammary gland 35. Spinal cord (cervical, thoracic and lumbarcords) 36. Sternum with marrow 37. Femurwith kneejOint 38. Skeletal muscle From the liver, each one slices ofthe Lobus dexter lateralis and the Lobus sinister lateralis were fixed in Carnoy's solution and embedded in paraplast.

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## 3.9.4. Histotechnical processing 

## I Examination by light microscopy and assessment offindings Fixation was followed by histotechnical processing, examination by light microscopy and assessment offindings according tothe table below: 

## Organs Dose group 

## 0 1 

## 2 

# 3 1. All gross lesions A2 

## A2 A2 A2 2. Brain A1 A1 3. Thyroids (with parathyroids) A1 A1 4. Thymus A1 A1

# ~hea A1 A1 A1 A1 7. Heart A1 A1 8. Liver A1 A1 9. Spleen A1 A1 10. Kidneys A1 A1 11.Adrenals A1 A1 12.Testes A1 A1 •13.Ovaries (with oviducts) i 

## A1 A1

14.Uterus A1 15.Vagina A1 A1 16. Epidymides A1 A1 17. Prostate A1 A1 18.Seminal vesicle A1 A1 19.Stomach (forestomach and glandular A1 A1 stomach) 20. Duodenum and ileum A1 A1 21.Jejunum (with Peyer's plaques) A1 A1

# ~2. Cecum, colon and rectum A1 A1 23. Urinary bladder A1 A1 24. Lymph nodes (mesenteric and axilla A1 A1 nodes) 25. Sciatic nerve A1 A1 26. Bone marrow (femur) A1 A1 27. Spinal cord (cervical, thoracic and lumbar A1 A1 cords)

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## Methods/Scope ofexaminations: A 

## = Hematoxylin-eosin 1

# =All animals/group 2 

## = All animals affected/group Animals that died orwere sacrificed in a moribund state were processed histotechnically and assessed like control animals. The immunorelevant organs and tissues were evaluated according to the following parameters: Thymus:**~** 

## Increased/decreased grade ofcortico-medullar ratio (related onlyto area)**~** 

## Increase ofstarry sky cells**~** 

## Changes ofcellular density in the cortex**~** 

## Changes ofcellular density in the medulla Spleen:**~** 

## Changes ofthe cellularity of PALS, lymphoid follicles, marginal zone, red pulp**~** 

## Altered cellular composition offollicles**~** 

## Altered number ofgerminal centers Lymph nodes (mesenteric and axillar lymph nodes):**~** 

## Changes in the cellularity offollicles, interfollicular area, paracortical area, medulla**~** 

## Altered cellular composition ofparacortex**~** 

## Altered numberofgerminal centers**~** 

## Hyperplasia ofhigh endothelial venules**~** 

## Apoptotic necrosis increased Peyer's patches (ofthejejunum):**~** 

## Changes ofthe cellularity offollicles (induding mantle zone and germinal centers)**~** 

## Changes ofthe cellularity of interfolliculararea**~** 

## Apoptotic necrosis increased Bone marrow:**~** 

## Changes ofthe cellularity**~** 

## Changes ofthe myeloid/erythropoid ratio Wheneverthe histopathologic evaluation ofthe immunorelevant organs and tissues did not reveal a morphologic alteration ofthese items and/orwhenever no other pathologicfinding was noted, these organs were diagnosed as "no abnormalities detected".

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# 3.9.5. Statistics of pathology Means and standard deviations were calculated. In addition, the following statistical analyses will be carried out:
! 

## Parameters Statisticaltest Markers in the tables References
Weight Non-parametric one-way * for HETTMANNSPERGER, T.P. parameters analysis using KRUSKALWALLIStest (two-sided). If the resulting p-valuewas equal or less than 0.05, a pairwise comparison ofeach P.5 0.05
** 

### for P.5 0.01 (1984):Statisticallnference based on Ranks, John Wiley & Sons NewYork, 132-140. International Mathematical and Statistical Libraries, Inc., 2500 Park dose groupwith the control group was performed using the WILCOXON testforthe hypothesis ofequal medians West Tower One, Houston, Texas 77042-3020, USA, nakl-1
- nakl-3 MILLER, R.G. (1981): Simultaneous Statistical Inference Springer-Verlag NewYork Inc., 165­ 167 NIJENHUIS, A. and S.W. WILF (1978): Combinatorial Algorithms, Academic Press, NewYork, 32-33

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# - 

# 4. RESULTS Throughout the chapter "results", the term "signifICant" implies thatthe inter-group differences have attained statistical

## significance (p .::: 0.05) when compared with the control group.

4.1. ANALYSES

4.1.1. Stabilityanalyses The stability ofthe test substance in the vehicle was demonstratedover a period of up to 96 hours at room temperature (see PART III). As the suspensionswere stored no longerthan this time period, the stability was guaranteed.

4.1.2. Homogeneitycontrol analyses Considering the low standard deviation in the homogeneity analysis, it can be concluded that 

# I**~as** distributed homogeneously in the vehicle.

4.1.3. Concentration control analyses The concentration control analyses ofall concentrations revealed that the values were in the expected range ofthe target concentrations, i.e. were always in a range ofabout 95.5­ 118.6% ofthe nominal concentrations. Although the concentration ofthe low dosewas 118.6% ofthe nominal concentration, the data are assessed to be valid. For details see PART III.

4.1.4. Food analyses On the basis ofduration ofuse and the analytical findings with respect to chemical and microbiological contaminantsthe dietwas found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 9, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*105

## /g food. Individual results can be found in the archives ofthel 

# I 

# I I

4.1.5. Drinkingwater analyses On the basis ofthe analytical findings the drinking water was found to be suitable. German 

# I Drinking Water Regulation (Trinkwasserverordnung) served as a guideline for maximum tolerable contaminants. Individual results can be found in the archives ofthe 

# I
1...-----1

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## 4.1.6. Bedding and enrichment analyses On the basis ofthe analytical findingsthe bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov.
- Dec. 1979, pp. 24
- 33, served as a guideline for maximum tolerable contaminants. Individual results are to befound in the archives ofthe 

# I I

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## 4.2. CLINICAL EXAMINATIONS Summary tables ofthe results are given inthe Appendix of PART I; individual values are given in PartA of PART II.



## 4.2.1. Mortality 

## 1 male and 1female ofgroup 3 (60 mg/kg bw'day) died prematurelyon day 6 and on day 

## 7, respectively; 1female ofgroup 3 (45 mg/kg bw/day) was sacrificed moribund on day 11. These cases ofdeathswere assessed as related tothe test-substance.



## 4.2.2. Clinical examinations (Tables IA- 

## 1 - IA-16) 

## High dose (60 mglkgbodyweight/day; from day

## 0 until day 7):

• Convulsions, clonicwere observed moderate in 4 females, severe in 2females, 

## epileptoid slight in 

## 1 male, moderate in 

## 1female, severe in 

## 2 malesand 

## 1female,

## tonic slight in 

## 1female and severe in 

## 1 male and 

## 2 females 

## • Hyperesthesia was observed slight in 2 males and 5 females, moderate in 4 females and severe in 1 male and 

## 2 females 

## • Hyperexcitabilitywas observed slight in 2 males and 5 females, moderate in 4 females and severe in 

## 1 male and 

## 2 females 

## • Nose, discharge, red was observed moderate in 

## 1female 

## • Piloerection was observed in 

## 2 females 

## • Salivation was observed slight in 

## 2 females, moderate in 

## 1 male and 

## 4 females and severe in 

## 1 male and female 

## • Skin, paleness was observed moderate in 

## 2 females 

## • Twitching was observed slight in 3 males and 5 females, moderate in 2 males and 3 females and severe in 

## 1 male and 3 females 

## High dose 

## (45 mglkgbodyweight/day; from day

## 8 until the endof

## the administration period):

• Anogenital region, smeared with urinewas observed severe in 1female 

## • Convulsions, tonic were observed slight, moderate and severe in each 1 female 

## • Hyperactivitywas observed in 1female 

## • Hyperesthesia was observed slight in 2 males and 1female 

## • Hyperexcitability was observed slight in 4 males and3females, moderate in 4 males and females and severe in 1 male and 2females 

## • Lateral position was observed in 1female 

## • Nose, discharge, red was observed slight in 1 female and severe in 2females 

## • Piloerection was observed in 2 males and 2 females 

## • Reduced general condition was observed slight in 1female and severe in 2 females 

## • Salivation was observed slight in 1female, moderate in 2 females and severe in 1female

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## • Twitchingwas observed slight in 4 males and 3 females, moderate in 1 male and 4 females and severe in 2 females Middose (30 mg/kg body weight/day):



## • Convulsions, tonicwere observed moderate in 1 male • Hyperactivitywas observed in 1female • Hyperesthesiawas observed slight in 1 male and 5females • Hyperexcitabilitywas observed slight in 5 males and 5 females, moderate in 3 males and 4females and severe in 1female • Piloerection was observed in 1 male • TWitching was observed slight in 5 males and females, moderate in 4 females and severe in 1female Mostofthe above-mentioned findings were obseNed on different days ofthe study, starting on day 0 aftertreatment and were present mostofthe time within the whole study period, alternating in occurrence in each individual animal. The findings were more pronounced in females (for more details see PART II, Tables IIA- 1 - IIA-16). Due to a clear dose-response relationship, these findings were assessed as related tothe test-substance.

4.2.3. Food consumption (Tables IA-17 -IA-20) Food consumption was reduced in all dosed animals; statistically significantly of -29.4% in males and -28.2% in females ofgroup 3 (60 mg/kg bw/day) on day 7; up to -11.9% in males and statistically significantly of -15.3% in females ofgroup 3 (45 mg/kg bw/day) on day 14; upto -9.8% in males and statistically significanUy of-18.0% in females ofgroup 2 (30 mg/kg bw/day) on day7 and up to -6.1% in males and stati&icallysignificantly of-12.3% in females ofgroup 1 (10 mg/kg bw/day) on day 14. DUe to a clear dose-response relationship, the influence on food consumption was assessed to be substance-related.

4.2.4. Waterconsumption (Tables IA-21
- IA-24) Waterconsumptionwas predominantly increased in all dosed animals, more pronounced in females; viz of+13.9% in males and statisticallysignificantly of+36.9% in females ofgroup 3 (60 mg/kg bw/day) on day 7; upto +8.1% in males on day 28 and +24.7% in females of group 3 (45 mg/kg bw/day) on day 14; up to +6.3% in males on day21, +26.4% in females on day 14 and statistically significantly of+24.6% in females ofgroup 2 (30 mg/kg bw/day) on day28 and up to +14.0% in males on day 21 and +27.3% in females ofgroup 1 (10 mg/kg bw/day) on day 14. Because ofthe high toxic effect ofthe test-substance in all dosed animals, this was assessed as substance-related.

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## Page 46 of368

## The decreased values of-8.5% in males group3 (45 mg/kg bw/day) and -13.6% in males of group 2 (30 mg/kg bw/day) on day 14, were assessed as outliers and therefore not substance-related.



## 4.2.5. Bodyweightdata (Figures 1 - 2; Tables IA-25
- IA-32) 

## Bodyweightwas statistically Significantly decreased of-13.9% in males and not statistically significantly decreased of-6.5% in females ofgroup 3 (60 mg/kg bw/day) on day 7; itwas also decreased during the administration period, up to -8.6% in males and -4.4% in females ofgroup 3 (45 mg/kg bw/day) on day 14, up to -7.7% in males on days 7 and 21 and -6.3% in females ofgroup 2 (30 mg/kg bw/day) on day 7 and upto -4.8% in males on days 7 and 28 and -4.3% in females ofgroup 1 (10 mg/kg bw/day) on day 7. In conclusion it can be stated that decreased body weight is more pronounced in males than in females. All above-mentioned body weight deviationsto control groups were assessed to be substance-related. 

## Bodyweight change was statistically Significantly decreased of-60.8% in males and -50.9% in females ofgroup 3 (60 mg/kg bw/day) onday 7; it was also decreased during the administration period in all dosed animals, upto -24.4% in males and -20.5% in females of group 3 (45 mg/kg bw/day) on day 14, statistically signficantly up to -37.7% in males and -48.7% in females ofgroup 2 (30 mg/kg bw/day) on day 7 and -20.9% in males and statistically Significantly of-35.3 in females ofgroup 1 (10 mg/kg bw/day) on day 7. All above-mentioned body weight change deviationsto control groups were assessed to be substance-related.



## 4.2.6. Food efficiency (Tables IA-33
- IA-36) Food efficiency was statistically significantly decreased in both sexes ofgroup 3 (60 mg/kg bw/day) on day 7, statistically Significantly increased in males ofgroup 3 (45 

## mg/kg bw/day) on day 14and not statistically increased in bothsexes during the administration period; food efficiency was also statistically significantlydecreased in both sexes ofgroup 2 (30 mg/kg bw/day) and in females ofgroup 1 (10 mg/kg bw/day) on day 7. Because ofa clear relationship to other clinical findings, like body weight data and food consumption, thisfinding was assessed as substance-related. On several occasions, food efficiency differed (increased/decreased values) in treatment groups 2 and 1from controls in malesand females on some days ofthe study. These findings were spontaneous in natureand therefore not substance-related.

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## 4.2.7. Functional observational battery 

## (Tables IA-37
- lA-52) Deviations from "zero values"were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. Besidethis, the following findings were observed and have to be assessed individually:

Home cage observations: 

## (Males see Table IA-37 and Females see Table IA-44) "Twitching" was observed in 

## 3/4 males and 3/3 females ofgroup 3 (45 mg/kg 

## bw/day). This was assessed as substance-related, because ofthe systemictoxicity ofthe test article.
Open field observations: 

## (Males see Tables IA-38
- IA-41 and Females see Tables IA-45
- IA-48) No substance-related findings were observed.

Sensorimotortests/ref/exes: 

## (Males see Tables IA-42
- IA-43 and Females see Tables IA-49
- lA-50) 'Twitching" was observed in 

## 2/4 males and 

## 2/3females ofgroup 3 (45 mg/kg 

## bw/day) This was assessed as substance-related, because ofthe systemic toxicity ofthetest article.

Quantitativeparameters: 

## (Males see Table lA-51 and Females see Table lA-52) "Rearing" was increased of +48.0% in males and +63.7% females of group 3 (45 mg/kg bw/day), of +26.3% in males and +21.4% in females of group 2 (30 mg/kg bw/day) and +10.5% in males and +14.3% in females ofgroup 1 (10 mg/kg 

## bw/day). Due to a cleardose-response relationship, this was assessed as substance-related. "Grip strength forelimbs" was reduced of -9.2% in males and -9.9% females of group 3 (45 mg/kg 

## bw/day), of-39.9% in females of group 2 (30 mg/kg 

## bw/day) and -8.8% in females of group 1 (10 mg/kg 

## bw/day). "Grip strength hindlimbs" was reduced of -16.8% in males and -5.9% females of group 3 (45 mg/kg bw/day), of-24.4% in males and -26.2% in females ofgroup 2 (30 mg/kg 

## bw/day) and -17.9% in males group 1 (10 mg/kg 

## bw/day). This was assessed as substance-related, because ofthe systemic toxicity ofthe test article.

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## Page 48 of368

# 4.2.8. Motoractivity measurement (Figures 3 - 4; Males see Tables lA-53 -lA-54 and Females see Tables lA-55
- lA-56) Regarding the overall motoractivity statistically significantly increased deviationswere measured in males and females ofgroup 3 (45 mg/kg bw/day), 2 (30 mg/kg bw/day) and 1 

## (10 mg/kg bw/day). This was assessed as being substance-related, caused by systemic toxicity. Comparing the single intervals with the control groups, interval 4 in all dosed males and interval 12 in group 3 male animals showeda statistically significantly increase. These findings were clearly associated tothetreatmentwith the test article and the above­ mentioned systemic toxicity.

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## Page 49 of368

# 4.3. CLINICAL PATHOLOGY Summary tables ofthe results are given intheAppendix of PART I; individual values are given in Part B of PART II.



# 4.3.1. Hematology (Tables IB- 1 - IB- 2 Hematological examinations) (Tables IB- 3 - IB- 6 Differential blood count) (Tables IB- 7 - IB- 8 Clotting analyses) After administration ofthe test substancefor 4 weeks therewas noticed a statisticallyweakly significant decrease ofthe haemoglobin concentration in the male 10 mg/kg bw/d as well as in the 45 mg/kg bw/d dose group. This decrease ofthe haemoglobin levels was not accompanied by a deviation ofany other red blood cell marker. Compared to the historical control data (min value: 9.1 mmollL, max. value 9.8 mmollL), the median ofthe control rats in this study was slightly higherthan the historical controls, whereas the values ofthe 10 mg/kg bw/d as well as the 45 mg/kg bw/d dose groups were within the historical controls. Therefore, this decrease ofthe haemoglobin concentration is not regarded as substancerelated.



# 4.3.2. Clinical chemistry (Tables IB- 9 - IB-10 Enzymes) (Tables IB-11 -IB-14 Blood chemistry) In the male rats therewas observed a decrease ofthe median ofthe total protein level beginning in the 30 mg/kg bw/d dose group. This decreasewas reflected in the decrease of the globulin fraction in the 45 mg/kg bw/d dose group. Even in thefemale rats ofthe 45 mg/kg bw/d dose group thetotal protein as well as the albumin and globulinfraction were decreased even not statistically significant. This decrease is accompanied by a slight but not statisticallysignificant decrease ofthe serum glucose and triglyceride values in the dosed animals ofboth sexes.


# 4.3.3. Urinalyses (Tables IB-15 -IB-18) Therewere notreatment-related changes in the urinalyses parameters measured.


# 4.3.4. Otherdeviations Additionally, there was found a decreased median ofthe potassium levels in the 10mg/kg bw/d as well as 30 mg/kg bw/d dose group ofthefemale rats (Table IB-13). This deviationwas inconsistent, when compared with the other sex, and lack dose-response relationship. Accordingly, this finding is considered to be ofno toxicological significance.

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## Page 50 of368

## 4.4. PATHOLOGY Summary tables ofthe results are to be found in the Appendix of PART I; individual tables areto befound in Part C of PART II.



## 4.4.1. Mortality One male high dose animal (animal no. 16) died 6 days after start of exposure and one female high doseanimal (animal no. 38) 7 days after start ofexposure. One female high dose animal (animal no. 36) has to be sacrificed in a moribund state 11 days afterstartof exposure.


## 4.4.2. Weight parameters (Tables IC- 1 - IC- 4)



## 4.4.2.1. Absolute organweights Therewere no treatment-related weight deviations recorded. The significant weight decrease ofthe thymus in dosegroup 2females is regarded to be incidental and ofno biological relevance (no dose-response relationship, no substanceinduced histopathological lesions in the high dose).



## 4.4.2.2. Relative organweights There were notreatment-related weight deviations recorded. The significant weight increaseofthe brain in dose group 2 females is regarded to be incidental and ofno biological relevance (no dose-response relationship, no substanceinduced histopathological lesions in the high dose).



## 4.4.3. Gross lesions (Tables IC- 5) Therewere no treatment-related findings noted.



## 4.4.4. Histopathology (Tables IC- 6) All findings noted are spontaneous or incidential in nature and not related to treatment.

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## Page 51 of368

# 5. DISCUSSION Regarding clinical examinations, the oral administration ofli by gavage for4 weeks caused severe signs ofgeneral systemictoxicity. Reduced f50crana increased water consumption, impaired bodyweight and bodyweight change, food efficiency, increased rearing, reduced grip strength in fore- and hildlimbs and increased deviations in overall motoractivity were obtained down to 10mg/kg body weight/day in both sexes. Moreover, 1 high dose animal ofeach sex was found dead within the first week ofthe administration period, Additionally, 1 high dose female was prematurely sacrificed moribund on study day 11. Due to this exceeding ofthe maximum tolerated dose level, the highest dosewas reduced from 60 to 45 rng/kg bodyweight from studyday 8 onwards. Regarding the clinical pathology, the decrease ofthe protein levels in the dosed rats of both sexes combined with slightly lowerglucose and triglyceride levels is regarded as a result ofa dysbalance between food intake and metabolic activity ofthe rats. There is no hint in clinical pathology for a failureofthe liver cells to synthesize proteins. Even pretended decreased protein levels because ofdecreased haematocritvalues can be excluded in the rats ofthis study. Regarding pathology, there were notreatment-related findings noted. All findings recorded are considered to be incidental in nature and not related totreatment.

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## Page 52 of368

# 6. CONCLUSION In conclusion, the oral administration

# o~ 

# Iby gavage over a period of4 weeks caused severe, substance-related as well as adverse dinical effects throughout all dose levels in both sexes. Whereas the treatment with the test article did not lead to any pathomorphological changes in respecttothe tissue and organs examined during necropsy, organ weight determinations and light microscopy. Therefore, the no observed adverse effect level (NOAEL) 

## underthe conditions ofthis study was below 10 mg/kg bodyweight/dayfor male as well as female Wistar rats.

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## Page 53 of368

# 7. APPENDIX
7.1. LIST OF ABBREVIATIONS USED IN TABLES IA (CLINICAL EXAMINATIONS) Food Cons. Water Cons. BWchange Food eff mg/MG kg/KG bw g M SD 

# N anm Rear GSF GSH FST Sum %dev cm Interv. Interr. 

## t d 

## = 

## = =



# = = = = 

## = =

= = = 

## = = 

# = = 

# = =



## = food consumption Waterconsumption bodyweight change food efficiency milligram kilogram body weight weight in gram mean value standard deviation numberofanimals for determining M and SD animal rearing grip strength forelimbs grip strength hindlimbs landing foot-splay test summary deviation in percent centimetre interval beam Interrupts twitching animal dead

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## Page 54 of368

# 7.2. LIST OFABBREVIATIONS USED IN TABLES IB (CLINICAL PATHOLOGy)

SD 

## = standard deviation N 

# = numberofvalues

HEMATOLOGY: WBC 

# = white blood cells (leukocytes) RBC 

# = red blood cells (erythrocytes) HGB 

# = hemoglobin HCT 

# = hematocrit MCV 

# = mean corpuscularvolume MCH 

# = mean corpuscular hemoglobin MCHC 

# = mean corpuscular hemoglobin concentration PLT 

# = platelets RETI 

# = reticulocytes NEUT 

# = polymorphonuclear neutrophils LYMPH 

# = lymphocytes MONO 

# = monocytes EOS 

# = eosinophils BASO 

# = basophils LUC 

# = large unstained cells NEUTA 

# = polymorphonuclearneutrophils (absolute) LYMPHA 

# = lymphocytes (absolute) MONOA 

# = monocytes (absolute) EOSA 

# = eosinophils (absolute) BASOA 

# = basophils (absolute) LUCA 

# = large unstained cells (absolute) HQT 

# = prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY: ALT 

# = alanine aminotransferase AST 

# = aspartate aminotransferase ALP 

## = alkaline phosphatase SGGT 

## = serum-y-glutamyltransferase NA 

## = sodium K potassium CL chloride INP inorganic phosphate CA 

## = calcium UREA 

## = urea CREA 

# = creatinine GLUC 

# = glucose

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## Page 55 of368

## TBll 

# = total bilirubin TPROT 

# = total protein ALB 

## = albumin GLOB 

# = globulins TRIG 

# = triglycerides CHOl 

# = cholesterol MG 

# = magnesium

URINAL YSIS:

Protein: 0 

# = negative 0.15 gil 

## 1 

# = 0.30 gil, 

## 0.60 gIl 

## 2 

# = 1.00gil, 

## 3.00 gil 

## 3 

# = > 

## 5.00 gil 

## Glucose: 0 

# = normal 1 

# = 2.8 mmol/l 2 

# = 5.6 mmol/l 3 

# = 11.1 mmol/l, > 

## 16.7 mmol/l Ketones: 0 

# = negative 1 

# = 1 mmol/l 2 

# = 5 mmol/l 3 

# = > 

## 15 mmol/l Urobilinogen: 0 

# = normal 1 

# = 17 IJmolil 2 

# = 68IJmoi/i 3 

# = 135 IJmol/l, > 

## 270 IJmol/l Bilirubin: 0 

# = negative 1 

# = 9IJmoi/i 2 

# = 25 IJmol/l 3 

# = 50 IJmol/l, 100 IJmolll, > 

## 200 IJmolll Blood (erythrocytes): 0 

# = negative 1 

# = 10 ery/lJl 2 

# = 50**ery/~.JI** 

## 3 

# = 150 ery/1J1, > 

## 250 ery/lJl

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## Page 56 of368

## Spec. gravity 

## = specific gravity Sediment: 

# 0 = none 

# 1 = few 

# 2 = many 

## 3 = masses

UNITS: mmol/l 

## = millimolelliter IJmolll 

## = micromole/liter gil, G/L 

## = gramlliter 

## III liter/liter 

## % = per cent fmol 

## = femtomole 

## = 10-15 

## mole fI 

## = femtoliter 

## = 10-15 

## liter terall 

## = teralliter 1012

## 11iter giga/l gigalliter 

## = 1 09 

## Iliter IJkatll 

## = microkatai/liter nkatll 

## = nanokatalliiter mg/kg bw/day 

## = milligram/kilogram bodyweightlday IJI 

## = microliter ML ml (milliliter)

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## Page 57 of368

## 7.3. LIST OF ABBREVIATIONS USED IN TABLES IC (PATHOLOGY)
F 

# = female animals F1 

# = final sacrifice groups (main groups) g 

# = weight determination in grams M 

# = male animals (under sex); mean value (on weight level) mg 

# = weight determination in rnilligrams
n 

# = numberofvalues measured forthe determination of mean value and standard deviation NAD 

# = numberofanimals without gross lesions ppm 

# = parts per million underdose level SD 

# = standard deviation s 

# = suspectweight (not included in the mean weights) 

## % 

# = percentage related to the reference weight in relative organ weight calculations 

## Codes forthe status at necropsy: 

## 1 

# = planned sacrifice 

## 2 

# = sacrificed in a moribund state 

## 3 

# = spontaneous death 

## Codes used at finding level: The codes are used for a grading system which takesinto consideration eitherthe severity or the numberorthe size ofa microscopicfinding. 

## Severity Number Size Grade 1 Minimal Veryfew Very small Grade 2 Slight Few Small Grade 3 Moderate Moderate number Moderate size Grade 4 Marked; severe Many Large Grade 5 Massive; extreme Extensive number Extensive size 

## Whenever a grading was not used, the microscopicfinding was indicated to be present (P).

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## Page 58 of368

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## '" 0 ii1 Print Date: 06-Nov-2006 Print Time: 09:43:45 OBSERVATIONS REPORT

- INCIDENCE Table IA study:

### ~I

# __________**~** Page 1 Sex: Male Treatment Group 

## o 1 2 3 Dose 

## o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 60 mg/kg bw/day Animal Count 5 5 5 5 Convulsions

epileptoid(behavior when removed from cage)

severe Incidence 2 ( 40%) Observed 3 Mean onset (Days) 5 epileptoid(behavior when removed from cage)

slight Incidence 1 ( 20%) Observed 1 Mean onset (Days) 4 tonic

severe Incidence 1 ( 20%) Observed 1 Mean onset (Days) 3 TOTALS Incidence 

### o 0%) 

## o 0%) 

## o 0%) 3 ( 60%) Observed 

### o o o 5 Mean onset (Days) 4 General observation

Nothing abnormal detected Incidence 5 (100%) 5 (100%) 5 (100%) 5 (100'!» Observed 130 130 129 112 Mean onset (Days) 0 0 0 0 TOTALS Incidence 5 (100'!» 5 (100'!» 5 (100'!» 5 (100%) Observed 130 130 129 112 Mean onset (Days) 0 0 0 0 Hyperesthesia

severeIncidence 1 ( 20%) Observed 1 Mean onset (Days) 2-l ::r (ii' 0. 

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# '" 0 :::I (fl 0 iil Print Date: 06-Nov-2006 Print Time: 09:4:3:45 OBSERVATIONS REPORT

- INCIDENCE Table IA Study: 

# I Page 2 Sex: Male
Treatment Group 0 1 2 :3 Dose 

### o mg/kg bw/day 10 rng/kg bw/day 30 mg/kg bw/day 60 mg/kg bw/day Animal Count 5 5 5 5

slightIncidence 2 ( 40%) Observed 2 Mean onset (Days) 2 TOTALS Incidence 0 0%) 0 0%) 0 0%) 3 ( 60%) Observed 0 0 0 3 Mean onset (Days) 2 Hyperexcitability

severeIncidence 1 ( 20%) Observed 1 Mean onset (Days) 2 slightIncidence 2 ( 40%) Observed 2 Mean onset (Days) 2
TOTALS Incidence 0 0%) 0 0%) 0 0%) :3 ( 60%) Observed 0 0 0 :3 Mean onset (Days) 2 Salivation

moderate Incidence 1 ( 20%) Observed 1 Mean onset (Days) 5

severeIncidence 1 ( 20%) Observed :3 Mean onset (Days) 2 TOTALS Incidence 0 0%) 0 0%) 0 0%) 1 ( 20%) Observed 0 0 0 4 Mean onset (Days) 4-; ::J" Vi" Co 0 n <: 3 (!l ;:!. Vi" 5' (!l "0 

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# ~ Vi" Ol 0" ::l c:r '< 5' (!l U) -0 0 ::l U) 0 Ul Print Date: 06-Nov-2006 Print Time: 09:43:45 OBSERVATIONS REPORT

- INCIDENCE Table IA study:

# I Page 3 Sex: Male
Treatment Group 

### o 1 2 3 Dose o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 60 mg/kg bW/day Animal Count 5 5 5 5 Twitching

moderate Incidence 2 ( 40%) Ol::>served 3 Mean onset (Days) 2

severeIncidence 1 ( 20'1;) Ol::>served 3 Mean onset (Days) 2 slightInoidenoe 1 ( 20%) 3 ( 60%) Observed 1 5 Mean onset (Days) 7 5 TOTALS Inoidence 

## o 0%) 

## o 0%) 1 ( 20%) 3 ( 60'1;) Observed 

## o o 1 11 Mean onset (Days) 7 4 Found dead Inoidenoe 1 ( 20%) Ol::>served 1 Mean onset (Days) 6 TOTALS Inoidence 

### o 0%) 

## o 0%) 

### o 0%) 1 ( 20%) Observed 

### o o 

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# I Sex: Male OBSERVATIONS REPORT INCIDENCE Print Date: print Time: Table Page 06-Nov-2006 10:07:52 IA 4

Treatment Group Dose Animal Count 0 o mg/kg bw/day 5 1 10 mg/kg bw/day 5 2 30 mg/kg bw/day 5 3 45 mg/kg bW/day 5 Convulsions

tonic

moderate Incidence Observed Mean onset (Days) 1 1 27 ( 20%)

TOTALS Incidence Observed Mea.n onset (Days) 0 0 0%) 0 0 0%) 1 1 27 ( 20%) 0 0 0%)

General observation

Nothing abnormal detected Incidence Observed Mean onset (Days) 5 335

8 (100%) 5 335

8 (100%) 5 285

8 (100%) 4 197

a ( ao%)

TOTALS InCidence Observed Mean onset (Days) 5 335

8 (100%) 5 335

8 (100%) 5 285

8 (100%) 4 197

8 ( aO%)

Hyperesthesia

slightIncidence Observed Mean onset (Days) 1 2 19 ( 20%) 2 3 20 ( 40%)

TOTALS Incidence Observed Mean onset (Days) 0 0 0%) 0 0 0%) 1 2 19 ( 20%) 2 3 20 ( 40%)

Hyperexcitability

moderate Incidence Observed Mean onset (Days) 3 8 10 ( 60%) 4 16 11 ( 80%)--I ::r (ii' Co 

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### 9' (I) til 'C 0 :J til 0 Cil Print Date: 06-Nov-2006 Print Time: 10 07:52 OBSERVATIONS REPORT

- INCIDENCE Table IA study: 

# I Page 5 Sex: Male Treatment Group 0 1 2 3 Dose 

### o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5

severeIncidence 1 ( 20%) Observed 3 Mean onset (Days) 11 slightIncidence 5 (100%) 4 ( 60%) Observed 19 14 Mean onset (Days) 11 12 TOTALS Incidence 0 0%) 0 0%) 5 (100%) 4 ( 60%) ObServed 0 0 27 33 Mean onset (Days) 11 II Piloerection Incidence 1 ( 20%) 2 ( 40%) Observed 1 3 Mean onset (Days) 15 12 TOTALS Incidence 0 0%) 0 0%) 1 ( 20%) 2 ( 40%) Observed 0 0 1 3 Mean onset (Days) 15 12 Twitching

moderate Incidence 1 ( 20%) Observed 1 Mean onset (Days) 13 slightIncidence 5 (100%) 4 ( 60%) Observed 27 46 Mean onset (Days) 14 12 TOTALS Incidence 0 0%) 0 0%) 5 (100%) 4 ( 60%) Observed 0 0 27 47 Mean onset (Days) 14 12-i ::;r iii· c. 0 n c: 3 (l)

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## '" Ul Print Date: 06-Nov-2006 print Time: 10:07:52 OBSERVATIONS REPORT

- INCIDENCE Table IA Study: 11.:=---­

_____---1 Sex: Kale Page 6 I
Treatment GrOUp 0 1 2 3 Dose o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day An1.mal Count 5 5 5 5 Sacrificed scheduled Incidence 5 (100%) 5 (100%) 5 (100%) 4 ( 80%) Observed 5 5 5 4 Mean onset (Days) 29 29 2!/ 29 TOTALS Incidence 5 (100%) 5 (100%) 5 (100%) 4 ( 80%) Observed 5 5 5 4 Mean onset (Days) 29 29 29 29BASF

# -'I....-

____...J Print Date: 06-Nov-2006

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# '" ;;: Print Time: 10:24:50 OBSERVATIONS REPORT

- INCIDENCE Table IA study:1 Page 7 Sex:**F~ema~~l~e----------~** Treatment Group 

### o 1 2 3 Dose 

## o mq/kg bw/day 10 mq/kg bw/day 30 mq/kg bw/day 60 mq/kg bw/day Animal Count 5 5 5 5 Convulsions

clonic

moderate Incidence 4 ( 80%) Observed 6 Mean onset (Days) 2 clonic

severe Incidence 2 ( 40%) Observed 2 Mean onset (Days) :3 epi1eptoid(behavior when removed from cage)

moderate Inc1dence 1 ( 20%) Observed 1 Mean onset (Days) 4 epileptoid(behavior when removed from cage)

severe Incidence 1 ( 20%) Observed 2 Mean onset (Days) 4 tonic

severe Incidence 2 ( 40%) Observed 3 Mean onset (Days) 6 tonic

slight Inc1dence 1 ( 20%) Observed 1 Mean onset (Days) 4 TOTALS Incidence 0 0%) 0 0%) 

### a 0%) 4 ( 80%) Observed 0 0 

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### cJ" :J C" '< 5' (J) fA "0 0 :J II> 0 ill BASF -I Print Date: 06-Nov-2006 Print Time: 10:24:50 OBSERVATIONS REPORT

- INCIDENCE Table IA page a Sex: Treatment Group 0 1 2 3 Dose 

## o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 60 mg/kg bw/day Animal Count 5 5 5 5 General observation

Nothing abnormal detected Incidence 5 (100%) 5 (100%) 5 (l00%) 5 (100%) Observed 130 130 130 93 Mean onset (Days) 0 0 0 0 TOTALS Incidence 5 (100%) 5 (100%) 5 (100%) 5 (100%) Observed 130 130 130 93 Mean onset (Days) 0 0 0 0 Hyperesthesia

moderate Incidence 4 ( 80%) Observed 7 Mean onset (Days) 2

severeIncidence 2 ( 40%) Observed 3 Mean onset (Days) 5 slight Incidence 5 (100%) Observed 6 Mean onset (Days) 1 TOTALS Incidence 0 0%) 0 0%) 0 0%) 5 (100%) Observed 0 0 0 16 Mean onset (Days) 2 Hyperexcitability

moderate Incidence 4 ( aO%) Observed 7 Mean onset (Days) 2

severeIncidence 2 ( 40%) Observed 3 Mean onset (Days) 5--l ::l" iii' c. 0 (') c 3 (I) 

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- Print Date: 06-Nov-2006 Print Time: 10:24:50 OBSERVATIONS REPORT

- INCIDENCE Table IA Study: 

# I Page 9 

### Sex: F
Treatment Group 0 1 2 3 Dose o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 60 mg/kg bw/day Animal Count 5 5 5 5

slightIncidence 5 (100%) Observeci 6 Mean onset (Days) 1 TOTALS Incidence 0 0%) 0 0%) 0 0%) 5 (100%) Observed 0 0 0 16 Mean onset (Days) 2 Nose, discharge

reci

moderate Incidence 1 ( 20%) Observed 1 Mean onset (Days) 5 TOTALS Incidence 0 0%) 0 0%) 0 0%) 1 ( 20%) Observed 0 0 0 1 Mean onset (Days) 5 Piloerection Incidence 2 ( 40%) Observed 5 Mean onset (Days) 6 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed 0 0 0 5 Mean onset (Days) 6 Salivation

moderate Incidence 4 ( BOlO) Observeci 6 Mean onset (Days) 5

severeIncidence 1 ( 20%) Observeci 3 Mean onset (Days) 3 

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# " '" 0 til BASF Print Date: 06-Nov-2006 

# -1 Print Time: 10:24:50 OBSERVATIONS REPORT

- INCIDENCE Table IA Study: Page 10 

# I Sex: Female
Treatment Group 0 1 2 3 Do

..e o mq/kg bw/day 10 mq/kg bw/day 30 mq/kg bw/day 60 mq/kg bw/day Animal Count 5 5 5 5

slight Incidence 2 ( 40%) Observed 2 Mean onset (Days) 7 TOTALS Incidence 0 0%) 0 0%) 0 0%) 4 ( BO%) Observed 0 0 0 11 Mean onset (Days) 5 Skin

palene

....

moderate Incidence 2 ( 40%) Observed 2 Mean on..et (Days) 5 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed. 0 0 0 2 Mean onset (Days) 5 Twitching

moderate Incidence 3 ( 60%) Observed 3 Mean onset (Days) 7

severeIncidence 3 ( 60%) Observed 7 Mean onset (Day

..) 3 slight Incidence 5 (100%) Observed. 14 Mean onset (Days) 3 TOTALS Incidence 0 0%) 0 0%) 0 0%) 5 (100%) Observed 0 0 0 24 Mean onset (Days) 4-I ::r 

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# " i!l ::> Ul Print Date: Print Time: OBSERVATIONS REPORT

- INCIDENCE Table Page Sex: Treatment Group 

### o 1 2 ::I Dose o mg/kg bw!day 10 mg/kg bw/day 30 mg/kg bw!day 60 mg/kg bw/day Animal Count 5 5 5 5 Found dead Incidence 1 ( 20%) Observed 1 Mean onset (Days) 7 TOTALS Incidence 

### o 0%) 

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## o 1 Mean onset (Days) 7 06-Nov-2006 10:24:50 IA 11--I :::T 00' a. 0 c 3 

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## '" 0 Ul Print Date: Il-Dec-2006 Print Time: 11:43:58 OBSERVATIONS REPORT

- INCIDENCE Table IA Study: 

# I Page 12 Sex: Female
Treatment Group 0 1 2 3 Dose 

### o mg/kg bw/day 10 mg/kg bW/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5 Anogenital region

smeared with urine

severe Incidence 1 ( 20%) Observed 15 Mean onset (Days) 9 TOTALS Incidence 0 0%) 0 0%) 0 0%) 1 ( 20%) Observed 0 0 0 15 Mean onset (Days) 9 Convulsions

tonic

moderate Incidence 1 ( 20%) Observed 1 Mean onset (Days) 28 tonic

severe Incidence 1 ( 20%) Observed 1 Mean onset (Days) 8 tonic

slight Incidence 1 ( 20%) Observed 1 Mean onset (Days) 19 TOTALS Incidence 0 0%) 0 0%) 0 0%) 3 ( 60%) Observed 0 0 0 3 Mean onset (Days) 18 General observation

Nothing abnormal detected Incidence 5 (100%) 5 (100%) 5 (100%) 4 ( 80%) Observed 335 335 255 109 Mean onset (Days) 8 8 8 8**~** ::T iii" 0. 0 (") c 3 <I> ;:l. iii" g: <I> "0 

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## 3 iii" VI 0" ::J 0­ '< g: <I> VI "0 0 ::J VI 0 Ci1 Print Date: 11-Dec-2006 Print Time: 11:43:58 OBSERVATIONS REPORT

- INCIDENCE Table IA study: 

# I Page 13 Sex: Female
Treatment Group 0 1 2 3 Dose 

### o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5
TOTALS Incidence 5 (100%) 5 (100%) 5 (100%) 4 ( 80%) Observed 335 335 255 109 Mean onset (Days) 8 8 8 8 Hyperactivity Incidence 1 ( 20%) 1 ( 20%) Observed 18 1 Mean onset (Days) 20 19
TOTALS Incidence 0 0%) 0 0%) 1 ( 20%) 1 ( 20%) Observed 0 0 18 1 Mean onset (Days) 20 19 Hyperesthesia

slightIncidence 5 (100%) 1 ( 20%) Observed 10 1 Mean onset (Days) 21 20
TOTALS Incidence 0 0%) 0 0%) 5 (100%) 1 ( 20%) Observed 0 0 10 1 Mean onset (Days) 21 20 Hyperexcitability

moderate Incidence 4 ( 80%) 4 ( 80%) Observed 6 20 Mean onset (Days) 13 11

severeIncidence 1 ( 20%) 2 ( 40%) Observed 1 11 Mean onset (Days) 15 10 slightIncidence 5 (100%) 3 ( 60%) Observed 18 28 Mean onset (Days) 12 10-i :::r iii' c. 0 n c:: 3 (!)**~** iii' 

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### :T (!) w "0 0 :::J w 0 iil Print Date: 11-Deo-2006 Print Time: 11:43:5B OBsERVATIONS REPORT

- INCIDENCE Table IA study:

# I Page U Sex: Female Treatment Group 0 1 2 3 Dose 

## o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5 TOTALS Inoidenoe 0 0%) 0 0%) 5 (100%) 4 ( BO%) Observed 0 0 25 59 Mean onset (Days) 13 10 Lateral position Incidence 1 ( 20%) Observed 1 Mean onset (Days) 11 TOTALS Incidence 0 0%) 0 0%) 0 0%) 1 ( 20%) Observed 0 0 0 1 Mean onset (Days) 11
Nose I discharge

red

severe Incidence 2 ( 40%) Observed 9 Mean onset (Days) 10 red

slight Incidence 1 ( 20%) Observed 2 Mean onset (Days) 9 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed 0 0 0 11 Mean onset (Days) 9 Piloerection Incidence 2 ( 40%) Observed 12 Mean onset (Days) 10 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed 0 0 0 12 Mean onset (Days) 10-I 03" iii" 0. 0 n <: 3 m ;:l iii" 

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# L Print Date: 11-Dec-2006 Print Time: 11:43:58 OBSERVATIONS REPORT

- INCIDENCE Table IA Page 15

# ~::L~"" Treatment Group 0 1 2 3 Dose 

## o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5 Reduced general**condit~on**

severe Incidence 2 ( 40%) Observed Mean onset (Days) 11 

# " slight Incidence 1 ( 20%) Observed 3 Mean onset (Days) 11 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed 0 0 0 7 Mean onset (Days) 11 Salivation

moderate Incidence 2 ( 40%) Observed 4 Mean onset (Days) 10

severe Incidence 1 ( 20%) Observed 2 Mean onset (Days) 8 slightIncidence 1 ( 20%) Observed 2 Mean onset (Days) 8 TOTALS Incidence 0 0%) 0 0%) 0 0%) 2 ( 40%) Observed 0 0 0 8 Mean onset (Days) 9 Twitching

moderate Incidence 

# " ( 80%) 4 ( 80%) Observed 6 18 Mean onset (Days) 16 9-j :T in" a. 0 0 c 3 

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"0 

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# L Print Date: 11-Dec-2006 Print Time: 11:43:58 OBSERVATIONS REPORT

- INCIDENCE Table IA study:

# be Page 16 Sex; Fema e Treatment Group 0 1 2 3 Dose 

## o mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day Animal Count 5 5 5 5

severeIncidence 1 ( 20%) 2 ( 40%) Observed 1 8 Mean onset (Days) 26 10 slightIncidence 5 (100%) 3 ( 60%) Observed 35 44 Mean onset (Days) 12 10 TOTALS Incidence 0 0%) 0 0%) 5 (100%) 4 ( 80%) Observed 0 0 42 70 Mean onset (Days) 15 9 Sacrificed moribund Incidence 1 ( 20%) Observed 1 Mean onset (Days) 11 TOTALS Incidence 0 0%) 0 0%) 0 0%) 1 ( 20%) Observed 0 0 0 1 Mean onset (Days) 11 Sacrificed scheduled Incidence 5 (100%) 5 (100%) 5 (100%) 3 ( 60%) Observed 5 5 5 3 Mean onset (Days) 29 29 251 251 TOTALS Incidence 5 (100%) 5 (100%) 5 (100%) 3 ( 60%) Observed 5 5 5 3 Mean onset (Days) 29 29 29 29Print Date: 06-Nov-2006 Print Time: 11:30:30

-I :;;; (ii" 0. 0 n c 3 <1l 

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## 3 (ii" (/) 0" ::> a­ '< 5' (!) (f) "0 0 :::J (f) 0 i.il FOOD CONSUMPTION Table IA study:

# L-L

___---' Page 17 Food Cons.

g/anm/day

Day 7



### o Male, 0 mg/kg bw/day Mean 20.1 SO 1.4 N 5 %dev Male, 10 mg/kg bw/day Mean 19.8 SO 1.7 N 5 %dev -1.5 Male, 30 mg/kg bw/day Mean 18.1 SO 1.1 N 5 %dev -9.8 Male, 60 mq/kg bw/day Mean 14.2** SO 3.0 N 4 %dev -29.4 Key: D 

### = Dunnett's test, Two-sided, • p 

### <= 0.050, •• P 

### <- 0.010 Experimental Unit

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## s: <l> CJ) "0 0 ::l CJ) 0 iii Print Date: 06-Nov-2006 Print Time: 11:01:21 FOOD CONSUMPTION Table IA studyfL_---

__________

## ~ Page 18 Food Cons.

g/arun/day Day 14 D Male, 0 mg/kg bw/day Mean 21.5 SO 2.0 N 5 %dev Male, 10 mg/kg bw/day
Mean 20.1 SD 1.5 N 5 %dev -6.1 Male, 30 mg/kg bw/day Mean 19.8 SD 1.6 N 5 '!!dev -7.6 Male, 45 mg/kg bw/day Mean 18.9 SD 3.1 N 4 %dev -11.9 Food Cons. g/arun/day

Day 21 D

21.6

1.3 5

21.4

1.8 5 -0.9

20.6

1.1 5 -4.9

21.0

2.3 4 -3.1 Food Cons. g/anm!day Day 28 D

20.4

1.6 5

19.7

1.7 5 -3.2

19.7

1.2 5 -3.3

19.7 2.1 4 -3.2 Key: D 

# = Dunnett's test, Two-sided, 

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### en print Date: 06-Nov-2006 Print Time: 11:31:38 FOOD CONSUMPTION Table IA Study:

# I Page 19
Food cons.

g/anm/day

Day 7

0 Female, 0 mg/kq DW/day
Mean 16.7 SD 1.3 N 5 %dev Female, 10 mg/kg DW/day
Mean 16.0 SO 1.1 N 5 %dev -4.1 Female, 30 mg/kg DW/day
Mean 13,7** SD 0.6 N 5 %dev -18.0 Female, 60 mq/kg DW/day Mean 12.0*· SD 1.0 N 5 %dev -2B.2 Key: D = Dunnett's test, TWo-sided, * p 

### <= 0.050, 

# *. P 

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## :a (i)" 5' <l> 

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### ~ :::! "0**~** 3 iii" !i!" 0 :::! c­ '< 5' 1II en "0 0 :::! en Q en FOOD CONSUMPTION Print Date: Print Time: Table Page 06-Nov-2006 11:02:12 IA 20 Food Cons.

g/anm/day Day 14 D Food Cons. g/anm/day Day 21 D Food Cons. g/a:run/day Day 2B D Female, 0 mg/kg bW/day Mean SD N %dev 17.S 1.7 5 17.1 1.2 5 16.3 0.9 5
Female, 10 mg/kg bW/day
Mean SD N "'<lev 15.4* 1.2 5 -12.3 lS.6 1.S 5 -B.S lS.4 0.5 5 -S.3 Female, 30 mg/kg bW/day Mean SD N %dev lS.7 0.3 5 -10.7 15.1 2.2 5 -11" 5 15.1 0.7 5 -6.9 Female, 45 mg/kg bW/day Mean SD N %<lev 14 .9* 0.7 3 -15.3 16.7 3.0 3 -2.4 15.4 2.6 3 -5.2 Key: D 

### = DUnnett's test, Two-sided, Experimental Unit 

# =Animal * p 

### <= 0.050, 

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### ;! iE' 0. 0 ('I c: 3 <1> 

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## '" '" Print Date; 06-Nov-2006 Print Time: 11:32:39 WATER CONSUMPTION Table IA Study: Page 21

water COns.

g/arun/day

Day 7

D Male, 

## o mg/kg bw/day Mean 1B.3 SD 2.2 N 5 %dev Male, 10 mg/kg bw/day Mean 20.1 SD 1.8 N 5 %dev 9.6 Male, 30 mg/kg bw/day
Mean 19.1 SO 2.0 N 5 %dev 4.1 Male, 60 mg/kg bw/day Mean 20.9 SO 2.4 N 4 %dev 13.9 Key: 0**~** Dunnett's test, Two-sided, 

## * p**<~** 0.050, 

### ** p <= 0.010 Experimental Unit 

# =AnimalPrint Date: 06-Nov-2006 Print Time: 11:02:45

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# '" Ul WATER CONSUMPTION Table IA study: ,-- 

# =:J Page 22
water Cons,

g/arun/day

Day 14 D Water Cons. g/anm/day

Day 21 D water Cons. g/anm/day

Day 28 D Male, 

### o mg/kg l::>w/day Mean SD N 20.8 3.5 5 18.7 0.7 5 18.9 2.2 5 %dev Male, 10 mg/kg bw/day Mean 21.6 21.3 19.4 SD 2.6 3.1 3.2 N 5 5 5 %dev 3.8 14.0 3.0 Male, 30 mg/kg bw/day Mean 17.9 19.8 19.9 SD 3.4 2.0 4.3 N 5 5 5 %dev -13.6 6.3 5.2 Male, 45 mg/kg bw/day Mean 19.0 19.5 20.4 SD 2.1 2.8 2.4 N 4 4 4 %dev -8.5 4.4 8.1 Key: D Dunnett s test, Two-sided, 

## * p 

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# ~ WATER CONSUMPTION Print Date: Print Time: Table Page 06-Nov-2006 11:33:37 IA 23
water Cons.

g/anm/day

Day 7

D Female, 0 mg/kg bw/day Mean SO N %dev 15.1 2.1 5
Female, 10 mg/kg bw/day Mean SD N %dev 17.6 2.9 5 16.4 Female, 30 mg/kg Qw/day
Mean SD N %dev 16.6 1.6 5 9.8 Female, 60 mg/kg Qw/day Mean SD N %dev 20.7** 2.7 5 36.9 Key: D 

### = Dunnett·s test, Two-sided, Experimental Unit 

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# I**~** WATER CONSUMPTION Print Date: Print Time: T;,ble Page 06-Nov-2006 11;03:33 IA 24 Water Cons.

g/anm/day

Day 14 D Water Cons. g/anm/day

Day 21 D Water Cons. g/anm/day

Day 28 D Female, 0 mg/kg bw/day
Mean SD N %dev 13.6 3.1 5 15,1 1.8 5 15.2 0.7 5
Female, 10 mg/kg bw/day Mean SD N %dev 17.3 3.2 5 27.3 16.0 3.4 5 6.1 17.9 2.0 5 17.9 Female, 30 mg/kg bw/day Mean SD N %dev 17.2 1.9 5 26.4 16.4 2.6 5 8.6 18.9* 2.2 5 24.6 Female, 45 mg/kg bw/day Mean SD N %dev 17.0 2.7 3 24.7 18.1 2.3 3 20.1 16.9 3.8 3 10.8 Key; D Dunnett's test, Two-sided, Experimental Unit 

### = Animal " P 

### <= 0.050, 

## ** P 

### <= 0.010-I :::r <ii' 0­ 0 t: 

# " 3 m ;3. <ii' 

## s: m "0 

# a "0 m ::1 '< 

### S. 

## s: m U) "0 0 ::J 0 

# '" Cil fi) "0 m Q, 

### "'D :::!l !ll m 0­ 

## co 0 

# ro ::J "0 

## co !l) --,J <0



# - W (1) 0
t: U) (1) 0'> 

## co CT '< ;? ::T**~** "0 !l) ::1 (ii' U) @ .0 t:**~.** IJ> 

### ="

## ~ ::J 

### lil 

## 3 <ii' IJ> 

### c' ::l CT '< 

## s: (1) til "0 0 ::l U) 0 iil Study:

# I**~** BODY Wl!.IGHT Print Date: Print Time: Table Page 06-Nov-2006 11:34:22 IA 25
Body Weight g Day 0

D Body Weight g Day 7

D Male, 0 mg/kg bW/day Mean SD N %dev 169.5 7.9 5 214.5 9.9 5
Male, 10 mg/kg bw/day Mean SO N 'Odev' 166.6 6.9 5 -0.5 204,2 9.9 5 -4.6 Male, 30 mg/kg bw/day
Mean SD N %dev 169.9 6.9 5 0.2 197.9 9.5 5 -7.7 Male, 60 mg/kg bW/day Mean SO N %deV' 167,7 6.9 5 -1.1 194.9** 16.6 4 -13.9 Key: D 

### = Dunnett's test, Two-sided, Experimental Unit 

## = Animal * p 

### <= 0.050, ** P 

### <= 0.010-I ::r iii" c. 0 <> c: 3 (l) 3­ iii" ':f (l) "0 

# a "0 (l) ::l­ 

# "" 

# a ':f (l) CII "0 0 ::J CII 0 in (j) "0 (l) Q" 

### "'U :::!l Q) (1) Q. (Q 0 CD ::J "0 

### ex:> 0> (0 

### ex:> (1) 0 

### ,,­ c 

# - (.,.) U> 0') (1) 

### ex:> cr 

# "" 0 ':f 9; "0 0> 

### ;:;, CI> U> Cil D C**~.** U> 

# g**~** :l "0 (1) 

## 3 en" U> 

## cr ::J cr 

# "" ':f (l) U> 

## "8 ::J U> 0 

### in study: BODlt" WEIGlIT Print Date: Print Time: Table Page 06-Nov-2006 11:04:03 IA 26
Body Weight g Day 14 D Body weight g Day 21 D Body weight g Day 28 D Male, 0 mg/kg bw/day Mean SD N %dev 247.5 13.5 5 278.0 19.0 5 293.7 21.0

5
Male, 10 mq/kg ow/day Mean SD N %dev 238.5 16.5

5 -3.6 265.6 20.9

5 -4.4 279.5 25.6

5 -4.8 Male, 30 mq/kg bw/day Mean SD N %dev 230.3 14.9 5 -6.9 256.6 13.9

5 -7.7 271 8 18.5 5 -7.5 Male, 45 rng/kg bw!day Mean SD N 'tdev 226.1 22.9

4 -8.6 256.7 23.6

4 -7.6 275.4 29.2

4 -6.3 Key: D 

### = Dunnett's test, Two-sided, Experimental Unit 

## = Animal 

### * p 

### <= 0.050, 

### ** p 

### <= 0.010-I :s iii' 0. 0 n c: 3 ro 

### ;a. iii' 5' (!) -0 

# a**~** '< 

### 9­ 5' ro -0 

## '" 0 :::I 0 

# '" (il

## ~ (!) n 

### "'0 In ::;; tu 0. 

## co 0 (l) :::I 0.:> "0



# - tn (0 

# '" In 0 ::­ c: W 

## en 

# '" ro 0.:> 0­ '< 0 5' 

### !!l -0 

## '" :l 

### ro' 

## '" 0; .0 c: 

### @' til**~** 

## ::: ro :::I "0 

## 3 (!) iii' til 0' :::I 0­ '< 5' (!) til 

## "'5 :::I til 0 (il 

# r
Study:

# I I I BODY WEIGHT Print Date: Print Time: Table Page 06-Nov-2006 11:34:43 IA 27
Body Weight 9 Day 0

D Body Weight 9 Day 7

D Female, 0 mg/kg bW/day Mean SD N %dev 135.5 9.5 5 161.9 16.0 5
Female, 10 mg/kg bw/day Mean SD N %dev 137.9 7.1 5 1.7 154.9 10.0 5 -4.3 Female, 30 mg/kg bw/day Mean SD N %dev 138.2 9.4 5 1.9 151.7 6.9 5 -6.3 Female, 60 mg/kg bw/day Mean SD N 'lsdev 139.4 10.1 5 2.1 151.3 12.4 5 -6.5 Key: D 

# = Dunnett's test, Two-sided, Experimental unit = Animal 

## * p 

### <- 0.050, 

## ** P 

### <= 0.010-; ::J" 

## or 0. 

# g 3 (I) 

### ;:. iii' :T (I) 'C 

# a 'lil**~** 

### So :T CD 

## 15 

# '"
:J**~** iii [j) 

## 'lil "U

### ~ !l)**~** 

## co 0 

### CD :J 

## c.oiil O'lil 

# o**~** 

# -

. W §i (j) (I)**co~**

### ~ g; 

## iil ;:l. (j) (J) CD .n c**~.** 

## "'**~**

# ~ 'lil 

## 3 00' 

## o 

# "' :J cr '< S' (I) 

# '" 15 :J

# ~ Study:L­

_____________ BODY WEIGHT Print Date: Print Time: Table Page 06-Nov-2006 11;04:48 IA 28
BOOy Weight g oay 14 

### o Body Weight g Oay 21 

### o Body Weight g Day 28 

### o Female, 0 mq/kg oW/day Mean SO N %cIev 173.1 12.0 5 187.4 12.7 5 194.4 11.4 5
Female, 10 mq/kg bW/day Mean SO N %cIev 172.4 12.1

5 -0.4 183.2 9.8 5 -2.3 191.9 7.1 5 -1.3 Female, 30 mq/kg bW/day Mean SD N %cIev 166.5 8.1 5 -3.8 179.9 5.7 5 -4.0 185.7 6.7 5 -4.5 Female, 45 mg/kg ow/day
Mean SD N 'Odev' 165.4 16.5 3 -4.4 180.1 21.6

3 -3.9 189.3 26.2

3 -2.6 Key: 0**~** Dunnett's test, Two-siQed, 

## * p 

### <= 0.050, 

### ** P 

### <= 0.010 Experimental unit = Animal-i ::T iii' a. 0 (') c: 3 (1) 

## a iii' 5' (1) '0

## ~ (1) ;J. '< 9, 5' (1) (Jl '0 0 ::l 

## '" 0 iil Ii> '0 (1) (') 

### "'U (1) ::;; Q) a. to 

### g CD t\) 

# c:o '0



# ..... <0 (1)



# .... c: W 0') (1) 

# '" 0:> a '<

### ~ !!; 

## 'B ;J. iif !Jl 

### iiJ Ll c:**~.** 00 

### ;Ji

### ~ ::l '0 (1) 

## '3 iii' 00 0' ::l a '< 5' ro 00 '0 0 :::> 00 Q (Jl Print Date: 06-Nov-2006 Print Time: 11:35:29 BODY WEIGHT CHANGE Table IA study:L-

__________**~** Page 29

BW change g Day 7

D Male, 0 mg/kg bw/day
Mean 45.0 SD 7.5 N 5 "'d.ev Male, 10 mg/kg bw/day Mean 35.6 SO 4.4 N 5 "'dev -20.9 Male, 30 mg/kg bW/day Mean 28.0"* SD 8.0 N 5 "'dev -37.7 Male, 60 mg/kg bw/day Mean 17.7"· SD 9.1 N 4 %dev -60,8 Key: 0 

## = Dunnett's test, Two-sided, 

## * p <= 0.050, 

### ** P <= 0.010 Experimental Unit 

# = Animal

## I Study:
...; ::r iii' a. 0 <> c 3 <t> ;:1i. (ii' Male, 

## s: <t> l1ean 1:1 

# a SD 1:1 <t> N ::I. '< %<lev Q. 

## s: l1ale, <t> 1:1 

# '" 0 l1ea.n ::> SD 

# '" N

## ~ (i) %<lev**~** l1ale, 

### "'0 Q. 

# "" OJ**~** (Q 0 l1ean CD ::> SD 1:1 

## co III N <0 N CD 

# - W %dev 

### 0 

### .,.... c l1ale, CD 0'> 

# '" cr 

## co '< l1ea.n 9­ SD !!; ::r N 1:1 %dev ;}, III <t> Key: 

# '" (il .0 C 

## ;' 

# '"**~** ;:;.. (b ::> 1:1 CD 

## 3 iir 

# '" cf ::> CT '< 

## s: CD 

## -g 

# '"
::> 0 

# '" iil BW change g Day 14 D 0 mg/kg hw/day 77.9 a.1 5

10 mg/kg hW/day69.9 10.6

5 -10.3 30 mg/kg hw/day60 4 13.4 5 -22.6 45 mg/kg hw!day59.0 15.5 4 -24.4 BODY WEIGHT CHANGE Print Date; Print Time; Table Page 06-Nov-2006 11:05:20 IA 30

BW change g Day 21 D 

### BW change g Day 2a D

10a.5

13.3

5 124.2 16.a

5

97.0 15.2 5 -10.5 110.9 20.0

5 -10.7

86.7 14.6

5 -20.1 101.8 la.6

5 -18.0

a9.6 16.6 4 -17.4 108.2 23.0

4 -12.9 D = Dunnett·s test, Two-sided, 

## * p 

### <= 0.050, 

### ** P 

### <= 0.010 Experimental unit = Animal____ 

# J Print Date: 06-Nov-2006 Print Time: 11:35:45

I BODY WEIGHT CHANGE Table IA

-I ::J" 

## or a. 

## g c:: 3 <I>**~** (i;' g: <I> "0 i3 "0 <I> ::l. '< 

# a g: <I> (J) 

## "8 :J 

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### n, "1J :;, <I> 

### ru a. 

### ee 0 CD :J 

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### a~ c:: W fJ) 0) <I> 

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## 1il d-, <I> (J) <il .n c::**~.** fJ) :E ::!, 

### $' ::> "0 (I) 

## 3 (i;' 

# '" 5' ::> c:r '< g: <I> (J) "0 0 

# a ::> fJ) Stuc1y:

l...1

### ~ Page 31

BW change g Day 7

D Female, 0 mg/kg bw/c1ay Mean 26.4 SD e.3 N 5 %c1ev Female, 10 mg/kg bw/c1ay Mean 17.1* SD 4.1 N 5 %dcv -35.3 Female, 30 mg/kg bw/c1ay Mean 13.5** SD 3.0 N 5 %c1ev -49.7 Female, 60 mg/kg bw/c1ay Mean 12.9** SD 3.1 N 5 %c1ev -50.9 Key: D = Dunnett's test, Two-sic1ec1, 

## * p <= 0.050, 

### ** P <= 0.010 Experimental Unit = Animal-1 ::r 

### iir a. 0 0 c 3 til**~** 00' 

## s: til 

# " 

# a 

# " til

### ~ Q. 

## s: (l) 

# '" " 0 ::::l 0 

## '" iil 

# "' (l) 

# " 0 

### -U 3i til til a. (Q 0 (I) ::::l <.0 

# " III .j:>. <0



# -. <I> 

# ­ 0 W U) c CJ) <I> 

### a:> C" '< 0 

## s:**~** 

## "E 

## a <I> Stud.y:

# I

Female, Mean SO N %dev Female, Mean SO N %dev Female, Mean SD N %d.ev Female, Mean SD N %dev BW change g Day 14 D 0 mg/kg bw/day 37.6

9.8 5

10 mg/kg bw/day 34.6

6.3 5 -8.1 30 mg/kq bw/day 28.4

6.7 5 -24.5 45 mg/kg bw/day 29.9

8.3 3 -20.5 BW change g Day 21 D BW change g Day 28 0 BODY WEIGHT CHANGE Print Date: Print Time: Table Page 06-NOV-2006 11:06:03 IA 32

51.9

8.2 5 58.9 6.9 5

45.3

6.5 5 -12.8

41.8

3.9 5 -19.5 54.0 4 5 5 -8.4

47.6

4.9 5 -19.3

44.5

8.6 3 -14.2 53.8 13.3 3 -8.7 Key: 0 

### = Dunnett's test, Two-sid.ed., 

## * p 

### <= 0.050, 

### ** P 

### <= 0.010 

## '" iil Experimental unit = Animal .0 C 

# a'
U)**~** 

# g'
::::l**~** 

## 3 00' 

# '" o· ::::l C" '< 

## s: <I> U) 

## "8 ::::l 

### g 

## '" '"

## I -I :r iir 0­ 0 (") c 3 co 

### ;a 1i>' 5' co "0 

# a "0 co 

## q g, 5' co "0 

## '" 0 :::! 0 

# '" til u; "0 co 

### 1::J (")

### ~ III 0­ (0 0 ('I) :::! "0 <0 Q) CJ'1 (Q <D 0 

## .... 

# -. w c 0) co 

# '" CXl**~** 0 5' 

### '!! "0 Q) ;::L 

### co' 

# '"**~** c 

# a' (/) ;<,i ;:: <D :J 

## Ttl 

## 3 w' U'1 0' :J**~** 5' <D 

## '" II :J (/) 0 (6 Print Date: 06-Nov-2006 Print Time; 11;53;44 Study:

# I
- ..**~** FOOD EFFICIENCY Table Page IA 33 Food eff 7 Day 7

D Male, 0 mg/kg bw/day Mean SD N 32,0 4,7 5 Male, 10 mg/kg bw/day Mean SD N 25,7 1.7 5 Male, 30 mg/kg bw/day
Mean SD N 22,2* 6.7 5 Male, 60 mg/kg bw/day
Mean SD N 17.0** 5.2 4 Key: D 

## = Dunnett·s test, Experimental Unit Two-sided, Animal * p**<~** 0.050, 

### ** P 

### <= 0,010

# [ print Date: OS-Nov-200S Print Time: 11:06:33

-1 ::r 0;' 0. 0 c 3 

# "
<1) 

### 2­ 0;' 5' <1) -0 (3 -0 <1) :::l. '<**~** 5' <1) -0 0 

# '"
::I 

### :g iil "iii' -0 <1) () :a; OJ 0. ""'CJ <1) (C 0 :J CD**(O~ O>~**  0 

## .... 

## -.. C VJ If> 0> <1) 

### ex> 0­ '< 0 5'**~** -0 

## a 

## '" <1) If> 

### <il .0 c**~.** 

# i**~** :J -0 <1) 

## 3 0;' If> 0' :J 0­ '< 5'­ CI> fh -0 0 

# " 0 

# '" iil FOOD EFFICIENCY Table IA study: 

### IL...

____ Page 34 Food eff 7 Food eff 7 Food eff 7

Day 14 Day 21 Day 28 D D D Male, 

## o mg/kg bw/day Mean 21.9 20.1 10.8 SD 5.2 3.4 3.6 N 5 5 5 Male, 10 mg/kg bw/day Mean 24.4 17.9 9.6 SD 4.8 2.2 3.2 N 5 5 5 Male, 30 mg/kg bw/day Mean 23.1 16.2 10.9 SO 

### 4.9 3.3 3.2 N 5 5 5 Male, 45 mg/kg bw/day Mean 31.3> 21.0 13.2 SD S.l 2.7 5.2 N 4 4 4 Key: D 

### = Dunnett's test, Two-sided, > p 

### <- 0.050, •• P 

### <= 0.010 Experimental Unit = Animal-I ::r iii" 0. 

### g c 3 (1) 3­ iii" ::;: (1) "0 (3 "0 (1)**~** 

# a ::;: (1) (I) "0 0 :::l 13 

### en 'ijJ "0**~** 

### "'C

### ~ Q) 0. CO 0 CD :::l <0 (Q


## ...... "0 

# '" (1) 

### 0 "::-' c 

# - W (1) 0'> 

# '" <Xl C" '< 0 

### ::;:**~** 

### 1i5 :::l. 

## m**~** c**~.** en**~** 

# "'" ro :::l "0 (1) 

## '3 iii" en 

## o· :::l C" '< 

### ::;: (1) tJ> "0 0 :J tJ> 0 

### en 

# [ 

# I Print Date: 06-Nov-2006 Print Time; 11;38:49 FOOO EFFICIENCY Table IA Study: Page 35 

# I I Food eff 7 Day 7

0 Female, 0 mg/kg ow/day Mean 22.3 SO 5.5 N 5 Female, 10 mg/kg oW/day Mean 15.2* SD 3.5 N 5 Female, 30 mg/kg ow/day Mean 14.2* SO 3.5 N 5 Female, 60 mg/kg ow/day Mean 15.4* SD 3.1 N 5 Key; D Dunnett's test, Two-sided, 

### * p <= 0.050, 

### ** P <= 0.010 Experimental Unit Animal-I ::T iii' a. a 

# '" (") 3 <1>::liii' g. <1> "0 

# a -g ;J. '< Q, g. <1> t.fl 

### ""8 :l t.fl a iil 

### u;­ "D <1> Q, 

### -0 ::!l Q) <1> a. 10 a CD :l 

## (Cal**o:>~**


# .... 0



# ..... W 

# '" (j) <1> 0:> '< 

# '" CT

### ~ 

## al**~** ;J. (6' t.fl 

### til .a 

# '"
en**~**

### ~ :l "D <II =3 iii 

## '" 0 :l CT '< g. <II t.fl "D a :l en 0 iil Study: 

# I
FOOD EFFICIENCY Print Date: Print Time: Table Page 06-Nov-2006 12:06:06 IA 36 Food eff 7 Food eff 7 Food eff 7

Day 14 D Day 21 D Day 28 D Female, 0 mg/kg ow/day Mean SD N 9.2 B.3 5 11.7 6.3 5 6.1 3.2 5 Female, 10 mg/kg bW/day Mean SD N 16.4 6.3 5 9.7 6.7 5 8.0 4.5 5 Female, 30 mg/kg bW/day Mean SD N 13.6 4.6 5 13.0 5.6 5 5.4 3.6 5 Female, 45 mg/kg ow/day Mean SD N 15.6 5.6 3 11.9 9.8 3 8.2 7.0 3 Key: D - Dunnett·s test, Two-sided, Experimental Unit 

## =Animal • P 

### <= 0.050, •• P 

### <= 0 010**~** ::r (ii' 

### Project No.

# C**~** c. 0 (') c: 3 male animals (» 

## a

# ~I HOME CAGE OBSERVATION -0 

# a posture -0 (» • animal is sitting orlying :::I. '< • animal is staying or moving 

### !2. • squatting posture 9' (» • abdominal position IJ> abdominal position with splayed limbs -0 C ::! lateral position IJ> 0 • oblique head posture (ij • opisthotonus Vi" "0 (» 

### tremors 

### lJ 

# a
· no tremors (0 c. OJ iii' • slighttremors (J) 

## g • moderate tremors 

## (,O-g severetremors (,Of!:> (» 

## Q.::-­ convulsions • no convulsions VJ en c: 0') (» • slightconvulsions (Xl 0­ '<
- moderate convulsions 0 · severe convulsions 

### !!l 9'
"0 

### abnormal movements :::I. 

# '" noabnormalities iii' en • manege movements**~** • head shaking .0 c: • excessive cleaning**~.** • frequent chewing !!j IJ> 

## a impairment ofgait (» ::! • animal is notwalking during observation "0 

## 3 (» • no impairmentofgait stiffgait (ii' U> o slight impairmentofcoordination, unsteady gait 0' :J o moderate impairmentofcoordination, shuffling gait 0' • severe impairment of coordination, dragging ofthe hindlimbs '< g: o severe impairmentofcoordination, with splayed limbs (»
- animal is unableto walk (abdominal or lateral position) IJ> "0 0 :J 

### otherfindings U> 0
- twitching Ul 

### FUNCTIONAL OBSERVATIONAL BATTERY



## omg/kg bw/day Rank N=5

0 

### 3 1 2 2 0 

### 3 0 4 0 

### 5 0 6 0 7 0

0 

### 5 1 

### 0 

### 2 0 3 0

0 

### 5 1 0 2 0 

### 3 0

0 

### 5 1 0 2 0 

### 3 0 4 

### 0

0 

### 3 1 

### 2 2 0 

### 3 0 4 0 

### 5 0 

### 6 

### 0 7 0 0 

### 5 t 0 

### Table IA· 37
Mean values day 26

10 mglkg bw/day 30 mg/kg bw/day 45 mg/kg bw/day N=5 N=5 N=4

1 1 0 4 4 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0



### 5 5 4 0 0 0 0 0 

### 0 0 0 0



### 5 5 4 0 0 0 0 0 

### 0 0 

### 0 0



### 5 5 4 0 

### 0 0 0 0 0 

### 0 0 0 0 0 0

1 1 0 4 4 4 0 0 0 0 

### 0 0 0 0 0 0 0 0 

### 0 0 

### 0 0 

### 0 0 

### 5 

### 5 1 

### 0 0 

### 3

# ~ Project No.1 FUNCTIONAL OBSERVATIONAL BATTERY Table IA - 38 

### g c: Mean values day 26

### ~ male animals 

## :a**~** 0 mg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day 

# ar OPEN FIELD OBSERVATIONS Rank N=5 N=5 N=5 N=4 "0 

# .g behaviourwhen removed from cage**~** - animal istense, but itshows no resistance against handling 0 5 5 5 4 

# '6
-animal shows aslight resistance againstthe handling 1 0 0 0 0 :: - animal shows no resistance againstthe handling butappears indifferent 2 0 0 0 0 

# ar -animal is difficultto handle, itshows aggressiveness 3 0 0 0 0**~** animal is very difficultto handle, itshows severe aggressiveness 4 0 0 0 0
::>

### ~ fur

### ~ - nothing abnormal detected 0 5 5 5 4 {ij

- discoloredfur 1 0 0 0 0 

### '1J**~<** urine staining ofanogenital region 2 0 0 0 0 Q) 

## W - - piloerection 3 0 0 0 0 

# <g**~** 

# -alopecia 4 0 0 0 0

.....**~** - reduced care on fur 5 0 0 0 0 

## o OJ 

## o 'lil skin O.

- nothing abnormal detected 0 5 5 5 4 ;:;

### ~ - discolored skin 1 0 0 0 0 (j)**~** . reddening 2 0 0 0 0 

### ex> '<
- paleness 3 0 0 0 0**~** - dehydration (exsiccosis) 4 0 0 0 0**~** - hypothermia (skin is cold during handling) 5 0 0 0 0 

## iii
- lesion(s) 6 0 0 0 0**~** - crust(s) 7 0 0 0 0 til**~** salivation**~<** no salivation 0 5 5 5 4

- 

## m -slight salivation (area around the mouth is moist) 1 0 0 0 0**~** - moderate salivation (wet mouth) 2 0 0 0 0**~** - severe salivation (mouth very wet, wet paws) 3 0 0 0 0
::> 

## 16 nose discharge 

## 3 - no discharge, dry nose 0 5 5 5 4**~<** clear discharge 1 0 0 0 0 

## g< - - reddish diSCharge 2 0 0 0 0**~** 

### :;: <l> -0 

# '" o ::>**~** Ul-1 ::r U;' 

### Project 

### NO,LI

_____...J 

### FUNCTIONAL OBSERVATIONAL BATTERY Table IA· 39 C. 

## g c 3 (I) male animals Mean values day 26

### :au;' 

## s: (I) 

### OPEN FIELD OBSERVATIONS Rank 

# omg/kg bw/day N=5 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day N=5 N=5 N=4 -0 (3 -0 

### lacrimation (I) ;:l '< Q. 

## s: (I) - no lacrimation slight lacrimation

- moderate lacrimation

- severe lacrimation 0 1 2 

### 3 5 0 0 0 5 0 0 0 5 0 0 0 4 0 0 0 (J) -0 0 :J (J) 0 til 'iii' 

### eyes/pupil size

- nothing abnormal detected, pupils contracted at room light

- chromodacryorrhea 0 1 

### 5 0 

### 5 0 

### 5 0 4 0 -0 (I) • exophthalmos 2 0 0 0 0 

# -a III <0 (1)



# ..... 0



# ..... n 3i (I) c. 0 ;;;J -0 Ql <Cl (I) - pupils dilated

- abnormal shape ofpupils

- oblique eye posture

- opacity

- cataract 

### 3 4 

### 5 6 

### 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

# - W 0') 00 -:­ c (J) (I) a '< 

### posture

- animal is sitting or lying

- animal isstaying or moving

squatting posture 0 1 

### 2 0 5 0 0 

### 5 0 0 5 0 0 4 0

### ~ - abdominal position 3 0 0 0 0**~** -0 Ql 

## a, (I) (J) (j) .t:J - abdominal position with splayed limbs

- lateral position

- oblique head posture

- opisthotonus 4 5 6 

### 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

c**~'** 

### palpebral closure (J) nothing abnormal detected 0 5 

### 5 5 4

### ~, 

## ar :J -0 - eyelid(s) slight closure

- eyelid(s) halfclosure

- eyelid(s) permanent closure 1 2 3 0 0 0 0 0 0 0 0 0 0 0 0 (I) 

## 3 iii' !!!, 0 :J a '< 

## s: (!) (J) -0 0 :::> Ul 0 til

# ~ Project

# NO.C**~** FUNCTIONAL OBSERVATIONAL BATTERY Table IA
- 40 

### g Mean values day 26 

# g male animals 

### ;::.**~** 0 mg/kg bw/day 10mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day 

## g- OPEN FIELD OBSERVATIONS Rank N=5 N=5 N=5 N=4 -0 

# .g respiration**~** - nothing abnormal detected 0 5 5 5 4 

# '6
-respiration labored 1 0 0 0 0 ;: - gasping/respiratory sounds 2 0 0 0 0 

## g- - respiration accelerated 3 0 0 0 0**~** - respiration irregular 4 0 0 0 0
:::> 

# Q tremors :::., - no tremors 0 5 5 5 4 .,og - slighttremors 1 0 0 0 0**'"O~.** 

### - moderate tremors 2 0 0 0 0 Ol 

## 15'

-severetremors 3 0 0 0 0 

### CO 0­ 

## ro 0 :::> convulsions

..... -0

. 

### 5 4 

## o**~** 

## - no convulsions 0 5 5 '" ctl

- slight convulsions 1 0 0 0 0 o.:;:!
- moderate convulsions 2 0 0 0 0

### ~**~** - severe convulsions 3 0 0 0 0 0'> ctl CO

### ~ abnormal movements/stereotypies**~** - no abnormalities 0 5 5 5 4 !!;

- manege movements 1 0 0 0 0 

## -g - head shaking 2 0 0 0 0 il:
- excessive cleaning 3 0 0 0 0

### ~ - frequent chewing 4 0 0 0 0

### ~**~.** Impairmentofgalt 

## m -animal is notwalking during observation 0 0 0 0 0 :;: - no impairment ofgait 1 5 5 5 4 

# a stiffgait 2 0 0 0 0 

### !g

- slight impairment of coordination, unsteady gait 3 0 0 0 0 

### 16
- moderate impairment ofcoordination, shuffling gait 4 0 0 0 0**~.** - severe impairment ofcoordination, dragging ofthe hindlimbs 5 0 0 0 0 

### ffi

- severe impairment of coordination, with splayed limbs 6 0 0 0 0 

# g -animal is unable to walk (abdominal or lateral position) 7 0 0 0 0**~** 

## s: ctl

## ~ :::> U) Q U)-I :::r 

### or 0­ 0 (") c:: 3 (1) 

### ;:;. iif 

### 5' (1) -0 

# a -0 (1) 

## q Q. 

### 5' (1) en -0 0 ::J Ul 0 til (jl -0 (1) (") 

### '"0 :3i t\) (1) <0 cCD 0 ::J



# ..... -0 OJ 0 <0 (1) W 0 c:: 

# - W 

## '" (1) (J) CO '< 0­**~** :::r**~** -0 OJ 3-. (1) 

# '" iil .c c**~.** 

# '" ::E

## ~ ::J -0 (1) 

## 3 iii·**~.** 0 ::J 0­ '< 

### 5' (1) (Jl 

## -g ::J (Jl 0 til Project No.1'----------' male animals

OPEN FielDOBSERVATIONS activity/arousal level

- normal exploration ofthe area

- reduced exploration ofthe area

- severe reduced exploration ofthe area. animal apathetic

- increased exploration ofthe area, sudden orjerky movements

- hyperactivity feces

- no defecation during observation period

- feces without abnormalities

- discolored feces

- crumbly feces

- softfeces

- muicid feces

- diarrhea urine

- no urination during observation period

- urinewithoutabnormalities (somewetareas on thefilter paper)

- discoloration ofurine

- polyuria (greatwet areas on thefilter paper) FUNCTIONAL OBSERVATIONAL BATTERY Table IA
- 41 Mean values day 26



## omg/kg bw/day 10 mg/kg bw/day 30 mglkg bw/day 45 mg/kg bw/day Rank N=5 N=5 N=5



### 0 5 1 0 2 0 3 0 4 0

0 

### 4 1 1 2 

### 0 3 

### 0 4 

### 0 5 0 

### 6 0

0 

### 1 1 4 2 0 3 0 5 5 4 0 0 0 

### 0 0 0 0 0 0 0 0 

### 0

4 5 3 

### 1 0 1 0 0 0 0 0 0 

### 0 0 0 

### 0 0 0 0 0 0



### 0 0 0 5 5 4 0 0 0 0 0 0-l :;;r iii' 0. 0 (") c: 3 <1> 

### ;a iii' 5' <1> "'0 <3 "'0 <1> ;:!. '< Q, 5' <1> "'0 0 

# '"
:J 

## '" 0 Ol Ol "'0 <1> 

### lJ Q. Q) <1> 

# "" 0. CO 0 

### ro :J

..... "'0 

## o OJ

### ~~ 0 -+\ ;;: <1> 

## w 

## '" 0')**a>~** 9­ ::J**~** "'0 OJ ;:!. ai' 

## '" a; .c c**~.** 

# '"**~** $ :J "'0 <1> 

## 3 vi" 5' 

# '" :J 0­ '< 5' <1> '0 

## '" 0 :J 

## '" 0 Ul 

### Project

# NOC··· FUNCTIONAL OBSERVATIONAL BATTERY Table IA·42 Mean values day 26 male animals 

## omgfkg bwfday 10mgfkg bwfday 30 mgfkg bw/day 45 mgfkg bwfday 

### SENSORIMOTOR TESTS/REFLEXES Rank N=5 N=5 N=5 N=4 

### approach response

- no reaction

- approaching to object

- escape reaction

- aggressive reaction and attacking ofobject 

### touch response

- no reaction

- orientationto the stimulus

- escape aftertouch

- aggressive reaction and attacking ofobject

- reaction to the stimulus but no ability to localize (e.g. turning to wrong side) 

### vision

- nothing abnormal detected (grasping with forelimbs)

- no grasping 

### pupillary reflex

- nothing abnormal detected, physiological adaptation ofthe pupil to light

- retarded adaptation ofthe pupil to light

- no adaptation ofthe pupil to light. pupils permanently contracted

- noadaptation ofthe pupil to light. pupils permanently dilated 

### pinna reflex

- immediate response to the stimulus

- no response to the stimulus 

### audition

- nothing abnormal detected, immediate normal response to the stimulus

- no response

- increased response

- hyperreaction 

### coordination ofmovements

- nothing abnormal detected, immediate righting response

- retarded righting response

- fails to turn into uprightposition, animal stays in lateral position

- no righting response, animal stays in dorsal position 0 

### 1 2 

### 3

0 

### 1 2 

### 3 4

0 

### 1

0 

### 1 2 

### 3


### 0 1

0 1 2 

### 3

0 1 

### 2 3 4 

### 1 0 0



### 5 0 0 

### 0 0

5 

### 0

5 0 0 0

5 

### 0

5 0 0 0


### 5 0 0 

### 0 3 2 0 0

4 1 0 0 

### 0

5 0

5 0 0 0

5 

### 0

5 

### 0 0 0


### 5 0 0 

### 0 3 2 0 0

4 1 0 0 

### 0

5 

### 0

5 0 0 0


### 5 0

5 0 0 0



### 5 0 0 0 

### 3 1 0 0

4 0 0 

### 0 0

4 0

4 0 

### 0 0

4 

### 0

4 

### 0 0 0

4 0 0 

### 0-l ::r 

## or 0. 0 n c 3 CT! 

## a w> 

## s: CT! 1:l 

# a CT! 

# "**~** 

### 9. 

## s: CT! (J) 1:l 0 ::l (J) 0 iil Vi' 1:l l1J 

### "1J 3-i Q) 

## :t <0 0 CD ::l -'" 1:l 

## o Q)

### CJ1~

...... 0 c 

# - w en CT! Q) 0­ CO '< 

## s: 0 

### !li 1:l Q) :::I.

### ~l

## ~ c 

### @. (J)**~**

### ~ ::l 1:l

# ~ or (J) ('5' ::l 0­ '< 

## s: CT! (J) 0 

# " ::l (J) 0 iil

# pr~ectNoiL __________

# ~ FUNCTIONAL OBSERVATIONAL BATTERY Table IA - 43 Mean values day 26 male animals 

# omg/kg bw/day 10 mglkg bw/day 30 mglkg bw/day 45 mg/kg bw/day SENSORIMOTOR TESTS/REFLEXES Rank N=5 N=5 N=5 N=4 behaviour during handling

- nonmal behavior, easyto handle, animal istense, but itshows no resistance easy to handle, animal is limply hanging in the hand

difficultto handle, animal shows a slight resistance to handle, animal shows a severe resistance vocalization

- no oronly sporadic vocalizations when touched

- very frequent vocalizations when touched

- vocalizations always when toUChed

- vocalization withouttouching pain perception

- nothing abnormal detected, immediate response tothe stimulus

- weak or retarded reaction to the stimulus

no response tothe stimulus

- hyperreaction tothe stimulus otherfindings

- twitching 0 1 2 3

0 1 2 3

0 1 2 3 

### 0 t 5 0 0 

### 0

5 

### 0 0 0

5 

### 0 0 0 5 

### 0 5 5 4 0 0 0 0 

### 0 0 

### 0 0 0

5 5 4 

### 0 0 

### 0 0 

### 0 0 0 0 

### 0

5 5 4 

### 0 0 0 0 0 

### 0 0 0 

### 0 5 5 

### 2 0 

### 0 2-I :; iii' a. 0 n c 3 <Il**~** iii' 

### g­ <Il u (3 u <Il ;J. '< Q, 

### g­ <Il v; u 0 :::> v; 0 iil 

## en u <Il 

### "1J Ol

# ~ CO a. (J) 0 :::>

..... u 

## o OJ

### O"l~ 0

## -~ v; W <Il O"l**co~**

### ~ !!; u OJ 

## a <1> v; 

### c;; .0 c 

## ;w v;**~**

### ~ :::> u <1> 

## 3 iii' (5' 

# '" :::> 0' '< 

### g­ <1> u 

# '" 0 :::> g) iil Project No.1 FUNCTIONALOBSERVATIONAL BATTERY Table IA-44 Mean values day 27 female animals 

## omglkg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mglkg bw/day HOME CAGE OBSERVATION Rank posture

- animal is sitting orlying

- animal is staying or moving

- squatting posture

- abdominal position

- abdominal position with splayed limbs

- lateral position

- oblique head posture

- opisthotonus tremors

- no tremors

- slighttremors

- moderatetremors

- severetremors convulsions

- no convulsions

- slightconvulsions

- moderate convulsions

- severe convulsions abnormal movements

- no abnormalities

- manege movements

- head shaking

- excessive cleaning

- frequent chewing impairmentofgait

- animal is notwalking during observation

- no impairment ofgait

- stiffgait

slight impairment ofcoordination, unsteady gait

- moderate impairmentofcoordination, shuffling gait

- severe impairmentof coordination, dragging ofthe hindlimbs

- severe impairmentof coordination, with splayed limbs

animal is unabletowalk (abdominal or lateral position) otherfindings

twitching 0 1 1 4 2 0 3 

### 0 4 

### 0 5 

### 0 6 

### 0 7 

### 0

0 5 1 0 2 

### 0 3 

### 0

0 5 1 

### 0 

### 2 0 3 

### 0

0 5 1 

### 0 

### 2 0 3 

### 0 4 

### 0

0 1 1 4 2 

### 0 3 

### 0 4 0 

### 5 0 6 

### 0 7 

### 0 0 5 t 0 0 

### 0 0 5 5 3 

### 0 0 

### 0 0 0 

### 0 0 0 0 0 

### 0 0 0 0 0 

### 0 0 0



### 5 5 3 

### 0 0 0 0 

### 0 0 0 0 0

5 

### 5 3 

### 0 0 0 0 0 0 0 0 0



### 5 5 3 

### 0 0 0 0 

### 0 0 0 0 

### 0 0 0 0

0 0 0 

### 5 5 3 

### 0 0 

### 0 0 0 0 0 0 0 

### 0 0 0 0 

### 0 0 0 

### 0 0 5 5 

### 0 0 0 3

## :i (ii' a. 0 n c 3 <1> ;:t (ii' :T <1> '0 

# a '0 II>

### ~ 

### S. :T (l) (I) '0 0 :J (I) 0 iil "iil '0 II> 

### '1J n**~** til 

### to CD OJ 0

..... '0 0'"**-..j~** 

## o ::­

..... c 

### W '" 0'> (l)**o:>~** 

## §:**~** '0 

## '" ::l­ ii)' (I) <i3 .0 c 

### @' (I)

### ~ 3:' II> OJ**~** 

## 3 (ii' (I) 6' OJ CT '< :T <1> '0 

# '" 0 ::l**~** ijl 

### Project

# No.1 FUNCTIONAL OBSERVATIONAL BATTERY Table IA - 45 Mean values day 27 female animals


### OPEN FIELD OBSERVATIONS Rank 

## omg/kg bw/day N=5 10 mg/kg bw/day 30 mg/kg bw/day 45 mglkg bw/day N=5 N=5 N=3 

### behaviour when removed from cage

- animal is tense. but itshows no resistance against handling 0 5 5 

### 5 3

- animal shows a slight resistance againstthe handling 1 0 0 0 0

- animal shows no resistance againstthe handling butappears indifferent 2 0 0 0 0

animal isdifficultto handle, it shows aggressiveness 3 0 0 0 0

- animal isvery difficultto handle. itshows severe aggressiveness 4 0 0 0 0 

### fur

- nothing abnormal detected 0 

### 5 5 5 3

- discolored fur 1 0 0 0 0

- urinestaining ofanogenital region 2 0 0 0 0

- piloerection 3 0 0 0 0

- alopecia 4 0 0 0 0

reduced care on fur 5 0 0 0 0 

### skin

- nothing abnormal detected 0 5 

### 5 5 3

- discolored skin 

### 1 0 0 0 0

- reddening 2 0 0 0 0

- paleness 3 0 0 0 0

- dehydration (exsiccosis) 4 0 0 0 0

- hypothermia (skin iscold during handling) 5 0 0 0 0

- lesion(s) 6 0 0 0 0

- crust(s) 7 0 0 0 0 

### salivation

- no salivation 0 5 5 

### 5 3

- slightsalivation (area around the mouth is moist) 

### 1 0 0 0 0

- moderate salivation (wet mouth) 2 0 0 0 0

- severe salivation (mouth very wet, wet paws) 3 0 0 0 0


### nose discharge

- no discharge, dry nose 0 5 

### 5 5 3

- clear discharge 1 0 0 0 0

- reddish discharge 2 0 0 0 0-I :T iii' Cl.. 

### Project

# NO.''--

____---' FUNCTIONAL OBSERVATIONAL BATTERY Table IA - 46 0 n c 3 <t> female animals Mean values day 27**~** 0;' 

## s: <t> 

### OPEN FIELD OBSERVATIONS Rank 

## omglkg bw/day N=5 10 mglkg bw/day 30 mg/kg bw/day 45 mg/kg bw/day N=5 N=5 N=3 "0 

### 0 "0 

### lacrimation <t> ;::l. '< Q. 

## s: <t> - no lacrimation

- slight lacrimation

- moderate lacrimation

- severe lacrimation 

### 0 1 

### 2 3 5 

### 0 0 0 5 

### 0 0 0 5 

### 0 0 0 3 

### 0 0 0 (/) "0 0 

# '" (/) Q III 

### eyes/pupil size

- nothing abnormal detected, pupils contracted at room light 0 5 

### 5 5 3 U> - chromodacryorrhea 

### 1 0 0 

### 0 0 "0 <t> - exophthalmos 

### 2 0 0 0 0 

### -U Q. :!> 

### ru <t> (Q Cl.. CD 0 

# '"**~** ""0 0 OJ <0 

### ex> <t> 

### 0 

# .... 

# - c W (/) (j) <t> cr 

### ex> '<**~** - pupils dilated

- abnormal shape ofpupils

- oblique eye posture

- opacity

- cataract 

### posture

- animal is sitting or lying

animal isstaying or moving squatting posture 

### 3 4 

### 5 6 7

0 1 

### 2 0 0 0 0 0



### 0 

### 5 0 0 0 0 0 0

0 5 0 0 0 0 0 

### 0

0 5 0 0 0 0 0 0

0 3 0 :T 

### !ll "0 OJ - abdominal position

- abdominal position with splayed limbs

lateral position 3 4 5 

### 0 0 

### 0 0 0 

### 0 0 

### 0 0 0 

### 0 0

### ~ 

## '" i11 oblique head posture nnill::.tnntnnl**J~** 

### 6 7 

### 0 0 0 0 0 

### 0 0 0 .c c**~.** 

### palpebral closure
(J) nothing abnormal detected 

### 0 5 5 

### 5 3**~** 

# i 

# '" "0 - eyelid{s) slight closure

- eyelid{s) half closure

- eyelid{s) permanent closure 

### 1 2 

### 3 0 0 0 0 

### 0 0 0 0 

### 0 0 0 0 <t> 

## 3 iii' (/) 

### o· 

# '" cr '< 

## s: <t> (II "0 0 

# '" til 0 Ul-1 ::>" 

## or Q. 0 c: 

## '" 3 <t> 

### ;a iii" 

## s= <t> "0 

# a "0 <t> ::l. '< 

# a s= (J) "0 

# '" 0 ::l 0 

# '" iil**~** "0 <t> 

### -U Q" ::"I !l) <t> CO C. 0 CD ::l -'" "0 

# o III

### c.o~ 0 

# -. ;" 

### W '" 0) <t>**co~**

## ~**~** 

## lil ::l. iii" 

# '" ill .c c**~"** 

# '"**~**

### ~ ::l 

## lil 

## 3 iii" 

## '" 0" ::l cr '< 

## s= <t> "0 

## '" 0 ::l 

# '"**~** Project No. FUNCTIONAL OBSERVATIONAL BATTERY Table IA
- 47 Mean values day 27 female animals 

# omg/kg bw/day 10 mg/kg bw/day 30 mglkg bw/day 45 mglkg bw/day OPEN FIELD OBSERVATIONS Rank N=5 N=5 N=5
respiratlon

- nothing abnormal detected

- respiration labored

- gasping/respiratory sounds

- respiration accelerated

- respiration irregular tremors

- notremors

- slighttremors

- moderatetremors

- severetremors convulsions

- no convulsions

- slightconvulsions

- moderateconvulsions

- severe convulsions abnormal movements/stereotypies

- no abnormalities

- manege movements

- head shaking

- excessive cleaning

- frequent chewing Impairmentofgalt

- animal is notwalking during observation

- no impairmentofgait

- stiffgait

- slight impairmentofcoordination, unsteady gait

- moderate impairmentofcoordination, shuffling gait

- severe impairment ofcoordination, dragging ofthe hindlimbs

- severe impairment

### ofcoordination, with splayed limbs

- animal is unableto walk (abdominal or lateral position) 

### 0 1 2 

### 3 4

0 

### 1 2 

### 3



### 0 1 2 

### 3



### 0 1 2 

### 3 4



### 0 1 2 

### 3 4 

### 5 6 7 5 

### 0 0 0 

### 0

5 0 

### 0 0



### 5 0 

### 0 0



### 5 0 0 

### 0 0

0 5 

### 0 0 0 0 

### 0 0 5 

### 0 0 

### 0 0

5 

### 0 0 0



### 5 

### 0 0 0

5 

### 0 0 0 0

0 

### 5 0 0 

### 0 0 

### 0 0 5 

### 3 0 0 0 

### 0 0 0 0 

### 0

5 3 0 0 0 

### 0 0 

### 0

5 3 

### 0 0 0 

### 0 0 

### 0

5 

### 3 

### 0 0 0 

### 0 0 0 

### 0 0

0 0 

### 5 3 0 0 

### 0 0 

### 0 0 0 0 0 0 0 0

## ..., :r fii" 0­ 0 () c: 3 (1) 3­ fii" 

### :::;: (1) "0 

# a "0 (1) ;::!. '< 

# a :::;: (1) 

# '" "8 ::J tJ) 0 

# '" iii

() "0 (1) 

### 1J ::;; Q) 16" 

## co 0­ 

## ro 0 :l



## ...... "0 Il.l 

## ...... (0 (1) 0 

### 0 -., c: W (1) 

# '" m 00 '< <:r 9­ :r**~** 

## 1il 

# a (1) 

# '" ro .0 c:**~.** 

# '"**~**

### ~ :l 

### 16 

## 3 (ii" i5 

## '" :l <:r '< 

### :::;: (1) "0 

# '" 0 :l 0 

## '" iii Project

# Noj 

# J FUNCTIONAL OBSERVATIONAL BATTERY Table IA
- 

### 48
Mean values day 27 female animals 

## omg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day OPEN FIELD OBSERVATIONS Rank N=5 N=5 N=5 N=3 activity/arousallevel

- normal exploration ofthearea

- reduced exploration ofthearea

- severe reduced exploration ofthe area, animal apathetic

- increased exploration ofthe area, sudden orjerky movements

- hyperactivity feces

- no defecation during observation period

- feces withoutabnormalities

- discolored feces

- crumbly feces

softfeces

- muicidfeces

- diarrhea urine

- no urination during observation period

- urinewithoutabnormalities (somewetareas on thefilter paper)

- discolorationof urine

polyuria (greatwetareas on thefilter paper) 0 5 1 0 2 0 3 0 4 0

0 

### 5 1 0 2 0 3 0 4 0 5 0 

### 6 0

0 4 1 1 

### 2 0 3 0 5 

### 5 3 0 0 0 0 0 0 0 0 0 0 0 0



### 5 5 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4 

### 2 0 1 3 3 0 0 0 0 0 0-I :or 

### or c. 0 (') c: 3 en**~** (h' g. en "0 

# a "0 en ::l. '< 

# a 5' m "0 

## '" 0 :> 

## '" 0 Ul Ii) "0 en Q, 

### "U :::!! 

### m m (Q c. ctl :> 0

..... "0



## ..... Jl



# ..... m 0

### -~ W '" (j) m**(X)~** 0 g.**~** "0 Ql ::l. iii' 

## '" Ql J::J c: @' 

# '"**~,** 

## it :> 

## 'li: 

## 3 v; 

## '" 0' :> c:r '< 5' m "0 

# '" 0 0 :> 

## '" Ul Project NO.C­ FUNCTIONAL OBSERVATIONAL BATTERY Table IA
- 49 Mean values day 27 female animals 

## omg/kg bw/day 10 mg/kg bw/day 30 mg/kg bw/day 45 mg/kg bw/day SENSORIMOTOR TESTS/REFLEXES Rank N=5 N=5 N=5 N=3 approach response

- no reaction

- approachingtoobject

- escape reaction

- aggressive reaction and attacking ofobject touch response

- no reaction

- orientation to the stimulus

- escape aftertouch

- aggressive reaction and attacking ofobject

- reaction tothe stimulus but no abilityto localize (e,g, turning to wrong side) vision

- nothing abnormal detected (grasping with forelimbs)

- no grasping pupillary reflex

- nothing abnormal detected, physiological adaptation ofthe pupil to light

- retarded adaptation ofthe pupilto light

- no adaptation ofthe pupilto light, pupils permanently contracted

- no adaptation ofthe pupil to light, pupils permanently dilated pinna reflex

- immediate response to the stimulus

- no response to the stimulus audition

- nothing abnormal detected, immediate normal response to the stimulus

- no response

- increased response

- hyperreaction coordination ofmovements

nothing abnormal detected, immediate righting response

- retarded righting response

- failstoturn into upright position, animal stays in lateral position

- no righting response, animal stays in dorsal position 

### 0 1 2 

### 3

0 1 2 

### 3 4



### 0 1

0 1 2 

### 3

0 1



### 0 1 2 3



### 0 1 2 

### 3 3 2 

### 0 0

5 

### 0 0 0 0



### 5 0



### 5 0 0 0

5 

### 0



### 5 0 0 0



### 5 0 0 

### 0 5 

### 0 0 0



### 5 0 0 0 0



### 5 0

5 

### 0 0 0

5 

### 0

5 

### 0 0 

### 0



### 5 0 0 0 3 2 

### 0 0

2 3 

### 0 0 0

5 

### 0

5 

### 0 0 0

5 

### 0



### 5 0 0 

### 0

5 

### 0 

### 0 0 2 

### 1 0 0

3 

### 0 0 0 0

3 

### 0

3 0 0 0

3 0

3 

### 0 

### 0 0

3 

### 0 0 0-i ::::; (ji 0. 

### g c 3 (l> ::t (ji" 5­ (l> "0 <3 "0 (l>**~** 

# a 5­ (l> "0 

# '" 0 :J 

## '" 0 <il Cii' "0 

### 1J 3i III (l> 

### to 0. CD 0 :J

...... "0 ru 

## ...... <0 N (l> 0 

# - ""'" c (l> 

### w 

# '" 0) c:T (Xl '<

## ~**~** "0 ru ;l. <n" 

# '" 

# 2 c**~"** 

## '"**~ ~**  (l> :J 

## -g 3 (ji" (5" 

## '" :J**~** 5­ (1) "0 

## '" 0 0 

## '" :J iil 

### Project No. FUNCTIONAL OBSERVATIONAL BATTERY Table IA - 50 Mean values day 27 female animals 

# omgfkg bwfday 10 mgfkg bwfday 30 mgfkg bwfday 45 mgfkg bwfday 

### SENSORIMOTORTESTS/REFLEXES Rank N=5 N=5 N=5 N=3 

### behaviour during handling

- normal behavior, easy to handle, animal istense, but it shows no resistance 0 5

- very easyto handle, animal is limply hanging in the hand 1 0

- slightly difficultto handle, animal shows a slight resistance against handling 2 0

- difficultto handle, animal shows a severe resistance against handling 3 0 

### vocalization

- no or only sporadic vocalizations when touched 0 5

- very frequent vocalizations when touched 1 0

- vocalizations always when touched 2 0

- vocalization without touching 3 

### 0 pain perception

- nothing abnormal detected, immediate response tothe stimulus 0 5

- weakor retarded reaction to thestimUlus 1 

### 0

no responsetothe stimulUS 2 

### 0

- hyperreaction to the stimulus 3 0 

### otherfindings 0 5

- twitching t 

### 0 5 0 0 0

5 0 0 

### 0

5 

### 0 0 

### 0 5 

### 0 5 0 0 0

5 0 0 0

5 

### 0 0 0 5 

### 0 3 0 0 0

3 0 0 0

3 0 

### 0 0 1 2Study:

# I**~** -i :;; iii' a. 

## g c 3 en 

### ;;:. iii' 5' Male, en '0 

# a Mean '0 en SD ::I­ '< N 

### So %dev 5' en Male, '0 

# '" 0 :::J Mean 0 

# '" SD Ul N 'iii' '0 ,"dev 

# ,., en 

### '"0 !l)

### ~ Male, (Q a. 0 

## ro :;J Mean 

# ..... '0

..... Q) SO 

## w'al



# - w N


# .... 0 %dev 

### 5i 0') en Male, CO '< cr 0 Mean 5'**~** SO '0 N III ::I­ %dev 

## il <i! Key: .0 C 

### @' 

# '" :Ii 3: CD**~** '0 (1) 

## 3 iii' fJ) 

### o· :;J cr '< 5' (1) fJ) '0 0 :;J en

### ~ FECES N Day 26 k 0 mg/kg bw/day 0.2 0.4 5

10 mg/kg bw/day 0.2 0.4 5 0.0 30 mg/kg bw/day 0.0 0.0 5 -100.0 45 mg/kg bw/day 0.3 0.5 4 25.0 Rear Day 26 k

7.6 4.6 5

8.4 4.3 5 10.5

9.6 2.5 5 215.3

11.3 5.9 4 48.0 k = Kruskal-wallis + Wilcoxon-test, Experimental Un1t = An1mal FUNCTIONAL OBSERVATIONAL BATTERY

FS T OSF GSH

em Newton Newton Day 215 Day 26 Day 26 k k k

11.6 4.7 5.8

1.3 1.9 0.9 5 5 5

12.2 4.8 4.7 1.6 2.1 1.4 5 5 5 5.2 3.0 -17.9

12.0 4.6 4.4 0.8 2.0 1.9 5 5 5 2.9 -2.15 -24.4

12.0 4.3 4.8

1.3 1.4 1.0 4 4 4 3.6 -9.2 -16.8 Two-s1ded, 

## * p <= 0.050, 

## ** P <= 0.010 Print Date: 06-Nov-2006 Print Time: 14:13:56 Table IA Page 51Study:

# I**~ ~**  ::r 0;­ 

## g a.
c: 3 (I) ;:t 0;­ :T Female, (I) -0 

# a Mean -0 (I) SO :::1. '< N Q. %dev :T (I) fJ) Female, -0 0 ::J fJ) Mean 0 

### ;;; SO (i) N -0 (I) %dev 

### lJ () 3i Il,) <1> Female, a. 0 

## co CD ::l Mean 

# ..... -0 Q) 

# ..... SO co .f;l.. (I) N 

### 0**~** %dev 

# -. c: W fJ) <1> Female, O'l CXl**~ ~**  Mean ::r 

### !ll SO -0 N Q) ::I. %deviiifJ) Key: NC

## ~ FECES N Day 27 k 0 mg/kg bw/day 0.0 0.0 5

10 mg/kg bw/day0.0 0.0 5 NC 30 mg/kg bw/day0.0 0.0 5 NC 45 mg/kg bw/day0.0 0.0 3 NC



# = Not Calculable FUNCTIONAL OBSERVATIONAL BATTERY Print Date: Print Time: Table Page 06-Nov-2006 14:38:23 IA 52

Rear Day 27 k FS T 

### am Day 27 k GSF Newton Day 27 k GSa Newton Day 27 k

11.2 4.9 5 10.0 1.2 5 6.2 1.8 5 4.5 0.9 5

12.8 3.9 5 14.3 10.9 0.9 5 9.5 5.6 0.9 5 -8.8 4.5 0.4 5 0.4

13.6 2.7 5 21.4 10.8 0.9 5 7.9 3.7 1.3 5 -39.9 3.3 1.1 5 -26.2

18 3 4.6 3 63.7 10.7 0.9 3 7.1 5.6 1.3 3 -9.9 4.2 0.9 3 -5.9

c: k 

# = Kruskal-Wallis + Wilcoxon-test, Two-sided, 

## * p 

### <"" 0.050, 

## ** P 

### <'" 0.010 

## III :;­ Experimental Unit 

# = Animal**~** 

# "" ro ::J 

### 16
0;- 

## 3

fJ) 

### o· ::J**~** :T (I) (I) 

## "8 ::J 

### rg ;;;

### -0 t\) 

## co CD**~ ~**  (]1 0 -., W Q) 0:> -j ::r iii' 0­ 0 c: 

# '" 3 <1> 

### ;a iii' 

## s: <1> '0 

# a '0 <1>

### ~ 

# a s: <1> '0 

# '" 0 ::J 0 Cil 

# '"**~** '0 

# '" <1>

# ~ 0. 0 ::J '0 ru to <1> ::­ c: 

## "' <1>**~** $? ::r**~** '0 ru 

# g. '" m J:l c**~.** :E**~** <1> ::J '0 <1> 

## 3 iii' 

## '" 0' ::J**~** 5' <1> (I) '0 0 ::J gJ iil 

### c:::=J Project Number

# I
GROUP MEANS MOTOR ACTIVITY DAY 26

MAL E S Interv. 1 Interv. 2 Interv. 3 Interv. 4 Interv. 5 Interv. 6 Beam Beam Beam Beam Beam Beam Interr. Interr. Interr. Interr. Interr. Interr. 

## o MG/KG M 60.4 47.2 36.2 27.0 35.0 14.0 SD 5.0 11.3 6.5 6.3 7.9 7.2

N 5 5 5 5 5 5

10 MG/KG
M 61.2 50.8 43.4 41.2 

## * 34.8 30.4 SD 15.4 8.8 7.4 8.2 6.4 9.3

N 5 5 5 5 5 5

30 MG/M
M 74.8 4B.6 42.4 43.0 

## * 37.2 37.8 SO 15.4 16.8 10.5 7.5 16.3 7.0

N 5 5 5 5 5 5

45 MG/KG
M 82.3 61.3 45.0 39.3 

### * 41.0 33.5 SO 7.9 6.7 13.1 9.5 11.9 22,9

N 4 4 4 4 4 4

Kruskal-Wallis + Wilcoxon-tests (two-sided): 

### * p<=0.05; 

### ** p<=0.01 (Statistical unit Interv. 7 Beam Interr.

19.2 19.2 5

38.8 13.6 5

30.8 10.7 5

39.0 31.3 4
Animal) TABLE IA

- 53

PRINT DATE 03NOV06

Interv. B

Beam Interr.

7.B 8.4

5

1B.0 15.0 5

30.B 12.0 5

19.0 14.B 4-; :::T (ii' 0. 

## g c 3 CD >l. (ii' g: CD -0 

# a -0 CD ::\. '< Q. g: CD -0 

## '" 0 ::J 

## '" 0 Cil 

## en -0 CD Q, 

### ""U t\) CD 

## "" CO 0. 0 CD ::J



### ....... -0 III


### ....... <0 0> CD


# - 0 .,..­ c UJ 

## '" CD 0> CT 

### ex> '< 0 g:**~** -0 III**~** CD 

# '" CD .0 C**~.** 

## '" ::;;

## ~ ::J -0 CD 

## 3 (ii' !!!, 0 ::J CT '< g: CD -0 

## '" 0 ::J 

## '" 0 Cil TABLE IA

- 54 

### [:::=::::J Project Number GROUP MEANS MOTOR ACTIVITY DAY 26 PRINT DATE 03NOV06

MAL E S Interv. 9 Interv.10 Interv.11 Interv.12 Interv.1-12 Beam Beam Beam Beam Sum Interr. Interr. Interr. Interr. Interr.


### o MG/KG M 1.2 4.0 0.8 0.6 253.4 SD 2.2 8.9 1.8 1.3 47.3 N 5 5 5 5 5

10 MG/KG M 15.0 10.4 6.8 3.4 354.2 

## * SD 21.8 12.4 11.7 7.6 83.0

N 5 5 5 5 5

30 MG/KG M 20.2 16.8 13.4 10.0 405.8 

## * SD 16.1 16.8 12.4 8.9 83.3

N 5 5 5 5 5

45 MG/KG M 22.3 32.8 20.5 18.0 

## * 453.8 

## * SD 27.9 24.8 14.5 20.4 121.0

N 4 4 4 4 4

Kruskal-Wallis + Wilcoxon-tests (two-sided): 

## * p<=0.05; 

## ** p<=O.Ol (Statistical unit Animal)-I :::T Cii· 0. 0 () c 3 CD ;:l. Cii·SCD "0 

## a "0 CD ;:l. '< 

### 8.SCD en "0 0 :::J en 0 

### en 

## en "0 CD 

### ""'0 () 3 Q) CD 0. 0 CO CD :::J



### ....... "0 OJ


### ....... CD 0 

# "" 10

c en 

# - W CD (j) CD '< 0" 0 S­**~** 

### iii ;:l. iii· en 

# 2 c**~.** en**~**

### ~ :::J "0 CD 

## 3 Cii· en 

### o· :::J 0" '< S' CD en "0 0 :::J en 0 

### en TABLE IA

- 55 

## c:::J Project Number GROUP MEANS MOTOR ACTIVITY DAY 27 PRINT DATE 03NOV06

FEMALES Interv. 1 Interv. 2 Interv. 3 Interv. 4 Interv. 5 Interv. 6 Interv. 7 Interv. 8 Beam Beam Beam Beam Beam Beam Beam Beam Interr. Interr. Interr. Interr. Interr. Interr. Interr. Interr.


### o MG/KG
M 66.0 46.2 32.6 35.2 28.8 16.6 7.8 1.4 SO 19.6 12.6 10.9 6.8 16.3 14.4 7.8 2.6

N 5 5 5 5 5 5 5 5

10 MG/KG
M 74.4 54.8 50.8 39.4 26.8 21. 6 16.6 18.4 SO 16.1 12.3 15.4 5.3 17.2 14.3 15.3 15.5

N 5 5 5 5 5 5 5 5

30 MG/KG M 70.2 52.4 41.2 32.4 38.2 23.0 28.8 29.2 SO 4.8 14.2 10.4 8.4 21. 8 6.9 14.6 21.8

N 5 5 5 5 5 5 5 5

45 MG/KG
M 96.0 48.3 49.3 35.7 39.3 22.3 27.3 23.0 SO 13.2 14.0 7.0 15.9 4.0 12.7 16.0 17.4

N 3 3 3 3 3 3 3 3

Kruskal-Wallis + Wilcoxon-tests (two-sided): 

## * p<=0.05; 

### ** p<=O.OI (Statistical unit = Animal)-I ::J" (ji' C. 0 c 3 

# "
CD**~** (ji' g. CD "0 

# a "0 CD 

# .z Q, g. CD II> "0 0 

# " :g Ul (j) "0 CD 

### -U :::;; 

# " Q) (ii' (C c. 0 

## eo 

# " -" "0 OJ -" CD 

## '" CD 0 Y C II> 

# - W CD 

### en CT CD '< 0 g.**~** "0 OJ 

# g II> CD .c c**~.** II> ::E**~.** 

# " "0 CD 

## 3 (ji' II> 

### o·
CT 

# " '< g. CD II> "0 0 

## :g 

# " Ul TABLE IA

- 56

gct Num,er

# I

GROUP MEANS MOTOR ACTIVITY DAY 27 PRINT DATE 03NOV06

F E MAL E S Interv. 9 Interv.10 Interv.11 Interv.12 Interv.1-12 Beam Beam Beam Beam Sum Interr. Interr. Interr. Interr. Interr.


### o MG/KG M 4.8 6.2 0.0 6.4 252.0 SD 10.7 13.9 0.0 10.1 51.6

N 5 5 5 5 5

10 MG/KG M 16.6 9.8 7.4 14.4 351.0 

## * SD 18.5 17.1 11.0 22.1 89.9

N 5 5 5 5 5

30 MG/KG M 24.2 17.6 22.4 16.8 396.4 

## * SD 13.8 15.2 23.9 12.6 118.3

N 5 5 5 5 5

45 MG/KG
M 17.3 7.3 8.7 21.7 396.3 

## * SD 14.6 9.3 12.5 20.6 77.5

N 3 3 3 3 3

Kruskal-Wallis + Wilcoxon-tests (two-sided): 

## * p<=0.05; 

### ** p<=O.Ol (Statistical unit Animal)

### "U Q) to CD



## ......

...... <0 0 

# -. W 0) 

### ex> -I :::T 

### en' 0. 0 (") c 3 CD 

### ;a. en' 

## s: CD "0 

### 0 "0 CD ::+ '< 

# a s: CD VI "0 0 ::J VI 0 U1 

## en "0 CD Q. :, CD 0. 0 ::J "0 co 

# '" CD


## -=­ c VI CD C" '< 0 

## s:**~** "0 ::+ 

# '" (ii' VI CD ..0 C**~.** VI**~**

### ~ ::J "0 CD 

## 3 en' VI 

### o· ::J C" '< 

## s: CD VI "0 0 ::J VI Q VI Study:

# I

WEC giga/l Day 29

k REC tera/l Day 29

k Male, 0 mg/kg bw/day
Mean SD N 6.26 1.38 5 7.66 0.32 5 Male, 10 mg/kg bw/day Mean SD N 5.94 1.62 5 7.55 0.15 5 Male, 30 mg/kg bw/day Mean SD N 7.08 0.86 5 7.51 0.31 5 Male, 45 mg/kg bw/day Mean SD N 7.03 0.36 4 7.52 0.32 4 Key: k 

## = Kruskal-Wallis + Wilcoxon-test, Experimental Unit 

## = Animal HGB mmol/l Day 29

k

9.9 0.3 5

9.5* 0.2 5

9.5 0.3 5

9.2* 0.3 4 Two-sided, HEMATOLOGICAL EXAMINATIONS

HCT MCV MCH 1/1 f1 fmol Day 29 Day 29 Day 29

k k k

0.410 53.5 1.29 0.009 1.3 0.02 5 5 5

0.396 52.5 1.25 0.009 0.7 0.03 5 5 5

0.400 53.3 1.26 0.011 1.3 0.06 5 5 5

0.396 52.7 1.22 0.008 1.3 0.03 4 4 4
* p 

## <= 0.050, ** P 

## <= 0.010 MCHC mmol/l Day 29

k

24.20

0.27 5

23.90

0.53 5

23.71

0.46 5

23.19

0.35 4 Print Date: 04-Jan-2007 Print Time: 08:51:36 Table IB Page 1

PLT RETI giga/l % Day 29 Day 29

k

936 2.2 114 0.4

5 5

917 2.0 118 0.5

5 5

889 2.4 110 0.5

5 5

930 2.7 110 0.3

4 4Print Date: Print Time: Table Page 04-Jan-2007 06:51:36 IB 2



### lJ 

## ru co 

## ro



# ..... N 0 

### 0 

## -. W (j) (Xl -I ::r 0;­ 0­ 0 C? c: 3 (!) 

### ;a. 0;­ :;: (!) '0 

## a -0 

## ! '< 

# a :;: (!) 0'> -0 0 ::J (fj 0 <il (i) 

## i n**~** (!) 0­ 0 ::J '0 <D 

## '" (!) ':­ c: 0'> (!) cr '< 0 

### :;: 

### !!l -0 ;1­(ii0'> 

## '"
iil £J c: 

# af 0'> 

### ;!i

### ~ ::J -0 

## '" 3 iii­ !!!. 0 ::J cr '< :;: 

## '" (/) '0 0 ::J 0'> 0 <il Study:

WEe REC giga/l tera/l Day 29 Day 29

k k Female, 0 mg/kg bw/d.ay Mean 4.95 7.35 SD 1.23 0.52 N 5 5 Female, 10 mg/kg bw/d.ay Mean 4.71 7.12 SD 0.93 0.23 N 5 5 Female, 30 mg/kg bw/d.ay Mean 4.29 7.20 SD 0.68 0.37 N 5 5 Female, 45 mg/kg bw!d.ay Mean 4.43 7.29 SD 0.15 0.56 N 3 3 Key: k = Kruskal-Wall1s 

### + Wilcoxon-test, Experimental unit Animal HGE mmol/l Day 29

k

9.3 0.2 5

9.2 0.3 5

9.2 0.3 5

9.1 0.3 3 TWO-Sided, HEMATOLOGICAL EXAMINATIONS

HCT MCV MeH 1/1 fl fmol Day 29 Day 29 Day 29

k k k

0.386 52.6 1.27 0.014 2.2 0.08 5 5 5

0.375 52.8 1.30 0.012 0.7 0.02 5 5 5

0.380 52 6 1.29 0.016 1.2 0.05 5 5 5

0.364 52.8 1.25 0.015 2.1 0.06 3 3 3 

### * P 

### <= 0.050, 

### ** P 

### <- 0.010 MCHC mmol/l Day 29

k

24.10

0.58 5

24.65

0.11 5

24.35

0.45 5

23.68

0.19 3 PLT g1ga/l Day 29

k

879 108

5

957 136

5

832

91

5

907

96

2 RETI % Day 29

2.2 0.4 5

2.3 0.7 5

2.4 0.3 5

2.7 0.8 3-i :r tir a. 0 (") c 3 <D 

## a. (ii' 5' <D 

# " a " <D ::l. '< Q, 5' <D 

# " 0 (/)

(J) 

# " Q IJ) 16 <D 

# " (") 

### '"0**~** III <D (Q a. (1) 0



# ..... " III N ([) 

# "

..... <D 0 ::­ 

# -. c 

## c..v (/) <D 0) 

# eo '< 0­ 0 5' 

### !li 

## " III d-. <D 

# '" Cil .0 c**~'** IJ)

### ~. 

## & 

# " <D 

# " 3 (ii' VI 

### o· 

# " 0­ '< 5' <D 

## '" 0 

# " " 0 Ul 

## '" DIFFERENTIAL BLOOD study: NEOT LYMPH MONO EOS BASO % % % % Day 29 Day 29 Day

# '"29 Da.y 29 Day 29 Male, 0 mg/kg bw/day Mean 12.0 84.5 1.2 1.4 0.5 SD 3.4 3 8 0.2 0.4 0.2 N 5 5 5 5 5 Male, 10 mg/kg bw/day Mean 13.1 83.3 1.0 1.6 0.5 SD 4.9 6.1 0.1 1.1 0.1 N 5 5 5 5 5 Male, 30 mg/kg bw/day Mean 16.7 79.0 1.9 1.4 0.5 SD 6.3 6.8 0.8 0.2 0.1 N 5 5 5 5 5 Male, 45 mg/kg bw/day Mean 13.4 83.0 1 3 1.4 0.4 SD 2.9 3.2 0.3 0.5 0.1 N 4 4 4 4 4 Key: Experimental Unit Animal COUNT Print Date: Print Time: Trible Page 06-Dec-2006 15:14:06 IB 3 LUC

% Day 29

0.5 0.1 5

0.5 0.2 5

0.5 0.2 5

0.5 0.1 4Print Date: 06-Dec-2006 Print Time: 15:14:06 DIFFERENTIAL BLOOD COUNT Table IB Study: Page 4
-I :T NEUT LYMPH MONO EOS BASO LUC iii" % % % % % % a. 0 n Day 29 Day 29 Day 29 Day 29 Day 29 Day 29 c 3 <t> 

### ;a Female, 0 mg/kg bw/day iii' Mean 11.4 84.8 1.4 1.6 0.5 0.4 SO <t> "tJ SO N 3.2 5 3.1 5 0.3 5 0.5 5 0.2 5 0.1 5 

# a "tJ CD ;:;. '< Female, 10 mg/kg bw/day 

# a SO CD Mean SO 11.1 3.7 85.4 3.8 1.0 0.3 1.8 0.1 0.4 

## o 1 0.4 0.1 

# '" "tJ N 5 5 5 5 5 5 0 ::l 

# '" 0 Female, 30 mg/kg bw/day iii Ci> Mean 11.8 84.1 1.3 1.9 0.4 0.4 "tJ <1l SD 1.2 1.6 0.5 0.4 0.1 0.2 

### ""C Q) n :3i CD N 5 5 5 5 5 5 (C 

## ro a. 0 ::l Female, 45 mg/kg bw/day -" "tJ N N 

## o OJ (Q CD 

### .;! Mean SD N 11.2 1.7 3 84.9 2.2 3 1.3 0.3 3 1.4 0.2 3 0,5 0.1 3 0.5 0.2 3

- W 0'> 

## co c 

## '" <1l CJ '< Key: Experimental Unit 

# = Animal
0 

## s:**~** 

## a? 

## a <1l 

# '" iD .Q C**~.** 

## '" 

# i

:::I "tJ <t> 

## :.I iii' !C, 0 ::l CJ '< 

## s: <t> 

## '" "tJ 0 ::l**~** iii

# I Print Date: 21-Dec-2006

-l ::r iir Cl. 0 ("') c:: 3 (I) 3­ in' 5' (I) '0 

# a '0 (I)

### ~ 

### S. 5' (I) 1ft '0 0 :> 1ft 0 iil 

### U> '0 (I) ("') 

### "'U ::; 

## ru (I) Cl. CD :> <.C 0



# ..... '0 N Q) <0 W (I) 0 -:­ 

# -. c:: (J) W (I) study:

# I

NEUTA giga/l Day 29 Male, 0 mg/kg bW/day Mean 0.71 SD 0.05 N 5 Male, 10 mg/kg bw/day Mean 0.75 SO 0.22 N 5 Male, 30 mg/kg bw/day Mean 1.21 SO 0.59 N 5 Male, 45 mg/kg bw/day Mean 0.94 SD 0.20 N 4 LYMPHA giga/1 Day 29

5.33 1.34 5

4.98 1.52 5

5.56 0.47 5

5.83 0.44 4 0') Key: Experimental Unit Animal 

## co '< 0' 

### g ::r 

### 'll '0 Q) ::l iii' 

# '" CD .0 c:**~.** (J)

# ~ (I) :::J '0 (I) 

## 3 (ii' 

# '" o· :::J**~** 5' (I) (J) '0 0 :> (J) 0 iil MONOA giga/l Day 29

0.07 0.01 5

0.06 0.02 5

0.14 0.07 5

0.09 0.02 4 DIFFERENTIAL BLOOD COUNT

EOSA g1ga/l Day 29 BASOA giga/l Day 29 LUCA g1ga/l Day 29

0.08 0.03 5 0.04 0.02 5 0.03 0.01 5

0.09 0.05 5 0.03 0.01 5 0.03 0.02 5

0.10 0.02 5 0.03 0.00 5 0.04 0.02 5

0.10 0.03 4 0.03 0.01 4 0.03 0.01 4 Print Time: 15:02:45 Table IB Page 5study:
-i NEUTA LYMPHA MONOA :r (ii' giga/l giga/l giga/l 0. Day 29 Day 29 Day 29 0 (') c: 3 Female, 0 mg/kg bw/day <D**~** iii' Mean 0.58 4.18 0.07 

## s: SD 0.27 0.98 0.01 <D N 5 5 5 '0 

# a '0 (l) Female, 10 mg/kg bw/day 

## q. 2­ Mean 0.51 4.04 0.05 

## s: SD 0.16 0.89 0.01 N 5 5 5 (l) '0 

## '" 0 ::J f/) Female, 30 mg/kg bw/day 0 til iii Mean 0.50 3.61 0.06 

## -g SD 0.06 0.61 0.02 

### '"0 (') N 5 5 5 =t !l) <b 

## co C. 0 Female, 45 mg/kg bw/day CD ::J



# ..... '0 I\.) tl> Mean 0,50 3.76 0.06 <0 ,f;. <b SD 0.08 0.13 0.02 0**~** N 3 3 3 co f/) 

# - W <b Key: Experimental Unit 

### = Animal 00 '< 

### 2­ 0> c:r

:r**~** '0 tl>**~** 

## '" <ii' til .J:J co @' 

## '"**~. ~**  ::J '0 <D 

## 3 (ii' f/) 0' ::J c:r '< 

## s: <D f/) '0 0 ::J f/) 0 til DIFFERENTIAL BLOOD COUNT

EOSA BASOA LUCA giga/l giga/l giga/l Day 29 Day 29 Day 29

0.08 0.02 0.02 0.03 0.01 0.01 5 5 5

0.08 0.02 0.02 0.01 0.01 0,01 5 5 5

0.08 0.02 0.02 0.02 0.01 0.01 5 5 5

0.06 0.02 0.02 0.01 0.00 0.01 3 3 3 Print Date: 21-Dec-2006 Print Time: 15:02:45 Table IB Page 6**~** :T (ii' C. 

## g c: 3 (!) 

## a (ii' 5' (!) "0 

### 3 "0 CI) ;:+ '< 

# a 5' CI) til 1J 0 ::> 0 

# '" ;;;; "'iii" "0 CI) 

### '1J 

## £. !ll 

# II co 0 CD ::>**~** "0 I\.) ru <0 U1 (!) 

### 0 

# - ::- c til W CI) 0'> 0­ 00 '< 0 5'**~** "0 ru 

## a (!) 

## '" m .0 c: 

## ai' 

# '"**~,** ::; CI) ::J 1J CI) 

## '3 (ii' 0' 

# '" ::> CT '< 5' (!) til 1J 0 0 ::> 

## '" ijl 

# 1--­ 

# I Print Date: 06-Dec-2006 Print Time: 15:15:23 CLOTTING ANALYSES Tilile IB study/

­ Page 7

Male, Mean SD N Male, Mean SD N Male, Mean SD N Male, Mean SD N Key: HOT seconds Day 29

0 mg/kg bw/day

10 mg/kg bW/daY

30 mg/kg bw/day

45 mg/kg bw/day k

37.3

1.2 5

315,4

1.4 5

37.0

1.2 5

36.3

0.6 4 k = Kruskal-Wallis + Wilcoxon-test, Two-sided, 

## * p 

### <= 0.050, 

## ** P 

### <- 0.010 Experimental Unit 

### = Animal-i or iii' 

## 8­ (') c: 3 <l> 

### 3­ (ii' 5' <l> "0 

# a "0 <l> 

## q Sl, 5' <l> !II "0 0 ::l !II Q (I) Ul "0 <l> 

### -0 (') III <l> 

# = a. 

### co CD ::l 0**~** "0 I'.) Q) 

## o>'lil 0 

# ­

....... c: W fJ> 0> <l> CXl**~** 0 5' !!; "0 Q)**~.** !II 

# 2 c:**~.** (I)**~**

### ~ ::l "0 <l> 

## 3 iii' (II 

### ci" ::l**~** 5' (1) (II "0 0 ::l (II 0 iii 

# [**.~** Print Date: 06-Deo-2006 Print Time: 15:15:23 CLOTTING ANALYSES Table IB Study:L-

______________ Page 6 HOT seconds Day 29 k Female, 0 mg/kg bw/day Mean 32.1 SD 1.1 N 5 Female, 10 mq/kg bw/day Mean 32.6 SD 1.3 N 5 Female, 30 mg/kg bw/day Mean 32.9 SO 2.6 N 5 Female, 45 mg/kg bw/day Mean 35.7 SO 1.0 N 3 Key: k**~** Kruskal-Wallis 

### + Wilcoxon-test, Two-sided, 

## * p 

### <= 0.050, 

### ** P**<~** 0.010 Experimental Unit**~** Animal-; :::r iii" a. 

### a (") c: 3 a> 

## a or 

## s:­ a> "0 

# a "0 a> ::1­ '< 

### 9" 

## s:­ a> tf> "0 

### a :l tf> Q tf> <il "0 <I> (") 

### "'U :; Q) <I> a. 

## m 0 :;, CO



# ..... "0 

# .. I'V (Q



### ....... a> 0 ::­ 

# - c: (.V a> 

## '" 0) 0­ CP '< 0 

## s:­
"0 

# ..**~** ::1­ (ii" 

## '" m .0 

### s" 

## m 

# '"**~ ~**  CD :l "0 a> 

## 3 iii" f1) 0" ::J 0" '< 

## s:­ CD "0 0 

# '"
:l tf> 

### a Cil Print Date: 06-Dec-2006 Print Time: 15:16:48 ENZYMES Table IB StUdy:! Page 9 ALT AST ALP SOOT ).\kat/l ).\kat/l llkat/l nkat/l Day 29 Day 29 Day 29 Day 29 k k k k Male, 

### o mg/kg bw/day Mean 0.56 1.76 2.53 7 SD 

## o 06 0.16 0.59 5 N 5 5 5 5 Male, 10 mg/kg bw/day Mean 0.53 1.73 1.96 2 SD 0.07 0.23 0.28 3 N 5 5 5 5 Male, 30 mg/kg bw/day Mean 0.82 2.11 2.17 1 SD 0.71 1.12 0.33 2 N 5 5 5 5 Male, 45 mg/kg bw/day Mean 0.54 1.72 2.74 9 SD 0.05 0.26 0.35 9 N 4 4 4 4 Key: Ie Krusleal-Wallis + Wilcoxon-test, Two-sided, 

## * p 

### <= 0.050, 

### ** P 

### <= 0.010 Experimental Unit = AnimalStudy:

# I

-l ::r (ii' a. 0 c: 

## '"' 3 (1) 

## a Female, (ii' S' Mean (1) SD "0 

# a N "0

### ~ '< Female, 

# a S' Mean <!) (fj SD "0 0 N ::I**~** Female, Ul Vi" "0 Mean (1) 12, ""'"Cl SD :::!l Q) (1) N (.Q a. 0 CD ::I Female, I\) 

# '" ..... "0 <0 

## co <1> Mean SD 

# a

# - c: N 

## w**~** 0) 0" ALl' Jlkat/l Day 29 k 0 mg/kg bw/day0.43 0.04 5 10 mg/kg bw/day 0.42 0.05 5 30 mg/kg by/day 0.43 0.06 5 45 mg/kg bw/day 0.43 0.06 3 AST Jlkat/l Day 29 k ALP Jlkat/l Day 29 k ENZYMES

SOOT nkat/l Day 29 k Print Date: Print Time: Table Page 06-Deo-2006 15:16:48 IB 10

1.60 0.26 5 1.33 0.31 5 15 7 5

1,42 0,17 5 1.50 0.39 5 11 5 5

1.37 0,16 5 1,03 0,17 5 6 9 5

1.49 0.25 3 1.43 0.09 3 13 6 3
CO'< Key: k 

### = Kruskal-Wallis + Wilooxon-test, T"Wo-sided, 

### * p 

### <= 0.050, 

### ** P 

### <= 0.010**~** Experimental Unit 

# = Animal ::r**~** "0 III 

## a. (1) (fj (i) J:l c:**~.** 

# '"**~** 

# 1i ::I 

# 1 (ii' 

# a· ::I 0" '< S' (1) "0 

# '" 0 ::I 0 

# '" Ul

### '1J til 

## co CD



# ..... N <.0 0


# .... W CTl 

## co -I ::y tn" e. 

## g c 3 (I) 

### ;a tn" 

### :T (I) "0 

# a "0 (I)

### ~ Q. 

### :T (I) It> "0 0 ::J 

## g; ill
"0 

# "'**~** 3i (I) 0­ 0 ::J "0 Q) <.0 (I)


# ... C It> (I) 0" '< 0 

### :T !!; "0 Q)

### ~ 

## '"**~** c 

# af 

# "'**~**

### ~ ::J 

## al 

## 3 tn" (J) 0" ::J 0" '< 

### :T (I) It> "0 0 ::J 

## :g til NA =1/1 Day 29 k K mmol/1 Day 29 k Male, 0 mg/kg ow/day Mean SO N 142.6 0.8 5 4.68 0.09 5 Male, 10 mg/kg ow/day Mean SO N 143.1 1 2 5 4.70 0.16 5 Male, 30 mg/kg oW/day Mean SO N 142.0 0.3 5 4.84 0.28 5 Male, 45 mg/kg ow/day Mean SO N 142.4 0.2 4 5.04 0.19 4 Key: k 

# = Kruskal-Wallis + Wilcoxon-test, Experimental Unit 

## = Animal CL mmol/1 Day 29 k

103.8

1.4 5

104.2

1.6 5

103.3

0.9 5

103.7

0.7 4 Two-sided, 

### * BLOOD CHEMISTRY

INP mmo1/1 Day 29 k

2.20 0.10 5

2.16 0.19 5

2.23 0.10 5

2.41 0.13 4 P 

### <= 0.050, 

## ** P CA mmol/l Day 29 k

2.62 0.02 5

2.59 0.08 5

2.62 0.09 5

2.62 0.03 4



### <= 0.010 UREA mmol/l Day 29 k

5.70 0.82 5

5.91 0.89 5

5.48 0.78 5

5.37 0.67 4 CREA JlIl101/1 Day 29 k

47.8

2.6 5

44 8

3.6 5

43.4

2.7 5

44.7

2.0 4 Print Date: Print Time: Table page GLUC mmol/1 Day 29 k

6.14 0.75 5

6.30 0.56 5

5.42 0.38 5

5.61 0.16 4 06-Deo-2006 15:17:46 IB 11-l ;:r (i)' c. 0 (') c: 3 <l> 

## a (i)' 

## s: <Il 

# a '0 <l> ::l. '< 

### So 

## s: <Il U> '0 0 :::J U> 0 

### ;;; (j) '0 (1) Q, 

### -0 Q) (1) 

# "" (0 c. 0 

### CD :::J



# ..... '0 W co III 0 (1) 

### 0 :­ 

# - c: W (1) 

## '" 0) 0' 00'<

### ~**~** 

## 1\**~** <Il 

# '" iil .0 c:**~.** :; ::l, ::: (1) :::J '0 <Il 

## 3 g;' 15 :::J 0' '< 

### ::; (1) 

## '" 

## '8 :::J 

## '" 0 

### ;;; 

# I

BLOOD CHEMISTRY Study:

# I

TBIL TPROT ALB GLOB TRIG pmol/l gil gil gil mmol/l Day 29 Day 29 Day 29 Day 29 Day 29 k k k k k Male, 

### o mg/kg bw/day Mean 1.98 62.86 36.72 26.14 0,55 SD 0.25 0.82 1.02 0.21 0,22 N 5 5 5 5 5 Male, 10 mg/kg bw/day Mean 1.62 61.50 35.B7 25.64 0.48 SD 0.33 0.93 0.47 0.84 0.32 N 5 5 5 5 5 Male, 30 mg/kg bw/day Mean 2.48 59,60* 35.39 24.20 0.35 50 1.12 2,30 1.10 1.60 0.10 N 5 5 5 5 5 Male, 45 mg/kg bw/day Mean 1.71 58.03* 34.74 23.29* 0.31 50 0.17 2.04 1.24 0.91 

## o 12 N 4 4 4 4 4 Key: k 

# = Kruskal-Wallis + Wilcoxon-test, Two-sided, * p 

### <= 0.050, ** P 

### <= 0.010 Experimental Unit 

## = Animal Print Date: Print Time: Table Page 06-Dec-2006 15:17:46 IB 12 CHOL mmol/l Day 29 k MG mmo1/1 Day 29 k

1.51 0.16 5 0.90 0.03 5

1.56 0.23 5 0.B6 0.05 5

1.69 0.23 5 0.91 0.06 5

1.81 0.17 4 0.90 0.02 4--i :::r (ji. Cl. 0 (") c 3 CD**~** (ji. g: CD "0 (3 "0 CD ;::). '< Q, g: CD "0 

## '" 0 ::J 

## '" 0 Ul 

# en "0 CD 

### -U (") :3i 

### ru CD CO Cl. 0 CD ::J



## ...... "0 W <0 

# '"

...... CD



### .,..... 0 

## -. c W 

## '" CD O'l eD '< 0­ 0 g:**~** "0 ;::). 

# '" CD· 

## '" CD .0 C**~.** 

## '" '!i 

## "" CD ::J 

## 16 

## 3 (ji. 

## '" o· ::J 0­ '< g: CD "0 

## '" 0 ::J 

## '" Q 

## '" Study:

# I

NA K mmol/l mmol/l Day 29 Day 29 k k Female, 0 mg/kg bw/day Mean 141.5 4.61 SD 1.0 0.21 N 5 5 Female, 10 mg/kg bw/day Mean 142.0 4.26* SD 1.4 0.17 N 5 5 Female, 30 mg/kg bw/day
Mean 141.5 4.25** SD 1.9 0.11 N 5 5 Female, 45 mg/kg bw/day
Mean 142.9 4.67 SD 2.2 0.25 N 3 3 Key: k 

### = Kruskal-Wallis + Wilcoxon-test, Experimental Unit 

### = Animal CL mmol/l Day 29 k

103.5

1.3 5

103.9

1.4 5

103.5

1.5 5

105.2

3.2 3 Two-sided, * BLOOD CHEMISTRY

INP mmol/l Day 29 k

1.90 0.19 5

1.88 0.12 5

1.77 0.17 5

2.07 0.07 3
p 

### <= 0.050, ** P CA mmol/l Day 29 k

2.64 0.06 5

2.65 0.02 5

2.62 0.05 5

2.60 0.08 3


### <= 0.010 UREA mmol/l Day 29 k

6.84 1.07 5

6.42 0.54 5

6.71 0.79 5

6.54 0.75 3 CREA )lI!Iol/l Day 29 k

46.8

3.7 5

47.7

2.5 5

48.7

1.3 5

48.1

2.4 3 Print Date: Print Time: Table Page 06-Dec-2006 15:17:46 IB 13 GLUC mmol/l Day 29 k

5.89 0.65 5

5.14 0.20 5

4.90 0.33 5

5.09 0.40 3-l :T 

### w" a. 0 <1 t: 3 <1> 

## a in' 

## s: <1> "0 

# a "0 <1> 

## q 9. 

## s: <1> "0 

## '" 0 0 ::> 

## '" in til' "0 <1> <1 

### 'lJ 3; Ol <1> (Q a. 0 CI.l ::> ...,\, "0 !\) W co N <1> 

### 0 

# ­ c: (Jl 

# - W <1> 

## en (X)**~** 0 

## s:**~** "0 1»

## ~ 

## '" @ J:l C 

### ca' 

## '"

# ~ <1> ::> 

### 1i1 

## 3 w' (Jl 

### o· ::>**~** 

## s: CD (Jl "0 0 ::> 0 

## '" (il Study:1

Female,
Mean SD N Female,
Mean SO N Female, Mean SD N Female,
Mean SD N TElL pmol/l Day 29 k


## o 1119"/lI:g bW/day2.18 0.55 5 10 mg/kg bw/day 2.32 0.52 5 30 1119"/11:9 bw/day 2.34 0.41 5 45 1119"/kg bw/day 2.23 0.12 3 TPROT gil Day 29 k

64.92 3.13 5

64.56

2.02 5

62.69

1.01 5

5B.46

3.42 3 Key: k = Kruskal-Wallis + Wilcoxon-test, Experimental Unit 

## = Animal Print Date: 06-Dec-2006 Print Time: 15:17:46 BLOOD CHEMISTRY Table IB Page 14
ALB GLOB TRIG CBOL MG gil g/l mmol/l mmol/l mmol/l Day 29 Day 29 Day 29 Day 29 Day 29 k k k k II:

39.37 25.55 0.33 1.70 0.94

2.08 1.09 0.05 0.33 0.04 5 5 5 5 5

38.80 25.76 0.26 1.59 0.98

1.09 1.03 0.03 0.29 0.08 5 5 5 5 5

37.53 25.16 0.27 1.53 0.94

O.BO 0.65 0.04 0.44 0.04 5 5 5 5 5

35.59 22.87 0.28 1.32 0.94

1.64 1.78 0.05 0.25 0.03 3 3 3 3 3 Two-sided, 

## * p <= 0.050, 

### ** P 

### <= 0.010

# ------------------------------------------------------------------------------------------------------------------------------------ -i ::r iii" c. 0 (") c 3 (l) 3­ iii" 5' (l) "0 

# a "0 CD

### ~ 

# a 5' (l) 00 "0 0 ::l 00 0 ii1**~** rn "0 

## l!l 'iJ Q)

### ~ 

## co c. ([) 0 ::l



# ..... "0 W 0> 

## w'f6



# - ":-' c: 0
rn W CD 0'>**o:>~**

### ~ !!l "0 0> ::l iii" 00 

## ro .c c**~.** 

## '"

## ~ m ::l "0 CD 

## 3 iii" (/) 5" ::l 0­ '< 5' (l) 00 "0 0 ::l 00 0 ii1 TABLE IB 15 

## c:::::J

PROJECT NUMBERLI

_____-1 SUMMARY TABLES URINALYSIS: CHEMICAL EXAMINATIONS PRINT DATE 22-DEC-2006
NOMINAL DAYS IN STUDY 25 MAL E S PARAMETER ASSESSMENTLIMITS GROUP 0 

## o MG/KG GROUP 1 10 MGIKG GROUP 2 :30 MGIKG GROUP :3 45 MG/KG PH-VALUE [2] 

### < 7 > = 8 5 0 5 0 5 0 4 0
PROTEIN [2] 

### < 

### > 2 3 5 0 5 0 5 0 4 0 GLUCOSE [1] 

### > 0 1 5 0 5 0 5 0 4 0 KETONES [1] 

### < 

### > 1 2 5 0 5 0 5 0 4 0 UROBILINOGEN [2] 

### < 

### > 1 2 5 0 5 0 5 0 4 0
BILIRUBIN [2] 

### < 

### > 1 2 5 0 5 0 5 0 4 0 BLOOD [1] 

### < 

### > 1 2 4 1 5 0 5 0 4 0 VOLUME (ML) MEAN SD N 5.8 2.0

5 6.0 1.7

5 4.7 1.3

5 4.6 1.1

4 SPEC.GRAVITY (GIL) MEAN SO N 1048 19 5 1047 12 5 1064 18 5 1063 17 4

Fishcr's exact tcst Fisher's exact test (one-sided) (two-sided) [1]: [2]: 

### *: *: p p 

### <= 0.05,



### <= 0.05, 

### **: **: p p 

### <= 0.01



### <= 0.01

# ------------------------------------------------------------------------------------------------------------------------------------ TABLE IB 16

-I :T 00' a. 0 (') c: 3 (1) 3. iii' 9' (1) 

# " 

# a**~** ::l. '< 

### 2. 9' (1) (/) 

# " 0 :::l**~** (il
'0 

# "' (1) Q. 

### '"0 :::!l Q) (1) (Q a. (\) 0 :::l



# ..... '0 W <0 

# "' .j::.. (1)


# - -::­ 

### 0 c:: (/) W (1) 

## m 

## oo '< c­

### ~**~** '0 III ;;L. (1) (/) 

### iil ..0 C 

## or (/)

### ~. 

### 1if :::l (1) 

# " 3**~.** 

### o· :::l c­ '< 9' (1) (/) '0 0 :::l (/) 0 Ul 

# D PROJECT NUMBERLI

_____...J SUMMARY TABLES URINALYSIS: SEDIMENT PRINT DATE 22-DEC-2006 NOMINAL DAYS IN STUDY 25 MAL E S PARAMETER ABSESSMENTLIMITs GROUP 0 o MG/KG GROUP 1 10 MG/KG GROUP 2 30 MG/KG GROUP 3 45 MG/KG CRYSTALS [2) 

### < 2 3 3 2 4 1 3 2 2 2 RENAL TUBULAR [1) 

### < 

### > 1 2 5 0 5 0 5 0 4 0 TRANSITIONAL [1) 

### < > 1 2 5 0 5 0 5 0 4 

### a SQUAMOUS [1) 

### < > 1 2 5 0 5 0 5 0 4 0 CASTS [1] 

### > 

## o 1 5 

### a 5 0 5 0 4 0 ERYTHROCYTES [1] 

### < > 1 2 5 

### a 5 0 5 

### a 4 0 LEUCOCYTES [1] 

### < > 1 2 5 

### a 5 0 5 0 4 0

F1sher's exact test (one-s1ded) [1]: *: p 

### <= 0.05, 

### **: p 

### <= 0.01 F1sher's exact test (two-sided) [2J: 

### *: p 

### <= 0.05, 

### **: p 

### <= 0.01-I ::r iii· e. 0 (") c 3 m 

## "­ 00· 

## s: <I> "0 

# a ." <I> ;:!. '< 

### !2. 

## s: <I> "0 

## '" 0 ::J 0 

## '" Ul (i;' ." m 

### '1J (") 5 !l.l m 

## co e. ct) 0 ::J



# ..... "0 W 0> 

### 0'1'&! 0 -::­ c (J) 

# - W <I> 0) cr 

## co '< 0 

## s: 9l "0 Ql

### ~ (J) (j) .0 5. (j) (/)**~**

### ~ ::J ." m 

### § 00· (/) 

### o· ::J cr '< 

## s: <I> (/) ." 0 ::J VI 0 <il TABLE IB 17 

# c:=J PROJECT NUMBER LI

____--l SUMMARY TABLES URINALYSIS: CHEMICAL EXAMINATIONS PRINT DATE 22-DEC-2006 NOMINAL DAYS IN STUDY 25**-------------------------------------------~---------- -----------------------------------------------------------------------------**  F E MAL E S PARAMETER ASSESSMENT- GROUP 0 GROUP 1 GROUP 2 GROUP 3 LIMITS o MG/KG 10 MG/KG 30 MG/KG 45 MG/KG PH-VALUE (2) 

### < 7 5 5 5 3 

### > 8 0 0 0 0 PROTEIN [2J 

### < 1 5 5 5 3 

### > 2 0 0 0 0 GLUCOSE (1] 0 5 5 5 3 

### > 1 0 0 0 0 KETONES [1] 

### < 1 5 5 5 3 

### > 2 0 0 0 0 UROBILINOGEN [2] 

### < 1 5 5 5 3 

### > 2 0 0 0 0 BILIRUBIN (2) 

### < 1 5 5 5 3 

### > 2 0 0 0 0 BLOOD [1) 

### < 1 5 5 5 3 

### > 2 0 0 0 0 VOLUME MEAN 3.9 4.2 4.1 3.1 (ML) SD 1.0 1.6 1.1 1.0 N 5 5 5 3 SPEC.GRAVITy MEAN 1058 1059 1058 1076 (GIL) SO 14 34 17 13 N 5 5 5 3

Fisher's exact test (one-Sided) [lJ: *: p 

### <= 0.05, 

### **: p 

### <'= 0.01 Fisher's exact test (two-sided) [2] : 

### *: p 

### <
.. 0.05, 

### **: p 

### <'= 0.01-1 ::r iii' 0­ 0 c: 3 

# "
CD 2­ iii' 5' <1> '0 

# a '0 <1> 

# .z g, 5' CD 

## '" 

## '8 :;, 0 

## '" <il (j) '0 <1> 

### 1J 

## g Q) <1> 0­ to 0 

## ro :;,**~** '0**W~** O'l CD 

## o ':-'

- c: W 

## '" CD O'l CT OJ '<:**~** ::r**~** '0 III 

## a <1> IJ) 

## ro -0 c: 

# or IJ)

## ~ ro :;, '0 <1> 

## 3 iii' 6' 

# '" :;, CT '<: 5' <1> II> '0 0 :;, 

## '" 0 <il T1IliILE IB 16 

# CJ PROJECT Ntn4BER 

# 1

. - -I

SUMMARY TABLES URINALYSIS: SEDIMENT PRINT DATE 22-DEC-2006 NOMINAL DAYS IN STUDY 25 FEMALES PARAMETER ASSESSMENTLIMITS GROUP 0 

## o MG/KG GROuP 1 10 MG/KG GROUP 2 30 MG/KG GROUP 3 45 MG/KG CRYSTALS [2) 

### < 2 3 4 1 5 0 4 1 3 0 RENAL TUBUI..AR [1] 

### < > 1 2 5 0 4 1 5 0 3 0 TRANSITIONAL [1

### J < > 1 2 5 0 4 1 5 0 3 0 SQUAMOUS [1] 

### < 

### > 1 2 5 0 5 0 5 0 3 0 CASTS (1

### J 

### > 0 1 5 0 5 0 5 0 3 0 ERYTHROCYTES [1] 

### < > 1 2 5 0 5 0 5 0 3 0 LEUCOCYTES [1] 

### < > 1 2 5 0 5 0 5 0 3 

# °

Fisher's exact Fisher's exact test test (one-sided) (two-sided) [1]: [2]: 

### *; 

### *: p p 

### <=

<= 0,05, 0.05, 

### **: 

### **: p p 

### <- 0.01

<= 0.01

# D PATHOLOGY REPORT Repeated Dose Oral Toxicity st;.Jdy in Wistar rats; Administration by gavage for 4 weeks ABSOLUTE WEIGHTS

- 

### MEAN VALUES (MALE) Sacrifice 

### Fl Sex M Group 

## o 1 2 

### 3 Terminal body weight g M 266.44 255.16 248.78 253.85 SD 19.433 22.387 14.781 29.447

n 5 

### 5 5 4 Adrenal glands mg M 68.0 64.2 71.6 63.75 SD 8.86 5.675 7.701 7.676



### n 5 

### 5 5 4 Brain 

### g M 1.92 1.894 1.948 1.925 SD 0.041 0.091 0.065 0.079



### n 

### 5 5 5 

### 4 Epididymides 

### g M 0.734 0.74 0.746 0.753 SD 0.046 0.03 0.055 0.01

n 

### 5 

### 5 5 4
Heart 

### g M 0.844 0.782 0.802 0.793 SD 0.06 0.075 0.065 0.105

n 

### 5 5 5 4 Kidneys 

### g M 2.07 2.018 2.032 1.94 SD 0.217 0.073 0.122 0.272



### n 5 5 5 

### 4 Liver 

### g M 7.25 6.934 6.758 6.823 SD 0.557 0.645 0.7 0.792



### n 

### 5 5 5 4 Spleen g M 0.532 0.488 0.484 0.488 SD 0.008 0.041 0.058 0.021

n 

### 5 

### 5 5 4 Testes 

### g M 2.98 2.87 2.892 2.95 SD 0.381 0.134 0.275 0.212



### n 

### 5 5 5 4 Thymus mg M 460.8 418.0 404.0 390.75 SD 80.051 56.303 80.165 42.649

n 

### 5 

### 5 5 

### 4 Thyroid glands mg M 18.8 17.0 19.4 18.0 SD 2.49 2.236 2.302 1.414

n 

### 5 5 5 4


### *: P 

### <= 0.05, 

### **: P 

### <= 0.01 Kruska1-Wallis H and Wilcoxon test, two sided

This document is the property ofthe sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

## Page 137 of368

### PATHOLOGY REPORT Repeated Dose Oral Toxicity study in Wistar rats; IC- 2/8 04.Jan.2007 

### WEKA Administration by gavage for 4 weeks ABSOLUTE WEIGHTS MEAN 

### VALUES Sacrifice Sex Group Terminal body weight

Adrenal glands

Brain

Heart

Kidneys

Liver

Ovaries

Spleen

Thymus

Thyrol.d glands

Uterus g M 

### SD

n 

### mg M 

### SD

n 

### g M 

### SD

n 

### g M 

### SD

n
g M 

### SD

n 

### g M 

### SD

n 

### mg M 

### SD

n 

### g M 

### SD

n 

### mg M 

### SD

n 

### mg M 

### SD

n 

### g M 

### SD

n 

### (FEMALE) F1

F 

## o 179.12

10.351 

### 5 83.8 14.55



### 5
1.

### 824 0.034 5 

### 0.626 0.057 

### 5 1.526 0.253 

### 5 5.026 0.573 

### 5 94.0 13.454

5 

### 0.376 0.086 

### 5 449.6

70.436

5 

### 15.4

3.286 

### 5 0.672 0.305 

### 5 1 

### 175.7

5.703 

### 5 

### 84.4

4.506 

### 5 1.812 0.086 

### 5 0.608 0.019 

### 5 1.508 0.092 5 

### 4.892 0.33 

### 5 95.6

5.941 

### 5 0.386 0.049 

### 5 381.0

68.953

5 

### 16.6 2.608 

### 5 0.53 0.201 

### 5 2 

### 169.78

5.598 

### 5 78.6

7.021 

### 5 1.86 0.066 

### 5 0.586 0.031 

### 5 1.338 0.141 5 

### 4.506 0.163 

### 5 83.0

7.81 5 

### 0.392 0.074 

### 5 348.8 

## *

29.004

5 

### 15.0

2.449 

### 5 0.646 0.285 

### 5 3 172 .2

25.827

3 

### 76.333 13.051 3 

### 1.927 0.145 3 

### 0.61 0.085 3 1.

### 41 0.128 3 

### 4.7 0.702 3 

### 89.333 26.633

3 

### 0.403 0.015 3 

### 327.0

88.017

3 

### 18.0

3.606 3 

### 0.817 0.133 3



### *: P 

### <= 0.05, 

### **: P 

### <= 0.01 

### Kruskal-Wallis Hand t\filcoxon test, two sided

This document isthe propertyofthe sponsors (specified on page 1). use byother parties requires written permission bythe sponsors 

## Page 138 of 368

### PATHOLOGY REPORT 

# D rr­

- 3/8

# I Repeated Dose Oral Toxicity st:J.dy in Wistar' rats; 04.Jan.2007 WEKA 

### Adminlstration by gavage for 4 weeks RELATIVE WEIGHTS

- MEAN VALUES (MALE) 

### sacrifice F1 

### Sex M 

### Group 

## o 1 2 3 

### Terminal body weight M 

### 100.0 100.0 100.0 100.0 

### n 5 5 

### 5 

### 4 

### Adrenal glands % M 

### 0.026 0.025 0.029 0.025 

### SD 0.003 0.003 0.002 0.001



### n 5 5 

### 5 

### 4 

### Brain % M 

### 0.724 0.745 0.785 0.765 

### SD 0.057 0.042 0.04 0.087

n 5 

### 5 

### 5 4 

### Epididymides % M 

### 0.276 0.292 0.301 0.299 

### SD 0.009 0.027 0.029 0.032

n 5 5 

### 5 

### 4 

### Heart % M 

### 0.317 0.306 0.323 0.312 

### SD 0.007 0.012 0.024 0.007

n 

### 5 5 

### 5 4 

### Kidneys M 

### 0.777 0.795 0.817 0.763 

### SD 0.059 0.064 0.023 0.019

n 5 5 

### 5 4 

### Liver M 

### 2.723 2.721 2.712 2.689 

### SD 0.133 0.162 0.143 0.098



### n 5 

### 5 

### 5 4 

### Spleen M 

### 0.201 0.192 0.195 0.193 

### SD 0.017 0.014 0.024 0.017



### n 5 5 

### 5 4 

### Testes M 

### 1.117 1.135 1.167 1.169 

### SD 0.098 0.149 0.135 0.101

n 

### 5 5 

### 5 4 

### Thymus % M 

### 0.174 0.164 0.163 0.154 

### SD 0.033 0.014 0.033 0.008

n 5 

### 5 

### 5 4 

### Thyroid glands M 

### 0.007 0.007 0.008 0.007 

### SD 0.001 0.001 0.001 0.001



### n 5 5 

### 5 4


### *: P <= 

### 0.05, 

### **: P <= 

### 0.01 

### Kruskal-Wallis H and Wilcoxon test, two sided

This document is the propertyofthe sponsors (specified on page 1), use byother parties requires written permission bythe sponsors 

## Page 139 of 368

# o PATHOLOGY REPORT IC- 4/8 

# I Repeated Dose Oral Toxicity study in wistar rats; 04.Jan.200J 

## WEKA Administration by gavage for 4 weeks RELATIVE WEIGHTS

- MEAN VALUES (FEMALE) Sacrifice F1 Sex F Group 

## o 1 2 3 Terminal body weight % M 100.0 100.0 100.0 100.0 n 

### 5 5 

### 5 3 Adrenal glands M 0.047 0.048 0.046 0.044 SD 0.007 0.002 0.004 0.002



### n 

### 5 5 5 3 Brain M 1.02 1.031 1.096** 1.129 SD 0.041 0.033 0.031 0.113

n 

### 5 5 5 3 Heart % M 0.349 0.346 0.345 0.355 SD 0.021 0.016 0.01 0.011

n 

### 5 5 5 3 Kidneys % M 0.85 0.859 0.787 0.824 SD 0.113 0.051 0.058 0.064



### n 5 5 

### 5 3 Liver M 2.804 2.785 2.656 2.73 SD 0.251 0.179 0.107 0.099



### n 5 5 

### 5 3 Ovaries % M 0.053 0.054 0.049 0.052 SD 0.008 0.002 0.004 0.015



### n 5 5 5 3 Spleen M 0.21 0.219 0.231 0.238 SD 0.047 0.025 0.046 0.038



### n 

### 5 5 

### 5 3 Thymus % M 0.25 0.217 0.206 0.188 SD 0.029 0.042 0.02 0.027

n 5 

### 5 5 3 Thyroid glands M 0.009 0.009 0.009 0.011 SD 0.002 0.002 0.001 0.002



### n 

### 5 5 5 3 Uterus % M 0.377 0.303 0.381 0.481 SD 0.17 0.117 0.166 0.113



### n 5 

### 5 5 3



# ...........................**~** 

## ..**~** 

# ............................. "' 

# ........................ *: P <= 0.05, 

### **: P 

### <= 0.01 Kruska1-Wa11is Hand "h1coxon test, two sided

This document is the propertyofthe sponsors (specified on page 1). use by otherparties requires written permission by the sponsors 

## Page 140 of 368

### PATHOLOGY REPORT Repeated Dose Oral Toxicity study in Wistar rats; Administration by gavage for INCIDENCE OF GROSS LESIONS Sacrifice Sex Group Animals in selected group No abnormalities Adrenal cortex Discoloration Axillary lymph nodes Enlarged Brain Discoloration Epididymides

Abscess Glandular stomach

Erosion/ulcer HypereIllla Iliac lymph nodes

Enlarged Liver Focus Lungs

Discoloration 

### 4 weeks

FI

M 

## o 5
4 I 5 5 2 5
;; 

### 3 5 2 F 

## o 5
5 I 

### 5 5

I 1

I

I 1

1 

### IC- 5/8 

# I I**O~ .Jan.2007**  

### WEKA

2 

### 3 5 

### 5
5 3

I

I



### 2

I



### 2

I

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## Page 141 of 368

# -

PATHOLOGY REPORT Repeated Dose Oral Toxicity study in Wistar rats; Administration by gavage for 4 weeks INCIDENCE OF ALL MICROSCOPIC FINDINGS Sacrifice F1 

### Sex M F 

### Group 

## o 1 2 3 

## o 1 

### 2 3 

### Animals in selected group 5 5 5 5 5 5 5 5 

### Adrenal cortex exam. 5 5 5 5 

### Congestion 1 

### Accessory cortical**tiss~e** 2 

### 1 

### Adrenal medulla exam. 5 5 5 

### 5 

### Axillary lymph nodes exanL 

### 5 5 5 5 

### Bone marrow (femur) exam. 5 5 5 5 

### Brain exam. 5 5 5 5 

### Cecum exam. 5 5 5 

### 5 

### Cervical cord exam. 5 5 

### 5 5 

### Colon exam. 5 5 

### 5 5 

### Duodemlln exam. 5 5 5 5 

### Epididymides exam. 5 5 

### Infiltration, lymphoid, (m)f 2 

### 1 

### Debris 1 

### Oligospermia 1 

### Granuloma, spermatogenic, (m) 

### 2 Forestomach exam. 5 5 5 5 

### Glandular stomach exam. 5 5 5 5



### Hyperemia, focal 1 

### Heart exam. 5 5 5 5 

### Ileum exam. 5 5 5 5 

### Iliac lymph nodes exam. 2



### Apoptotic necrosis, (m)f 1 

### Sinus histiocytosis 1 

### Hyperplasia, lympho-reticulo 1 

### Jejunum exam. 5 5 5 

### 5



### Apoptotic necrosis, Peyer's 1 

### Mineralization, Peyer's p. 1 1

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### PATHOLOGY REPORT IC- 7/8 

# D I I

Repeated Dose Oral Toxicity study in Wistar rats; 04.Jan.2007 WEKA Administration by gavage for 4 weeks INCIDENCE OF ALL MICROSCOPIC FINDINGS Sacrifice FI Sex M F Group 0 

### I 2 3 

## a I 2 3 

### Animals in selected group S S 5 5 5 5 5 5



# . ..

# . .

# .............. ... .. 

# . . .

# ....... ...... "

# .

# . ... 

## .

# . . .. . .

# . .. 

## . . .

# . . . . .. .

# .. ... 

# ..

# .. .. 

### .

## . .

# .. .

# ....**~ ~** **~** 

### Kidneys exam. S S 5 5



### Congestion 1 1 

### Cyst(s) 1 

### Mineralization, medulla, (m) 3 2 

### Tubules, basophilic, (m)f 3 2 1 2 

### Liver exam. 5 

### 1 5 5 5 

### Congestion 1 

### 1 

### Infiltration, lymphoid, (rn) f S 

### 3 3 3 

### Hematopoiesis, extramedullar 1 

### Necrosis, (IT.ulti)focal 1 1 1 

### Fatty change, single cell I 

### LUITbar cord exam. S 5 S 5 

### I,ungs exam. S 5 S 5 

### Congestion 1 1 

### Hyperplasia, neuroendocr. c. I 

### Eosinoph. cell infiltr., (m) 2 1 1 2 

### Histiocytosis, alveolar, (m) 1 1 

### Mesenteric lymph n. exam. 5 5 S 5



### Apoptotic necrosis, (m)f 1 

### Ovaries exam. 5 5 

### Oviducts exam. 5 

### 5 

### Parathyroid glands exarn. 5 S 4 5 

### Prostate exam. 5 

### 5



### Atrophy, diffuse 1 

### Rectum exam. S 5 5 5 

### Sciatic nerve exam. S S 5 

### 5 

### Seminal vesicle exa:TI. 5 

### 5



### Atrophy, diffuse 1 

### Spleen exam. S 5 S S



### Hematopoiesis, extramedullar 1 

### Testes exam. S 5



### Debris in sem. tubules, (m)f 1

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## Page 143 of 368

### PATHOLOGY REPORT Repeated Dose Oral Toxicity study in Wistar rats; Administration by gavage for 

### 4 weeks INCIDENCE OF 

### ALL MICROSCOPIC FINDINGS Sacrifice Sex Group**~~imals ~n**  

### selected group Fl M 

## o 5 1 

### 5 2 

### 5 3 5 

### Thoracic cord exam. 5 5 

### Thymus exam. 5 5 

### Cyst(s) 1 

### Thyroid glands exam. 5 

### 5 Ectopia, thymic tissue Cyst, branchiogenic 1 

### Trachea exam. 5 5 

### Urinary bladder exam. 5 

### 5 Uterus exam. Dilation of horn(s) Vagina exam. 

# If 8/8 

### 04.Jan.2007 WEKA

F 

## o 5 5 5 1 

### 5 2 

### 5 3 5 5 5

5 

### 5 1



### 5
5 

### 5 

### 2 5 5 

### 5 5 

### 4 5

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## Page 144 of 368