PMN Submission 


For 


TS-EV07J3 


Sanitized Copy 


Book II ofII 

Attachment #19 

STUDY TITLE 

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 of the Sponsor(s). It is the property of the Sponsor(s) and may be used 

a encies actin 

only for that purpose for which it was intended by the Sponsor(s). Every other or additional use. exploitation, reproduction, 

publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory 

within the limits of their administrative authorit . 

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

Page 1 of 99 
THIS PAGE IS INTENTIONALLY LEFT BLANK 


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

Page 2 of 99 

GLP COMPLIANCE STATEMENT 


This study was conducted in accordance with the OECD Principles of Good Laboratory 

Practice and the GLP Principles of the German "Chemikaliengesetz" (Chemicals Act) which 

meet the United States Environmental Protection Agency Good Laboratory Practice 

Standards [40 CFR Part 160 (FIFRA) and Part 792 (TSCA)], with the exception that 

recognized differences exist between the GLP Principles/Standards of OECD and the 

Principles/Standards of FIFRA and TSCA. 

To be filled for USA EPA submission only: 

Sponsor 

Date 

Submitter 

Date 

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

Page 3 of 99 
Statement for US EPA submissions only: 

FLAGGING CRITERIA 


The flagging criteria of 40 CFR 158.34 are not applicable for this study type 


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

Page 4 of 99 
SIGNATURE PAGE 

Study Director: 

Management: 

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

Page 50f99 
CONTRIBUTORS TO THE STUDY I SUPERVISORY LABORATORY PERSONNEL 


Head of Experimental Toxicology and 


Ecology: 


Study Director: 


Data processing: 


Statistics: 


Analytical Chemistry: 


Quality Assurance Unit (QAU): 


Genotoxicity: 


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

Page 6 of 99 
-


STATEMENT OF THE QUALITY ASSURANCE UNIT 

The Quality Assurance 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 of study 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 of study: 07-14-2006 07-14-2006 

Report: 11-09-2006 11-09-2006 

~~~~~---.--

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

Page 70f99 
GLP·CERTIFICATE (FROM THE COMPETENT AUTHORITY) 

Gute Laborpraxls I Good Laboratory Practice 

GLP·Bescheinigung 

(gem./according to 

I Statement 

§ 19 Abs. 1 Chemikaliengesetz) 

of GLP Compliance •

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 Test facility 

PrQfung nach Kategorien / Areas of Expertise 

(gem. I according ChernVwV-GlP Nr. 5.3/OECD guidance) 

1,2,3,4,5,8,9 

Kat. 9 - Biochemische und pathologische Untersuchungen zu Wirkmechanismen / 


Biochemical and pathological examinations conceming mode of action 


Datum der Inspektion / Date of Inspection 


(Tag.Monat Jahr 1day.month year) 


08.06.2005 und 25. bls 27.07.2005 

Die genannte PrOfeinrichlung befindet slch im The above mentioned test facility 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 of GLP. 


gefUhrt werden kOnnen. 


55116 Mainz 

~1llilw;;1:dU:!a1LIIlL1..SlQllll1lure. Date 

landesamt fur Umwelt. Wasserwirtschaft und Gewerbeaufsicht 

Kaiser-Friedrich-SIraBe 7 

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

Page 8 of 99 
CONTENTS 

TITLE PAGE 

BLANK PAGE 

GLP COMPLIANCE STATEMENT 

FLAGGING CRITERIA 

SIGNATURE PAGE 

CONTRIBUTORS TO THE STUDY I SUPERVISORY LABORATORY PERSONNEL 

STATEMENT OF THE QUALITY ASSURANCE UNIT 

GLP-CERTIFICATE (FROM THE COMPETENT AUTHORITY) 

CONTENTS 

1. SUMMARY 

2. INTRODUCTION 

2.1. OBJECTIVES 

2.2. SELECTION OF DOSES/CONCENTRA TIONS 

2.3. TEST GUIDELINES 

2.4. STUDY DATES 

2.5. RETENTION OF RECORDS 

3. MATERIAL AND METHODS 

3.1. TEST SU BST ANCE 

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 VEH ICLE, TEST GROUPS AND DOSES 

3.4.1. Choice of the vehicle 

3.4.2. Pretests for dose selection 

3.4.3. Test groups and doses - 1st experiment 

3.4.4. Test groups and doses - 2nd experiment 

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

Page 9 of 99 
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 OF TEST SUBSTANCE PREPARATION 

EXPERIMENTAL PROCEDURE 

Cell cycle time 

Sampling times 

Control articles 

Preparation of test cultures 

Treatment of test cultures 

Preparation of metaphase spreads 

EVALUATION 

Mitotic index 

Cell counts 

Cell morphology 

Treatment conditions 

Chromosome analysis 

Acceptance criteria 

Assessment criteria 

Statistics 

RESULTS 

CHROMOSOME ANALYSIS - 1 ST EXPERIMENT 

Assay without S-9 mix; 4-hour exposure. 18-hour sampling time 

Assay with S-9 mix; 4-hour exposure, 18-hour sampling time 

CHROMOSOME ANALYSIS - 2ND EXPERIMENT 

Assay without S-9 mix; 18-hour exposure. 18-hour sampling time 

Assay without S-9 mix; 18-hour exposure, 28-hour sampling time 

Assay with S-9 mix; 4-hour exposure. 28-hour sampling time 

MITOTIC INDEX 

CELL COUNTS 

CELL MORPHOLOGY 

ASSESSMENT OF THE SLIDES 

TREATMENT CONDITIONS 

DISCUSSION 

CONCLUSION 

REFERENCES 

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

Page 10 of 99 
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 

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

Page 11 of 99 
1. SUMMARY 

The substancel ~as assessed for its potential to induce structural chromosomal 

aberrations (clastogenic activity) and/or changes in the number of chromosomes 

(aneugenic activity) in V79 cells in vitro both in the presence and in the absence of a 

metabolizing system. 

According to an initial range-finding cytotoxicity test for the determination of the highest 

experimental doses, the test substance did not exhibit any pronounced toxicity up to the 

highest recommended doses, i.e. 1 500 IJg/mL (-10 mM), the following doses were tested 

and the test groups in bold type were evaluated: 

• 1st experiment 

4-hour exposure, 18-hour sampling time, without 5-9 mix 


0; 187.5; 375; 750; 1 500 IJg/mL 


4-hour exposure, 18-hour sampling time, with 5-9 mix 


0; 187.5; 375; 750; 1 500 IJg/mL 


• 2nd experiment 

For confirmation of the results of the 1st experiment including continuous exposure and 

a second sampling time, the following doses were tested and the test groups in bold type 

were evaluated: 

18-hour exposure, 18-hour sampling time, without 5-9 mix 


0; 187.5; 375; 750; 1 500 IJg/mL 


18-hour exposure, 28-hour sampling time, without 5-9 mix 


0; 375; 750; 1 500 IJg/mL 


4-hour exposure, 28-hour sampling time, with 5-9 mix 


0; 187.5; 375; 750; 1 500 IJg/mL 


This document is the property of the sponsors (specified on page 1 j. use by other parties requires written permission by the sponsors 

Page 12 of 99 
About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells at a 

metaphase-like stage of mitosis (c-metaphases). After preparation of the chromosomes 

and staining with Giemsa, 100 metaphases for each culture in the case of the test substance, and vehicle controls, or 50 cells for each culture in the case of the concurrent 

positive controls, were analyzed for chromosomal aberrations. 

The negative controls (vehicle controls) gave frequencies of aberrations within the range 

expected for the V79 cell line. 

Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected 

increase in the number of cells containing structural chromosomal aberrations. 

On the basis of the results of the present study, the test SUbstance did not cause any 

increase in the number of structurally aberrant metaphases inc!. and excl. gaps at both 

sampling times either without S-9 mix and/or after adding a metabolizing system in two 

experiments performed independently of each other. 

No increase in the frequency of cells containing numerical aberrations was demonstrated 

either. 

Thus, under the experimental conditions of this assay, is considered not to be a 

chromosome-damaging (clastogenic) agent under in vitro conditions in V79 cells. 

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

Page 13 of 99 
2. INTRODUCTION 

2.1. OBJECTIVES 

The aim of the present study was to assess the potential of the test substancd lor its 

metabolite(s) to induce structural chromosomal aberrations as a consequence of clastogenic 

activity and/or polyploidy including endoreduplicated cells due to an aneugenic mode of 

action. For this purpose, an in vitro cytogenetic assay was carried out in V79 cells (1, 2, 3). 

Most substances are not mutagenic or carcinogenic themselves, but only after metabolic 

transformation, which in vivo is catalyzed mainly by the enzyme systems of the liver. 

Therefore, the tests are carried out not only directly, but also in the presence of an 

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/concentrations were selected on the basis of the non-GLP range finding part 

of the study (see also section 3.4.). The raw data of this part are archived together with the 

raw data of this 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.1 0 (19 May 2000) 

• EPA/OPPTS 870.5375 (August 1998) 

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

Page 14 of 99 
2.4. STUDY DATES 

Test dates: Study initiation date: 06 Jul2006 

Experimental starting date 

(seeding of cells, 1 st experiment): 12 Jul2006 

Experimental completion date 

(draft report to QAU): 19 Oct 2006 

2.5. RETENTION OF RECORDS 

GLP-relevant records and materials are stored at I Jfor at least the 

period of time specified in the GLP principles. Details concerning responsi ilities or locations 

of archiving can be seen from the respective SOPs and from the raw data. 

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

Page 15 of 99 
3. MATERIAL AND METHODS 

3.1. TEST SUBSTANCE 

The analyses of the test substance will be I were carried out at the I I 	 I ~--~ 

Name of test substance: 

Test substance No.: 

Batch identification: 

CAS No.: 

Purity: 	 99.9 g/1 00 9 (see test substance characterization; 

Project No.1 ~ 

Homogeneity: 	 The homogeneity of the test substance was guaranteed 

on account of the high purity and was ensured by mixing 

before preparation of the test substance preparations. 

Storage stability: 	 The stability of the test substance under storage 

conditions throughout the study period will be verified by 

reanalysis on completion of the experimental phase of 

the study or will be substantiated otherwise. The results 

will be reported in an amendment to this report. 

ADDITIONAL TEST SUBSTAI\JCE INFORMATION 

Date of production: 


Molecular weight: 


Physical state, appearance: 


Storage conditions: 


1995 

148.19 glmol 

Solid, beige 

Room temperature 

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

Page 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 of about 12 - 16 hours) 

• high plating efficiency 90%) 

• stable karyotype (modal number of 22 chromosomes). 

Stocks of the 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 for the cytogenetic 

experiments was checked for 

• mycoplasma contamination 

• karyotype stability 

• plating efficiency (= colony forming 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-hour treatment), MEM medium was used without 

FCS supplementation. 

3.2.3. Cell culture 

Deep-frozen cell suspensions were thawed at 3rC in a water bath, and volumes of 

0.5 mL were transferred into 25 cm

2 plastic flasks containing about 5.0 mL MEM (minimal 

essential medium incl. glutamine), supplemented with 10% FCS (fetal calf serum) 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 after dispersion with 0.25% trypsin solution (about 1.0 mL). 

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

Page 17 of 99 
3.3. EXOGENOUS METABOLIC ACTIVATION 

3.3.1. S-9 fraction 

The S-9 fraction was prepared according to AMES et al. (4). 

At least 5 male Sprague-Dawley rats (200 - 300 g, Charles River Laboratories, Germany 

GmbH) received a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 200 mg/mL 

solution in corn oil) per kg body weight 5 days before sacrifice. 

During this time, the animals were housed in Makrolon cages and were accommodated in 

fully air-conditioned rooms in which central air conditioning guaranteed a range of 

temperature of 20 - 24"C and a relative humidity of 30 - 70%. The day/night rhythm was 

12 hours (12 hours light from 6.00 -1B.00 hours and 12 hours darkness from 1B.00 - 6.00 

hours). 

Standardized pelleted feed and tap water from bottles were available ad libitum. 

5 days after administration, the rats were sacrificed and the livers were prepared (all 

preparation steps for obtaining the liver microsome enzymes were carried out using sterile 

solvents and glassware at a temperature of +4"C). The livers were weighed and washed in a 

weight-equivalent volume of a 150 mM KCI solution, then cut into small pieces and 

homogenized in three volumes of KCI solution. After centrifugation of the homogenate at 

9 000 x g for 10 minutes at +4"C, 5-mL portions of the supernatant (so-called S-9 fraction) 

were stored at -70"C to -BO"C. 

3.3.2. S-9 mix 

The S-9 mix was prepared freshly prior to each experiment (4). For this purpose, a sufficient 

amount of S-9 fraction was thawed at room temperature, and 1 volume of S-9 fraction was 

mixed with 9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, 

was kept on ice until used. The concentrations of the cofactors in the S-9 mix were: 

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 NaH2P04 

solution at a ratio of about 4 : 1. 

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

Page 18 of 99 
3.4. SELECTION OF VEHICLE, TEST GROUPS AND DOSES 

3.4.1. Choice of the vehicle 

Due to the limited solubility of the 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 

for which historical control data are available. 

3.4.2. Pretests for dose selection 

The doses for the 1st experiment (18-hour sampling time) were determined from appropriate 

range-finding cytotoxicity tests with cultures exposed for the duration of 4 hours to a wide 

dose range of the test article, i.e. 1 IJg/mL - 1 500 IJg/mL culture medium both without S-9 

mix and after adding a metabolizing system. In these pretests, various parameters were 

checked for all or at 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 nontoxic test substances 

should not exceed a limit of 5 mg/mL or 10 mM, as recommended by the EC, OECD and by 

an ICPEMC Task Group (6). 

According to the results of the pretests, the test substance is nontoxic after an exposure time 

of 4 hours up to the highest recommended dose of 10 mM (- 1 500 IJg/mL). 

On the basis of the findings from the pretests, the top dose in the 1 st experiment was 

selected after an exposure period of 4 hours with and without S-9 mix taking into account 

the current guidelines (see item 1.2.) or recommended criteria (6). 

The results of the pretests after continuous treatment applied in the 2nd experiment are 

given in the table on page 22. 

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. 

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

Page 19 of 99 
Table 1: 


4-hour exposure, 18-hour sampling time, 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 key to abbreviations and explanations see page 23. 

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

Page 20 of 99 
Table 2: 

4-hour exposure, 18-hour sampling 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 key to abbreviations and explanations see page 23. 

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

Page 21 of 99 
Table 3: 


18-hour exposure, 18-hour sampling time, without 5-9 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. 

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

Page 22 of 99 
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 of good quality 

2 = reduced number of analyzable metaphases 

3 = reduced number of analyzable metaphases, some of poor quality 

R = evaluation possible but with reservations 

= considerably reduced number of analyzable metaphases 

2 = considerably reduced number of analyzable metaphases, some of poor quality 

3 = evaluation of metaphases influenced by precipitation of the 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 of the 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 

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

Page 23 of 99 
3.4.3. Test groups and doses - 1 st experiment 

The number of test groups selected on the basis of the pretests and evaluated in the 

1st main cytogenetic experiment can be seen from the following table. Duplicate cultures 

were used for all 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 - 375.0 fJg/mL 200

4 18 - 750.0 fJg/mL 200 

4 18 - 1 500.0 fJg/mL 200 

4 18 - 500.0 fJ9/mL EMS 100·

4 18 + Vehicle control (DMSO) 200 

4 18 + 187.5 fJ9/mL ** 

4 18 + 375.0 fJ9/mL 200 

4 18 + 750.0 fJ9/mL 200 

4 18 + 1 500.0 fJ9/mL 200 

4 18 + 0.5 fJg/mL CPP 100* 

* 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 

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

Page 24 of 99 
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 meta phases 

analyzed was reduced from the planned 200 mitoses to 100. 

** No evaluation 

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

Page 25 of 99 
3.5. TEST SUBSTANCE PREPARATION 

The substance was dissolved in DMSO. 


To achieve a solution of the test substance in the vehicle, the test substance preparation was 


shaken thoroughly. 


All test substance formulations were prepared immediately before administration. 


3.6. ANALYSIS OF TEST SUBSTANCE PREPARATION 

The stability of the test substance at room temperature in the vehicle DMSO and in water 

each over a period of 4 hours was verified analytically. 

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

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

Page 26 of 99 
3.7. EXPERIMENTAL PROCEDURE 

3.7.1. Cell cycle time 

The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours [last measurement 

based on the BrdU method of SPEIT, G. et al. (12): July 2006] under the selected culture 

conditions. Thus, the selected 1st sampling time of 18 hours (see item 3.7.2.) was within the 

1 - 1.5 x the normal cell cycle time, as recommended by the OECD Guideline No. 473 "In 

Vitro Mammalian Chromosome Aberration Test". The later sampling time of 28 hours was 

chosen to cover a possible cell cycle delay. 

3.7.2. Sampling times 

Chromosomal aberrations were generally analyzed in the first metaphase after they had 

formed to avoid loss during mitoses or conversion of the initial aberrations into more complex 

derivatives during subsequent cell cycles. 

Since aberrations are induced by the majority of chemical clastogens during DNA replication, 

the sampling time must allow cells to progress through the S-phase after treatment so that 

initial DNA damage is converted to chromosome alterations visible at mitosis. 

Since V79 cells are asynchronous and different chemicals may affect different stages of the 

cell cycle, more than one sampling time is necessary. Furthermore, mitotic delay may result 

from clastogen exposure and thus considerably delay the first post-treatment mitosis. 

Therefore, samples were taken at 18 hours and 28 hours covering the intervals at which 

maximum aberration frequencies were expected. 

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

Page 27 of 99 
--~--------------------------------------------------------------~ 

3.7.3. Control articles 

• 	 Vehicle control 

The vehicle controls with and without S-9 mix only contained the vehicle for the test 

substance at the same concentration and volume used in the test culture. 

• 	 Positive controls 

The following positive control substances for clastogenicity were used to demonstrate the 

sensitivity of the test method and the activity of the 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 is well-defined under the selected culture conditions, since 

both positive control articles are well-established reference clastogens. 

3.7.4. Preparation of test cultures 

• 	 Logarithmica"y growing cultures (each after the 2nd passages in the 1st and 2nd 

experiments) which were more than 50% confluent were trypsinized (0.25% trypsin 

solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Prior to trypsin treatment, 

the cells were 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 chamber of 

Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture. 

• 	 The Quadriperm dishes were incubated with 5% CO2 at 3rC and ~ 90% humidity. 

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

Page 28 of 99 
-


3.7.5. Treatment of test cultures 

About 24 - 30 hours after seeding 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 or without 

1 mL S-9 mix. Concurrent negative and positive controls (see item 3.7.3.) 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 or 24 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 of a sampling 

time of 28 hours incubated again for another 10 hours. 

3.7.6. Preparation of metaphase spreads 

• Cell harvest and preparation of slides 

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 hypotonic treatment, 5 mL of a 0.4% KCI solution which was at 3rC was added 

for about 20 minutes. 

Subsequently, 5 mL of fixative (methanol: glacial acetic acid/3 : 1) which was added 

at 4°C and kept for at least 15 minutes and 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 of the 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. 

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

Page 29 of 99 
3.8. EVALUATION 

3.8.1. Mitotic index 

A mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in 

both experiments. 

3.8.2. Cell counts 

For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) 

were treated in the same way as in the main experiment. Growth inhibition was estimated by 

counting the number of cells in the dose groups in comparison with the concurrent vehicle 

control at the end of the culture period using a counter (easy 1). 

3.8.3. Cell morphology 

About 3 hours and 16 - 18 hours after test substance treatment, cultures of all test groups 

were checked for cell morphology, which is an indication of attachment of the cells to the 

slides. 

3.8.4. Treatment conditions 

pH values and osmolarity were measured. The solubility of the test substance in the vehicle 

used and in the aqueous culture medium at the beginning and at the end of treatment was 

checked to ensure proper culturing and to avoid extreme treatment conditions (6). 

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

Page 30 of 99 
3.S.S. Chromosome analysis 

As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for 

all test groups, and if cells had 20 - 22 chromosomes, they were 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 gap and chromosome gap (isochromatid gap) 


Unstained regions (so-called achromatic lesions.:::: width of one chromatid) without 


dislocation of the segment which appears to be separated 

- S' and S" 

Chromatid break and chromosome break 

Visible discontinuity in chromatid or chromosome structure with lateral or longitudinal 

dislocation of the 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 of a segment at the level of chromatids or chromosomes 


- m.A. 


Multiple aberrations 


Metaphases with 5 or more aberrations excl. gaps 


- Disintegration of chromosomal structure (pulverization = P) 

The chromosomes are present as irregular particles and a chromosomal structure can 

no longer be detected. 

-	 Exchanges (trans locations) 


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


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

Page 31 of 99 
Int' and Int" 

Intrachanges at the level of chromatids and chromosomes 

The joining of broken 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 of chromatids and chromosomes 


The joining of broken ends capable of reuniting two or several chromosomes. They are 

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 

Metaphases with absent (hypoploid) or additional (hyperploid) chromosomes 

Only hyperploid metaphases are registered 

- Polyploidy 


Changes in the number of chromosomes by whole chromosome sets 


Endopolyploidy 

Tetraploid metaphases with diplo-chromosomes (products of endomitotic chromosome 

reduplication) 

Slides were coded before microscopic analysis. If only a few cells were found or if the 

metaphases were of low quality, a chromosome analysis was not carried out. 

If there is a clear increase in chromosomally damaged cells, the number of meta phases to 

be analyzed is reduced from the planned 200 mitoses/test group. 

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

Page 32 of 99 
3.8.6. Acceptance criteria 

The V79 in vitro cytogenetic aSiay is considered valid if the following criteria are met: 

• 	 The quality of the slides must allow the identification and evaluation of a sufficient number 

of analyzable metaphases. 

• 	 The numbers of cells with structural! numerical aberrations in the negative control (vehicle 

control) has to be within the normal range of the historical negative control data (see 

Appendix 5 and 6). 

• 	 The positive control chemicals both with and without 8-9 mix have to induce a significant 

increase in the number of cells with structural aberrations (see Appendix 7). 

3.8.7. Assessment criteria 

The test chemical will be assessed as "positive" in this assay if the following criteria are met: 

• 	 A dose-related and reproducible significant increase in the number of cells with structural! 

numerical chromosomal aberrations. 

• 	 The number of aberrant cells exceeds both the concurrent negative control range and the 

highest value of the negative historical control range. 

A test substance generally will be considered as "negative" if the following criteria are met: 

• 	 The number of cells with structural! numerical aberrations in the dose groups is not 

significantly above the concurrent negative control and is within the historical control data. 

3.8.8. Statistics 

the statistirl evaluation of the data was carried out using the MUCHAN program system 

The proportion of metaphases with aberrations was calculated for each group. 

A comparison of each dose group with the vehicle control group was carried out using 

Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm 

corrected versus the dose groups separately for each time and was performed one-sided. If 

the results of this test are significant, labels (* p < 0.05, ** P ~ 0.01) are printed in the tables. 

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

Page 33 of 99 
4. RESULTS 

4.1. 	 CHROMOSOME ANALYSIS - 1ST EXPERIMENT 

(Tables 1 - 12; Appendix 1) 

• Clastogenic mode of action 

After a treatment time of 4 hours no increase in the number of chromosomally damaged 

cells was observed either without 8-9 mix or after the addition of a metabolizing system. 

The types and frequencies of aberrations observed are given on pages 35. - 36. 

• Aneugenic mode of action 

No increase in the number of cells with changes in the number of chromosomes was 

demonstrated either without 8-9 mix or after the addition of a metabolizing system. 

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

Page 34 of 99 
4.1.1. Assay without 8-9 mix; 4-hour exposure, 18-hour sampling time 

Vehicle control: 

8 (4.0%) meta phases 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%) meta phases 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. 

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

Page 35 of 99 
4.1.2. Assay with 5-9 mix; 4-hour exposure, 18-hour sampling 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 x 2Ex 


1 500 IJg/mL: 

13 (6.5%) metaphases inc!. gaps 

8 (4.0%) meta phases 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. 

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

Page 36 of 99 
4.2. CHROMOSOME ANALYSIS - 2ND EXPERIMENT 

(Tables 13 - 28; Appendix 2) 

• Clastogenic mode of action 

Both with and without 8-9 mix, no increase in the number of structurally damaged 

metaphases was observed either after a treatment time of 4 hours or after a continuous 

treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours. 

The types and frequencies of aberrations are given on pages 38 - 40. 

• Aneugenic mode of action 

No increase in the number of cells with changes in the number of chromosomes was 

demonstrated either without 8-9 mix or after the addition of a metabolizing system. 

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

Page 37 of 99 
4.2.1. Assay without S-9 mix; 18-hour exposure, 18-hour sampling time 

Vehicle control: 

19 (9.5%) meta phases 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. 1 x 8"; 1 X 0"; 1 x Ex 


750 IJg/mL: 

17 (8.5%) meta phases incl. gaps 


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


1 500 lJg/mL: 

17 (8.5%) meta phases inc!. gaps 


8 (4.0%) metaphases excl. gaps, i.e. 1 x 8'; 1 x 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. 

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

Page 38 of 99 
4.2.2. Assay without S-9 mix; 18-hour exposure, 28-hour sampling time 

Vehicle control: 

7 (3.5%) metaphases incl. gaps 

2 (1.0%) meta phases 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"; 1 X 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. 

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

Page 39 of 99 
4.2.3. Assay with 5-9 mix; 4-hour exposure, 28-hour sampling time 

Vehicle control: 

4 (2.0%) metaphases incl. gaps 


2 (1.0%) meta phases 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 8f 1 x 8n ; ; 2 x 2Ex 


7S0 J.lg/mL: 

4 (2.0%) meta phases incl. gaps 


2 (1.0%) meta phases excl. gaps, i.e. 1 x F'; 1 x Ex 


1 SOO J.lg/mL: 

5 (2.5%) meta phases incl. gaps 

3 (1.5%) meta phases 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. 

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

Page 40 of 99 
4.3. MITOTIC INDEX 

The mitotic index is based on 1 000 cells per culture for the different test groups without and 

with metabolic activation. The number of mitotic cells in the samples scored are given in 

Appendix 3. 

According to the results of the determination of the mitotic index, no suppression of the 

mitotic activity was observed under all experimental conditions. 

4.4. CELL COUNTS 

The results of the cell count are given in Appendix 3. According to the results of the cell 

count, no growth inhibition was observed under all experimental conditions. 

~5. CELL MORPHOLOGY 

Observations on cell morphology (attachment to slides) are given in Appendix 4. 

Cell attachment was not influenced at any dose evaluated for structural chromosomal 

aberrations. 

4.6. ASSESSMENT OF THE SLIDES 

Details on the quality of metaphases are given in Appendix 4. 

4.7. TREATMENT CONDITIONS 

The osmolarity and pH values and the observations on the solubility of the test substance 

are given in Appendix 4. 

According to this, osmolarity and pH values were not influenced by test substance treatment. 

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

Page 41 of 99 
5. DISCUSSION 

According to the results of the present in vitro cytogenetic study, the test substancell 

did not lead to an increase in the number of structural chromosomal aberrations inc~ excl. gaps either without S-9 mix or after the addition of a metabolizing system in two 

experiments performed independently of each other selecting different exposure times 

(4 hours treatment and continuous treatment) and sampling times (18 and 28 hours); the 

types and frequencies of aberrations were close to the range of that of the concurrent 

negative control values at both sampling times and in the range of the historical control data 

(see Appendix 5). 

No increase in the number of cells containing numerical chromosomal aberrations was 

demonstrated. 

The increase in the frequencies of chromosomal aberrations induced by the positive control 

agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the 

metabolic activity of the S-9 mix employed. 

6. CONCLUSION 

Thus, under the experimental conditions chosen here, the conclusion is drawn thatD is 

not a chromosome-damaging (clastogenic) agent under in vitro conditions using V79 cells. 

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

Page 42 of 99 
7. 	REFERENCES 

1. 	 BRADLEY, M.O.; BHUYAN, B.; FRANCIS, M.C.; LANGENBACH, R; PETERSON, A.; 

HUBERMAN,E.: 

Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis in 

literature. A report of the 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 of the 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.; 

T AALMAN, RD.F.: 

Aneuploidy: a report of an ECETOC task force. 

Mut. Res .. 410, 3 - 79 (1998) 

4. 	 AMES, B.N.; McCANN, J.; YAMASAKI, E.: . 

Methods for detecting carcinogens and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. 

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 report from ICPEMC Task Group 9. 

Mut. Res .• 257. 147 - 204 (1991) 

7. 	 SCHMID, W.: 

A technique for in situ karyotyping of primary amniotic fluid cell cultures. 

Humangenetik, 30, 325 - 330 (1975) 

8. 	 SPEIT, G.; HAUPTER, S.: 

Cytogenetic effects of penicillamine. 

Mut. Res., 190,197 - 203 (1987) 

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

Page 43 of 99 
9. 	 EVANS, H.J.; O'RIORDAN, M.L.: 

Human peripheral blood lymphocytes for the analysis of chromosome 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; ausgearbeitet von der "Arbeitsgruppe der 

Industrie, Cytogenetik", 1987 

12. 	SPEIT, G.; HAUPTER, S.; VOGEL, W.: 

Characterization of mitosis with sister chromatid differentiation (SCD) and consequences 

for the analysis of proliferation kinetics and sister chromatid exchanges in 

asynchronously growing cells. 

Hum. Genet., 71,358 - 360 (1985) 

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

Page 44 of 99 
Table 1: 

Table 2: 

Tables 3 -7: 

Tables 8 - 12: 

APPENDIX 1 


1ST EXPERIMENT 


(TABLES 1 -12) 


Summary table without S-9 mix: 

4-hour exposure, 18-hour sampling time 

Summary table with S-9 mix: 

4-hour exposure, 18-hour sampling time 

Individual cultures without S-9 mix: 

4-hour exposure, 18-hour sampling time 

Individual cultures with S-9 mix: 

4-hour exposure, 18-hour sampling time 

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

Page 45 of 99 
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. 

co 0

:::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 

TABLE 1

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. 

co 0

: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 TIME 
ANALYSIS OF CHROMOSOMES -I 

:::T 

(ii' PROJECT-NO, 	 INDIVIDUAL DATA C. 

0 SUBSTANCE NAME ; ,----I-------' 

n 

c 

3 

CD S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: 18 h

~ 

TABLE 3 I 

16-0CT-2006 

(ii' 

~ CULT. NO. OF CD 

-0 NO. METAPH,

a Dose -0 

CD 

~ 

'< 

g, 

~ Vehicle DMSO CD 

(f) 

-0 

0 004 0100 ::J 

(f) 

0 

Cil 

en 010 0100 -0 

CD 

Q, 


::!l
 "'U 	 CD 

Q) 	 C. 


CO 0


::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 0.0 
.l\.NALYSIS OF CHROMOSOMES 4 -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 0.0 
ANALYSIS OF CHROMOSOMES 5 -; 

:::T 

iii' PROJECT-NO, INDIVIDUAL DATA n. 

0 SUBSTANCE NAME 0 

c 

Cl>

3 S-9 MIX: WITHOUT EXPOSURE TIME: 4 h SAMPLING TIME: lS h

;a 

iii' 

S' CULT. NO. OF Cl> 

"0 NO. METAPH.

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 0.0 
6 -I

::r 

(ii' PROJECT-NO, 0­

0 

C1 

SUBSTANCE NAME ~ c 

(1) 

3 S-~ MIX: WITHOUT

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) 	 ~ 

co 0


: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 0.0 
7 

-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 

(i) 008 0050

" <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 0.0 
ANALYSIS OF CHROMOSOMES 8 --I 

::r 

or PROJECT-NO. INDIVIDUAL DATA co. 

0 SUBSTANCE NAME (")

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 0.0 
ANALYSIS OF CHROMOSOMES 9 -i 

:T 

00" !?ROJECT-NO. INDIVIDUAL DATA 0. 

0 SUBSTANCE NAME () 

c: 

3 

<D S-9 MIX: WITH EXPOSURE TIME: 4 b SAMPLING TIME: 18 b

;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 

~ 

ti> 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

<D 


Q" 


" 

'1J 

t\) ~ c.o 0

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 
ANALYSIS OF CHROMOSO~~S I -; 

::T 

(ii' PROJECT-NO, INDIVIDUAL DATA a. 

0 SUBSTANCE NAME

c: 

3 " 

<t> S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 18 h

~ 

.. 

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 

:> 

~ 

iii 
ANALYSIS OF CHROMOSOMES --! 

::T 

iii· PROJECT-NO. INDIVIDUAL DATA a. 

0 SUBSTANCE NAME ~,----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) 


Summary table without S-9 mix: 

18-hour exposure, 18-hour sampling time 

Summary table without S-9 mix: 

18-hour exposure, 28-hour sampling time 

Summary table with S-9 mix: 

4-hour exposure, 28-hour sampling time 

Individual cultures without S-9 mix: 

18-hour exposure, 18-hour sampling time 

Individual cultures without S-9 mix: 

18-hour exposure, 28-hour sampling time 

Individual cultures with S-9 mix: 

4-hour exposure, 28-hour sampling time 

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

Page 58 of 99 
-1

::r 

(ii' 

g. 

(") 

c 

3 

<I> 

~ 

(ii' 

5' 

<I> 

't:l 

a 

't:l 

<I> 

~ 

a 

5' 

<I> 

(J) 

't:l 

0 

::l 

til 

0 

en 

(j) 

't:l 

<I> 

0, 

"'0 15' 

0­ III 

c.c 0

::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) cto 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 0.0 
ANALYSIS OF CHROMOSOMES -i

:r 

00' PROJECT-NO. INDIVIDUAL DATA e. 

0 SUBSTANCE NAME [ (")

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 

(i) 037 0100 "0 

'" Q.

'"0 ::!1 


Q) '" e. 


CO 0

::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. 

co 0

(J) :J 

"0 

0) Ql 

<0 .j':l. <l> 

0 -,.­

-c:: <0 <II

(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 

750.0 	ug/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 0.0 
ANALYSIS OF CHROMOSOMES -l 

~ PROJECT-NO. INDIVIDUAL DATA a. 

0 SUBSTANCE NAME n 

c: 

3 

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

~ 

(ii' 

SO CULT. NO. OF en 

"0 NO. METAPH.

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" 

m OJ

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

CC 0

::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' 

::fro 

"'"a 

"'" ro 

::l. '< 

8­

::fro 

'" "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 

'3en' 

0' '" 

:J 

cr 

'< 

::fro 

'" 

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 0.0 
ANALYSIS 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) 

OJ 0.

co 0

: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 '" iil 
ANALYSIS OF CHROMOSOMES -i

:::r 

(ii' PROJECT-NO. INDIVIDUAL DATA a. 

0 SUBSTANCE NAME <>

c: 

(J) 

3 S-9 MIX: WITHOUT EXPOSURE TIME: 19 h SAMPLING TIME: 28 h

;::. 

(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' 

OJ a.

<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 0.0 
ANALYSIS OF CHROMOSOMES TABLE 24 -I

:::r 

iii" PROJECT-NO. INDIVIDUAL DATA 0.. SUBSTANCE NAME [ g

c: 

3 

m S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h 16-0CT-2006

::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 0.0 
ANALYSIS OF CHROMOSOMES -i 

=r iir PROJECT-NO" : 1-- l INDIVIDUAL DATA a. 

0 SUBSTANCE NAME : I (") 

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 0.0 
ANALYSIS OF CHROMOSOMES -I 

~ 

0;' PROJECT-NO. INDIVIDU1\L DATA a. 

0 SUBSTANCE NAME (") 

c 

3 

(1) S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h

~ 

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 

Ol 
ANALYSIS OF CHROMOSOMES -I

::r 

iii" PROJECT-NO. 	 INDIVIDUAL DATA a. 

0 SUBSTANCE NAME C'l 

'"3 

(1) S-9 MIX: WITH EXPOSURE TIME: 4 h SAMPLING TIME: 28 h

:a 

<Ii 

s: 

(1) CULT. NO. OF

NO. METAPH. 

{3 

'"0 

Dose '"0 

(1) 

::l 

'< 

So 

s: 1 500.0 ug/ml (1) 

((j 


'"0 


0 049 0100 ::J 

lS 

til 

C;; 052 0100 '"0 

(1) 

C'l 

3i 1J 

ell 

CO 0

::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 0.0 
ANALYSIS OF CHROMOSOMES -I 

::r 

0;' PROJECT-NO. 	 INDIVIDUAL DATA Q. 

0 SUBSTANCE NAME 

c: 

3 " 

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

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. 


co 0


:::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 property of the 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 1 000 cells per culture for the different test 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 controls which are set at 100%. 

• 1st experiment 

Time (hours) 

Exposure Sampling 

4 18 

Test groups 

Vehicle control 

DMSO 

S-9 

mix 

-

1st culture 

% 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 

-= not determined 

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

Page 76 of 99 
• 2nd experiment 

Time (hours) 

Exposure Sampling 

Time (hours) 5-9 11 st 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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 77 of 99 
CELL COUNT 

The results of the cytotoxicity test cell count can be seen in the following tables. The number 

of cells counted in the dose groups are expressed as a percentage of the concurrent vehicle 

control value . 

• 1st experiment 

Time (hours) Test groups S-9 mix I Cell count 

No. of cells 

Exposure Sampling 1 x 105/mL Rei % 

4 18 Vehicle control - 3.03 100.0

DMSO 

4 18 187.5 - 2.94 97.0 IJg/mL 

4 18 375.0 3.37 111.2 IJg/mL -

4 18 750.0 3.02 99.7 IJg/mL -

18 1 500.0 3.12 103.0 4 IJg/mL -

4 18 Vehicle control + 3.25 100.0 

DMSO 

4 18 187.5 IJg/mL + 3.58 110.2

4 18 375.0 IJg/mL + 3.51 108.0

4 18 750.0 + 3.68 113.2 IJg/mL 

4 18 1 500.0 IJg/mL + 3.20 98.5 I I 

not determined 

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

Page 78 of 99 
• 2nd experiment 

Time (hours) Test groups S-9 mix Cell count 

No. of cells 

Exposure Sampling 1 x 105

/mL Rei % 

18 18 Vehicle control - 2.32 100.0

DMSO 

18 18 187.5 IJg/mL 3.09 133.2 -

18 18 375.0 2.94 126.7 IJg/mL -

18 18 750.0 2.70 116.4 1J9/mL -

18 18 1 500.0 2.56 110.3 1J9/mL -

18 28 Vehicle control - 5.03 100.0

DMSO 

18 28 375.0 IJg/mL 4.36 86.7 -

18 28 750.0 IJg/mL - 4.40 87.5

18 28 1 500.0 IJg/mL 3.81 75.7 -

4 28 Vehicle control + 3.97 100.0 

DMSO 

4.15 104.5 

4 

4 28 187.5 IJg/mL +

28 + 
 4.04 101.8

4 


375.0 1J9/mL 

28 + 
 4.05 102.0

4 


750.0 IJg/mL 

28 1 500.0 3.68 92.7 1J9/mL + 

- = not determined 

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

Page 79 of 99 
APPENDIX 4 

CELL MORPHOLOGY, ASSESSMENT, 


TREATMENT CONDITIONS 


This document is the property of the 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 

• 1st experiment 

Time (hours) 

Exposure Sampling 

Test groups S-g 

mix 

Cell morphology I 

Attachment to 

slides 

Assessment 

of the 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 

1 500.0 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 

1 500.0 IJg/mL 

+ 

+ 

+ 

+ I 

+ 

+ 

+ 

+ I 

E1 

E1 

E1 

E1 

E 

N 

= evaluation possible 

1 = sufficient metaphases of good quality 

2 = reduced number of analyzable metaphases 

3 =reduced number of analyzable metaphases, some of poor quality 

evaluation not possible 

1 =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 meta phases strongly influenced by the precipitation of the 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 

(-) = not determined =assessment not possible due to test substance precipitation 

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

Page 81 of 99 
• 2nd experiment 

Time (hours) 

Exposure ISampling 

Test groups 5-9 

mix 

Cell morphology I 

Attachment to 

slides 

Assessment 

of the slides 

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 

1 500.0 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 

1 500.0 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 

1 500.0 tJ9/mL 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

E1 

E1 

E1 

E1 

E :: evaluation possible 

1 :: sufficient metaphases of good quality 

2 :: reduced number of analyzable metaphases 

3 = reduced number of analyzable metaphases, some of poor quality 

N = evaluation not possible 

1 =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 meta phases strongly influenced by the precipitation of the test 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 due to test substance precipitation 

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

Page 82 of 99 
TREATMENT CONDITIONS 

The osmolarity and pH values and the observations on the solubility of the test substance 

can be seen from the following tables. 

• 1st experiment 

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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 83 of 99 
• 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 1 500.0 1J9/mL + 7.4 392 S S S S 

S = solution 

Su = suspension 

E emulsion 

p = preci pitation 

Ma = macroscopically 

Mi = microscopically 

mOsm = milliosmolar 

= not determined 

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

Page 84 of 99 
APPENDIX 5 


HISTORICAL NEGATIVE CONTROL DATA - CLASTOGENICITY 


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

Page 85 of 99 
Historical Negative Control Data 


Summary 


Negative Control - Without 5-9 Mix 

Exposure I Sampling 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 I Sampling 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. of expo 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 property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 86 of 99 
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. of expo 63 14 

Exposure I Sampling 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. of expo 15 16 

Untreated Control - With S-9 Mix 

Exposure I Sampling 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 property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 87 of 99 
--L-________________________________________________________________~ 

Historical Negative Control Data 

Vehicle Control DMSO - 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 4.0 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. of expo 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. of expo 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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 88 of 99 
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. of expo 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. of expo 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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 89 of 99 
Historical Negative Control Data 

Vehicle Control Acetone - Without 5-9 Mix 

Exposure I Sampling 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. of expo 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 I Sampling 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 

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

Page 90 of 99 
APPENDIX 6 


HISTORICAL NEGATIVE CONTROL DATA - ANEUGENICITY 


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

Page 91 of 99 
Historical Negative Control Data 


Summary 


Negative Control- Without 5-9 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. of exp.* 147 147 63 22 22 

Endopoly· 

ploidy 

0.0 

0.0 

0.0 

1 

Exposure I Sampling 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. of exp.* 80 80 I 34 65 65 33 

Negative Control - With 5-9 Mix 

Exposure I Sampling 4/18 h 4/28 h 

Aneu- Pol ndopoly· Aneu· Poly. Endopoly Aberrations [%] ploidy ploi ploidy ploidy ploidy ploidy 

Mean 0.2 0.3 0.2 0.4 0.0

Min 0.0 0.0 0.0 0.0 0.0 0.0 

0.7 

Max 5.0 4.2 2.4 1.5 3.4 0.5

No. of exp.* 182 182 63 88 88 34

* differentiation between polyploidy and endoploidy since January 2000 

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

Page 92 of 99 
-


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. of exp.* 45 45 17 10 10 1 

Exposure I Sampling 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 I Sampling 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. of exp.* 59 59 17 31 31 11 

• differentiation between polyploidy and endoploidy since January 2000 

This document is the property of the sponsors (specified on page 1), use by other 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. of exp.* 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. of exp.* 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- IEn~: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. of exp.* 111 111 41 49 49 20 

* differentiation between polyploidy and endoploidy since January 2000 

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

Page 94 of 99 
Historical Negative Control Data 

Vehicle Control Acetone - Without 5-9 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. of exp.· 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. of exp.· 5 5 3 5 5 3 

Vehicle Control Acetone - 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. of exp.· 9 9 5 6 6 3 

* differentiation between polyploidy and endoploidy since January 2000 

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

Page 95 of 99 
Historical Negative Control Data 

Vehicle Control Ethanol - Without 5-9 Mix 

Exposure I Sampling 4/18 h §4/28h 

Aberrations [%] ploidy 

Aneuploidy 

Poly- Endopolyploidy ploidy 

Poly­ ~ 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. of exp.* 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. of exp.* 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. of exp.* 3 3 0 2 2 0 

* differentiation between polyploidy and endoploidy since January 2000 

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

Page 96 of 99 
APPENDIX 7 


HISTORICAL POSITIVE CONTROL DATA 


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

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. of expo 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. of expo 159 50 

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

Page 98 of 99 
Historical Positive Control Data 

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

Exposure I Sampling 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. of expo 10 10 

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

Page 99 of 99 
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 document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be used 

only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, 

publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory 

a encies actin within the limits of their a,....d...m...in...is"'tr....a"'tiv...ec-:a...ut...h"'or"'itL·____________________-1 

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

Page 1 of 5 
GLP COMPLIANCE STATEMENT 

This study was conducted in compliance with the OECD Principles of Good Laboratory 

Practice and the GLP Principles of the German "Chemikaliengesetz" (Chemicals Act) which 

meet the United States Environmental Protection Agency Good Laboratory Practice 

Standards [40 CFR Part 160 (FIFRA) and Part 792 (TSCA)], with the exception that 

recognized differences exist between the GLP Principles/Standards of OECD and the 

Principles/Standards of FIFRA and TSCA. 

This document is Ihe property of the sponsors (specified on page 1). use by other parties requires written permission by the sponsors 

Page 2 of5 
SIGNATURE PAGE 

Study Director: 

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

Page 30f5 
-


STATEMENT OF THE QUALITY ASSURANCE UNIT 

The Quality Assurance 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 of study Date of inspection 

(mm-dd-yyyy) 

Reported to Study Director 

and to Management 

(mm-dd-yyyy) 

Study Plan: 07-06-2006 07-06-2006 

Conduct of study: 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 property of the sponsors (specified on page 1l. use by other parties requires written pennisslon by the sponsors 

Page 4 of 5 
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 property of the sponsors (specified on page 1). use by other parties requires written permission by the sponsors 

Page 5 of 5 
Attachment #21 

STUDY TITLE 

Report 


Repeated Dose Oral Toxicity study in Wistar rats; 


Administration by gavage for 4 weeks 


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 SUMMARY TABLES) 


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

only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, 

publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory agencies 

actin within the limits of their administrative authorit . 

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

Page 1 of 368 
THIS PAGE IS INTENTIONALLY LEFT BLANK 


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

Page 2 of 368 

GLP COMPLIANCE STATEMENT 

This study was conducted in accordance with the OECD Principles of Good Laboratory 

Practice and the GLP Principles of the German "Chemikaliengesetz" (Chemicals Act). which 

meet the United States Environmental Protection Agency Good Laboratory Practice 

Standards [40 CFR Part 160 (FIFRA) and Part 792 (TSCA)]. with the exception that 

recognized differences exist between the GLP Principles/Standards of OECD and the 

Principles/Standards of FIFRA and TSCA. 

Study Director .l:1..I!!.f..L~..J•.?f!J7­

Date 

To be filled for USA EPA submission only: 

Sponsor 

Date 

Submitter 

Date 

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

Page 3 of 368 
THIS PAGE IS INTENTIONALLY LEFT BLANK 


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

Page 5 of 368 
--L-__________________________________________________________________~ 

CONTRIBUTORS TO THE STUDY I SUPERVISORY 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 of the sponsors (specified on page 1). use by other parties requires written permisSion by the sponsors 

Page 6 of 368 
,-. 

STATEMENT OF THE QUALITY ASSURANCE UNIT 

The Quality Assurance 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 of study Date of inspection Reported to Study Director 

and to Management 

(mm-dd-yyyy) (mm-dd-yyyy) 

Study Plan: 10-05-2006 10-05-2006 

Conduct of study: 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 is the property of the sponsors (specified on page 1). use by other parties requires written permission by the sponsors 

Page 7 of 368 
GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) 

Gute Laborpraxls I Good Laboratory Practice 

GLP-Bescheinlgung I Statement of GLP Compliance 

(gem./according to § 19 Abs. 1 Chemikaliengesetz) • 

Eine GLP-Inspektion zur Oberwachung und Assessment of conformity with GLP according 


der Einhaltung der GLP-Grundsalze gema~ to Chemikaliengesetz and Directive 8813201 


Chemikaliengesetz bzw. Richtlinie 881320IEG EECat:: 


wurde durchgefOhrt in: 


PrOfeinrichtung / Test facility 

PrOfung nach Kategorien / Areas of Expertise 


(gem.! aa:o<dlng ChernVwII.GLP Nr. 5.3IOECD guidance) 


1,2,3,4,5,8,9 


Kat. 9 - Biochemische und pathologische Untersuchungen zu Wirkmechanismen / 


Biochemical and pathological examinations concerning mode of action 


Datum der Inspektion / Date of Inspection 


(Tag.Monat Jahr 1 day.month year) 


08.06.2005 und 25. bls 27.07.2005 


Die genannte PrOfeinrichtung beflndet sich im The above mentioned test facility 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 com pliance with 


unter Einhaltung der GLP-Grundsatze durch­ the Principles of GLP. 


gefOhrt werden kOnnen. 


Landesamt fOr Umwelt, Wasserwirtschafl und Gewerbeaufslcht 


Kaiser-Friedrich-StraBe 7 


55116 Malnz 


"cN~';;~·;,;;;iAd~d;,;:Gt:p:o;';;,:.;;.;;j;~;;;';;;ho;.d·~·i· Landesamt fUr ( J Name and ad..... of !he GLP Moniloring Authority) Umwelt. Wasserwirtschaft 

und Gewerbeaufsicht 

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

Page 8 of 368 
-


CONTENTS OF PART I (REPORT SECTION AND SUMMARY TABLES) 

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 QUALITY ASSURANCE UNIT 

GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) 

CONTENTS OF PART I (REPORT SECTION AND SUMMARY TABLES) 

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. TEST ANIMALS 

3.2.1. Species and strain 

3.3. HOUSING AND DIET 

3.4. TEST GROUPS AND DOSES 

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

Page 9 of 368 
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 PREPARATIONS AND PREPARATION FREQUENCY 

ANALYSES 

Analyses of the test substance preparations 

Analytical methods 

Food analyses 

Drinking water analyses 

Bedding and enrichment analyses 

EXPERIMENTAL PROCEDURE 

Mortality 

Clinical observations 

Detailed clinical observations (DCO) 

Food consumption 

Water consumption 

Body weight data 

Food efficiency 

Functional observational battery (FOB) 

Motor activity assessment 

Statistics of clinical examinations 

CLINICAL PATHOLOGY 

Hematology 

Clinical chemistry 

Urinalysis 

Statistics of clinical pathology 

PATHOLOGY 

Necropsy 

Organ weights 

Organ I Tissue fixation 

Histotechnical processing I Examination by light microscopy and 

assessment of findings 

Statistics of pathology 

RESULTS 

ANALYSES 

Stability analyses 

Homogeneity control analyses 

Concentration control analyses 

Food analyses 

Drinking water analyses 

Bedding and enrichment analyses 

CLINICAL EXAMINATIONS 

Mortality 

Clinical examinations 

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

Page 10 of 368 
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 

Motor activity 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) 

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

Page 11 of 368 
Part A: 


CLINICAL EXAMINATIONS (MEAN VALUES AND SUMMARY TABLES) 


Body weight 

Motor activity 

Observations Report - Incidence 

Food consumption 

Water consumption 

Body weight 

Body weight change 

Food efficiency 

Functional observational battery 

Motor activity 

Part B: 


CLINICAL PATHOLOGY (MEAN VALUES AND SUMMARY TABLES) 


Hematological examinations 

Differential blood count 

Clotting analyses 

Enzymes 

Blood chemistry 

Urinalyses 

Part C: 


PATHOLOGY (MEAN VALUES AND SUMMARY TABLES) 


Absolute weights - mean values (male) 


Absolute weights - mean values (female) 


Relative weights - mean values (male) 


Relative weights - mean values (female) 


Incidence of gross lesions 


Incidence of all microscopic findings 


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 

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

Page 12 of 368 
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 

Body weight 

Functional observational battery 

Motor activity 

Part B: 


CLINICAL PATHOLOGY (INDIVIDUAL VALUES) 


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 

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

Page 13 of 368 
CONTENTS OF PART III (SUPPLEMENT) 

TITLE PAGE 

TABLE OF CONTENTS 

Final Report 


Chemical and spectroscopic characterization of~or the notification according to the German Chemi~ 

I I 

Final Report 


Characterization ofi Iafter termination of 


toxicological studies'-------' 


I 

ANALYTICAL REPORT 


STABILITY ANALYSIS OF 


Homogeneity and Concentration Control Analysis o~,-__...... 


in Drinking water with 0.5% Carboxymethylcellulose 


Functional Observational Battery (FOB): 


Detailed description of examinations, ranking and documentation 


procedures 


HISTORICAL CONTROL DATA OF CLINICAL PATHOLOGY TESTING 

THIS REPORT CONSISTS OF PART I, II AND III. 

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

Page 14 of 368 
1. SUMMARY 

1.1. METHODS 

I Iwas administered to groups of 5 male and 5 female Wistar rats at dose levels of 0 

(vehicle control; group 0), 10 (group 1), 30 (group 2), 60 (group 3; from day 0 till day 7) and 

45 (group 3; from day 8 till day 28) mg/kg body weight/day over a period of 4 weeks. 

The animals of group 3 received doses of 60 mg/kg bw/day from day 0 till day 7 and 45 

mg/kg bw/day from day 8 till the day 28, because of severe toxic effects at 60 mg/kg bw/day. 

1.2. OBSERVATIONS 

Food consumption, water consumption and body weight were determined weekly. The 

animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical 

examinations in an open field were conducted prior to the start of the administration period 

and weekly thereafter. In addition, all animals were checked daily before and about 1 hour as 

well as 3 - 4 hours after administration for any clinical signs. A functional observational 

battery (FOB) and measurement of motor activity was carried out after 26 days of treatment 

in males and 27 days of treatment 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. 

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

Page 15 of 368 
1.3. RESULTS 

1.3.1. Analytics 

The various analyses: 

• 	 Demonstrated the stability of the test substance preparations over a period up to 96 hours 

at room temperature 

• 	 Showed the homogeneous distribution of the test article in the vehicle 

• 	 Showed the correctness of the prepared concentrations 

1.3.2. Findings 

The following test substance-related adverse findings were noted: 

60 mg/kg body weight/day (from day 0 until day 7) 

• 	 1 male and 1 female were found dead on day 6 and on day 7, respectively 

• 	 Convulsions, clonic were observed moderate in 4 females, severe in 2 females, epileptoid 

slight in 1 male, moderate in 1 female, severe in 2 males and 1 female, tonic slight in 1 

female 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 

• 	 Hyperexcitability was 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 1 female 

• 	 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 

• 	 Food consumption in both sexes was statistically significantly reduced of -29.4% in males 

and -28.2% in females on day 7 

• 	 Water consumption 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 

• 	 Body weight change in both sexes was 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 

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

Page 16 of 368 
-


45 mg/kg body weight/day (from day 8 until the end of the administration period) 

• 	 1 female was prematurely sacrificed moribund on day 11 

• 	 Anogenital region, smeared with urine was observed severe in 1 female 

• 	 Convulsions, tonic were observed slight, moderate and severe in each 1 female 

• 	 Hyperactivity was observed in 1 female 

• 	 Hyperesthesia was observed slight in 2 males and 1 female 

• 	 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 1 female 

• 	 Nose, discharge, red was observed slight in 1 female and severe in 2 females 

• 	 Piloerection was observed in 2 males and 2 females 

• 	 Reduced general condition was observed slight in 1 female and severe in 2 females 

• 	 Salivation was observed slight in 1 female, moderate in 2 females and severe in 1 female 

• 	 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 of day 14 (-8.5%) in males and a maximum 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 

• 	 Hyperactivity was observed in 1 female 

• 	 Hyperesthesia was observed slight in 1 male and 5 females 

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

Page 17 of 368 
• 	 Hyperexcitability was observed sUght in 5 males and 5 females, moderate in 3 males and 4 

females and severe in 1 female 

• 	 Piloerection was observed in 1 male 

• 	 Twitching was observed slight in 5 males and females, moderate in 4 females and severe 

in 1 female 

• 	 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 day 7 

• 	 Water consumption in both sexes was increased during the administration period, with the 

exception of day 14 (-13.6%) in males and a maximum of +6.3% in males on day 21. a 

maximum of +26.4% in females on day 14 and s1atistically significantly of +24.6% in 

females on day 28 

• 	 Body weight in both sexes was decreased during the administration period, with a 

maximum of -7.7% in males on days 7 and 21 and -6.3% in females on day 7 

• 	 Body weight 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 day 7 

• 	 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 hindlimbs of -39.9% and -26.2% in females 

• 	 Motor activity was 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 day 21 and +27.3% in females on day 14 

• 	 Body weight in both sexes was decreased during the administration period. with a 

maximum of -4.8% in males on days 7 and 28 and -4.3% in females on day 7 

• 	 Body weight 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 

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

Page 18 of 368 
In conclusion, the oral administration o~ Iby gavage over a period of 4 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 

respect to the tissue and organs examined during necropsy, organ weight determinations and 

light microscopy. 

Therefore, the no observed adverse effect level (NOAEL) under the conditions of this study 

was below 10 mg/kg body weight/day for male as well as female Wistar rats. 

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

Page 19 of 368 
2. 	 INTRODUCTION 

2.1. OBJECTIVES 

The aim of the study was to determine the toxicolq;Jical profile of the test substance including 

the target organs and the "no observed adverse effect level" (NOAEL) after 4 weeks of oral 

administration by gavage. 

2.2. SELECTION OF DOSES 

By request of the sponsor, the following dose levels were selected for the present study: 


60 mg/kg body weight/day: as high dose with expected toxic effects 


30 mg/kg body weight/day: as mid dose 


10 mg/kg body weight/day: as low dose 


Due to severe clinical signs of toxicity, 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 suitable for the detection of a 


toxicological hazard. 


2.3. TEST GUIDELINES 

The study was conducted according tol based on the following test guidelines: 

• 	 OECD Guidelines for Testing of Chemicals; Method No. 407: Repeated Dose 28-day 

Oral Toxicity Study in Rodents; adopted July 27, 1995 

• 	 EC Commission directive 96/54/EEC of September 30, 1996; Part B: Methods for the 

determination of Toxicity; Toxicity after 28-day administration (oral); Official Journal of the 

European Communities No. L 248, pp. 213-217, 1996 

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

Page 20 of 368 
--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 

October 04,2006 

October 05, 2006 

October 06, 2006 

October 30,2006 

October 31 , 2006 

November 01, 

2006 

November 02, 2006 

November 03, 2006 

November December 2006 

January 25, 2007 

Phase of study I Examination 

Experimental starting date: Arrival of the 

animals and beginning of the adaptation period 

Randomization of the animals 

Sampling for analyses 

Start of the 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 of food) of about 16 to 20 hours before necropsy 

FOB = Functional observational battery 

M = Measurement of motor activity 

1...2 = Identification number of the specific examination 

2.5. RETENTION OF RECORDS 

GLP-relevant records and materials are stored a or at least the 

period of time specified in the GLP principles. Detal s concerning responsi llities or locations 

of archiving can be seen from the respective SOPs and from the raw data. 

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

Page 21 of 368 
3. MATERIAL AND METHODS 

3.1. TEST SUBSTANCE 

The analyses of the test substance have been carried out at thel I 

Name of test substance: 

Test substance No.: 

Batch identification: 

Purity: 

Homogeneity: 

Stability: 

I ~----~ 

99.9 g/1 OOgl'-_____--1 

given 

stable until January at 

room temperature 

ADDITIONAL TEST SUBSTANCE INFORMATION 


Date of production: 1995 

Physical state/Appearance: 

Storage conditions: room temperature 

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

Page 22 of 368 
3.2. TEST ANIMALS 


3.2.1. Species and strain 

Test species and strain: Rat; Crl:WI(Han) 

Reason for species selection: Rats were 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, 

Sandhofer Weg 7, 97633 Sulzfeld 

Arrival in the testing facility: September 26,2006 

Identification: The rats were identified clearly by ear tattoo 

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 dust free embedding, supplied by SSNIFF, 

Soest). Motor activity measurements were conducted in Polycarbonate cages with wire 

covers from Ehret, Emmendingen (floor area about 800 crrf) and small amounts of absorbent 

material (see above). For enrichment wooden 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 of 20 ­

24°C for temperature and of 30 -70% for relative humidity. The day/night cycle was 12 hours 

(12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.). 

Deviations from these ranges did not occur. The animal room was completely disinfected 

prior to the study using a disinfector ("AUT EX" , fully automatic, formalin-ammonia-based 

terminal disinfector). The floor and the walls were 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. 

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

Page 23 of 368 
3.4. TEST GROUPS AND DOSES 

Males 

Dose group 

(color of the 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 

! (color of the 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 to the body weight determined most recently. 

2) Vehicle: drinking water with 0.5% carboxymethylcellulose 

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

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 of test substance was weighed out depending on the desired concentration. Then the 

vehicle (0.5% carboxymethylcellulose in drinking water) was filled up to the desired volume, 

subsequently mixed using a magnetic stirrer. During application the test substance 

preparations were kept homogeneous using a magnetic stirrer. The test substance 

preparations were prepared twice a week. 

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

Page 25 of 368 
3.6. ANALYSES 

3.6.1. Analyses of the test substance preparations 

The anal ses of the test substance preparations were carried out at th 

The stability of the test substance in the vehicle over a period of up to 96 hours was proven 

before the start of the studyl I 

Before the beginning of the 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 sample was 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_...., 

sent to the 

3.6.2. Analytical methods 

The methods used for analytical investigations of the 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 water analyses 

3.6.5. Bedding and enrichment analyses 

The bedding and the enrichment are regularly assayed for contaminants (chlorinated 

hydrocarbons and heavy metals). 

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

Page 26 of 368 
3.7. EXPERIMENTAL PROCEDURE 

On the day of arrival the animals were subjected to an acclimatization period during which 

they received ground diet and drinking water ad lit)tum. Prior to the first detailed clinical 

observation, the animals were distributed according to weight among the individual test 

groups, separated by sex. The weight variation of the animals used did not exceed ±20 

percent of the mean weight of each sex. The list of randomization instructions was compiled 

with a computer. 

At the start of the administration period (day 0) the rats were 42 ±1 days old. 

The test substance was administered daily by gavage for about 28 days. Control animals 

received the vehicle only. The volume to be administered was 10 ml/kg body weight/day 

(related to the body weight determined most recently in each case). At the end of the 

administration period the animals were sacrificed after a fasting period (withdrawal of food) 

for at least 16 - 20 hours. 

3.7.1. Mortality 

A check for moribund and dead animals was made twice daily on working days and once 

daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they 

were sacrificed and necropsied. 

3.7.2. Clinical observations 

All animals were checked daily before, about 1 hour after and 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 prior to the administration period 

and thereafter at weekly intervals. The findings were ranked according to the degree of 

severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with 

sides of 25 cm high). The following parameters were examined: 

1. abnormal behavior during "handling" 

2. fur 

3. skin 

4. posture 

5. salivation 

6. respiration 

7. activity/arousal level 

8. tremors 

9. convulsions 

10. abnormal movements 

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

Page 27 of 368 
11. impairment of gait 

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 over a period of 7 days and calculated 

as mean food consumption grams per animal and day. 

3.7.5. Water consumption 

Individual water consumption was determined weekly over a period of 4 days and calculated 

as mean water consumption in grams per animal and day. 

3.7.6. Body weight data 

Body weight was determined before the start of the administration period in order to 

randomize the animals. During the administration period the body weight was determined on 

day 0 (start of the administration period) and thereafter at weekly intervals. The difference 

between the body weight on the respective day of weighing and the body weight 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 body weight 

and food consumption: 

x 100 = Food efficiency for day x 


FCytox 


BWx = body weight on day x (g) 


BWy = body weight on day y (last weighing date before day x) (g) 


FCytox = mean food consumption from day y to day x; calculated as mean daily food consumption on day x, 


multiplied with the number of days from day y to day x (g) 

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

Page 28 of 368 
-


3.7.8. Functional observational battery (FOB) 

A functional observational battery was performed in all animals at the end of the 

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 sensorirrotor tests as well as reflex tests. The 

findings were ranked according to the degree of severity, if applicable. The observations 

were performed at random. A detailed description of the methods, the ranking and 

documentation system can be found in Part III (Supplement). 

Home cage observations: 

The animals were observed in their closed home cages; any disturbing activities (touching 

the cage or rack, noise) were avoided during these examinations in order not to influence the 

behavior of the animals. Attention was paid to: 

1. posture 

2. tremor 

3. convulsions 

4. abnormal movements 

5. impairment of gait 

6. other findings 

Open field observations: 

The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and 

observed for at least 2 minutes. Following parameters were examined: 

1. behavior when 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 of gait 

15. activity/arousal level 

16. feces (number of fecal pellets/appearance/consistency) within two minutes 

17. urine (appearance/quantity) within two minutes 

18. number of rearings within two minutes 

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

Page 29 of 368 
Sensorimotor Tests/Reflexes: 

The animals were removed from the open field and subjected to following sensorimotor or 

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. behavior during "handling" 

9. vocalization 

10. pain perception ("tail pinch") 

11. grip strength of forelimbs 

12. grip strength of hind limbs 

13. landing foot-splay test 

14. other findings 

3.7.9. Motor activity assessment 

Motor activity was measured on the same day as FOB was performed. The measurement 

was performed in the dark using the 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 animals were put into the cages in a 

randomized order. The measurements started at about 2.00 p.m. The number of beam 

interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not 

commence at the same instant for all cages; the period of assessment for each animal 

started when the first beam was interrupted by pushing the cage into the rack (staggered 

start). Measurements ended exactly 60 minutes thereafter. During the measurements the 

animals received no food and no water. 

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

Page 30 of 368 
3.7.10. Statistics of clinical examinations 

Means and standard deviations of each test group were calculated for several parameters 

(see tables). Further statistical analyses were performed according to following tables: 

Parameters Statistical test Markers in the References 

tables 

food consumption, A comparison of each * for p ~ 0.05 DUNNETI, C.w. (1955): A multiple 

water consumption, 

body weight, body 

group with the control 

group was performed 

** for p 0.01 comparison procedure for comparing 

several treatments with a control. JASA, 

weight change, food using DUNNETT's test Vol. 50, 1096-1121 

i efficiency (two-sided) for the 

hypothesis of equal DUNNETT, c.w. (1964). New tables for 

means multiple comparisons with a control. 

Biometrics, Vol. 20, 482 - 491 

feces, rearing, grip Non-parametric one­ • for p 0.05 SIEGEL, S. (1956): strength forelimbs, way analYSis using .. for p ~ 0.01 Non-parametric statistics for the 

grip strength KRUSKAL-WALLIS test behavioural sciences. McGraw-Hili New 

hindlimbs, foot­ (two-sided).lf the York 

splay test, motor resulting p-value was 

activity equal or less than 0.05, 

a pairwise comparison 

of each dose group with 

the control group was 

performed using 

. Wilcoxon-test (twosided) for the 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 

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

Page 31 of 368 
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 subsequent analysis of the blood and serum samples 

were carried out in a randomized sequence. For urinalysis the individual animals were 

transferred to metabolism cages (withdrawal of food and water) and urine was collected 

overnight. The urine samples were evaluated in a randomized sequence. 

The assays of blood and serum parameters were performed under internal laboratory quality 

control conditions with commercial reference controls to assure reliable test results. 

The results of the clinical pathology examinations are expressed in units of the International 

System (SI). 

The following examinations were carried out in all surviving animals per test group and sex at 

the end of the 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 Wright without 

being evaluated. 

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

Page 32 of 368 
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) MCV x erythrocytes 

Mean corpuscular fI RBC/PL T 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 

Reticu I 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) 

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

Page 33 of 368 
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 UV test, 

340 nm; 3rC 

Recommendations of the German 

• Society for Clinical Chemistry: 

' "Standardization of methods for 

determining enzyme activities in 

biological liquids" . 

Aspartate aminotransferase 

(AST) 

(L-aspartate: 2-oxoglutarate 

aminotransferase; 

EC 2.6.1.1.) 

\Jkat/I kinetic UV test, 

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 color test. 

415 nm, 3rC 

Roche working instructions 

,(-Glutamyltransferase 

(GGT) 

('( -glutamyl) peptide: aminoacid-,(­

glutamyl-transferase; 

EC 2.3.2.2.) 

nkat/I kinetic color test, 

415 nm. 3rC 

Szasz. G. et aI., 

J. Clin. Chem. Clin. Biochem. 12.228 

(1974) 

Roche working instructions 

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

Page 34 of 368 
Blood 

Chemistry 

Parameter 

Unit Method References 

Sodium 

(NA) 

mmol/l 

Potassium ion selective electrodes (ISE) Hitachi 917 - working instructions 

(K) 

mmol/l 

Chloride 

(Cl) 

mmol/l 

Inorganic 

phosphate 

(INP) 

mmol/l molybdate reaction Henry, R.J. in: "Clinical Chemistry", Harper 

and Row Publishers, New York (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 determination with the 

ureasel glutamate 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. et aI., 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 

T riglycerides 

(TRIG) 

mmolll enzymatic color test with lipase 

esterasel glycerokinasel glycerol3-phosphate oxidase/4-aminophenazone 

mod. method by Wahlefeld, A.w., in 

"Methoden der enzymatischen Analyse" 

[Methods of enzymatic analysis] 

(Berg meyer, H.U., ed.) Vol. II, 3rd ed., 

Verlag Chemie Weinheim, GERMANY, pp. 

1878-1882 (1974); Roche working 

instructions 

Cholesterol 

(CHOl) 

mmol/l enzymatic determination with 

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) -

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

Page 35 of 368 
--L-________________________________________________________________________~ 

3.8.3. Urinalysis 

With the exception of volume, 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 or Atago operating instructions 

: Sediment microscopy Hallmann, L., [Clinical Chemistry and 

Microscopy] 12, ed., 233-246, Georg 

Thieme, Stuttgart, Germany (1966) 

Color, turbidity by visual evaluation 

Volume graduated tubes 

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

Page 36 of 368 
-


3.8.4. Statistics of clinical pathology 

Means and standard deviations of each test group were calculated for several parameters 


(see tables). 


Further statistical analyses were performed according to following tables: 


Parameter Statistical test 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) for the equal medians 

* for p ~ 0.05 

** for p ~ 0.01 

SIEGEL, S. (1956): 

Non-parametric statistics 

for the behavioural 

sciences. 

McGraw-Hili New York 

Parameter Statistical test Markers in the 

tables 

References 

Urinalysis, 

except volume, 

color, turbidity 

and specific 

gravity 

Pairwise comparison of each dose 

group with the control group using 

FISHER's exact test for the hypothesis 

of equal proportions 

* for p < 0.05 

** for p 0.01 

SIEGEL, S. (1956): 

Non-parametric statistics 

for the behavioural 

sciences. McGraw-Hili New 

York 

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

Page 37 of 368 
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 after their 

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 I Tissue 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 

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

Page 38 of 368 
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 glandular stomach) 

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 lumbar cords) 

36. Sternum with marrow 

37. Femur with knee jOint 

38. Skeletal muscle 

From the liver, each one slices of the Lobus dexter lateralis and the Lobus sinister lateralis 

were fixed in Carnoy's solution and embedded in paraplast. 

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

Page 39 of 368 
3.9.4. 	Histotechnical processing I Examination by light microscopy and assessment 

of findings 

Fixation was followed by histotechnical processing, examination by light microscopy and 

assessment of findings according to the 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) 

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

Page 40 of 368 
Methods/Scope of examinations: 


A = Hematoxylin-eosin 


1 =All animals/group 


2 = All animals affected/group 


Animals that died or were 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 of cortico-medullar ratio (related only to area) 


~ Increase of starry sky cells 


~ Changes of cellular density in the cortex 


~ Changes of cellular density in the medulla 


Spleen: 


~ Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp 


~ Altered cellular composition of follicles 


~ Altered number of germinal centers 


Lymph nodes (mesenteric and axillar lymph nodes): 


~ Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla 


~ Altered cellular composition of paracortex 


~ Altered number of germinal centers 


~ Hyperplasia of high endothelial venules 


~ Apoptotic necrosis increased 


Peyer's patches (of the jejunum): 


~ Changes of the cellularity of follicles (induding mantle zone and germinal centers) 


~ Changes of the cellularity of interfollicular area 


~ Apoptotic necrosis increased 


Bone marrow: 


~ Changes of the cellularity 


~ Changes of the myeloid/erythropoid ratio 


Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not 


reveal a morphologic alteration of these items and/or whenever no other pathologic finding 


was noted, these organs were diagnosed as "no abnormalities detected". 


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

Page 41 of 368 
3.9.5. Statistics of pathology 

Means and standard deviations were calculated. In addition, the following statistical analyses 

will be carried out: 

! Parameters Statistical test Markers 

in the 

tables 

References 

Weight Non-parametric one-way * for HETTMANNSPERGER, T.P. 

parameters analysis using KRUSKALWALLIS test (two-sided). If 

the resulting p-value was 

equal or less than 0.05, a 

pairwise comparison of each 

P.5 0.05 

** for 

P.5 0.01 

(1984):Statisticallnference based on 

Ranks, John Wiley & Sons New York, 

132-140. 

International Mathematical and 

Statistical Libraries, Inc., 2500 Park 

dose group with the control 

group was performed using 

the WILCOXON test for the 

hypothesis of equal medians 

West Tower One, Houston, Texas 

77042-3020, USA, nakl-1 - nakl-3 

MILLER, R.G. (1981): 

Simultaneous Statistical Inference 

Springer-Verlag New York Inc., 165­

167 

NIJENHUIS, A. and S.W. WILF (1978): 

Combinatorial Algorithms, Academic 

Press, New York, 32-33 

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

Page 42 of 368 

I 
-


4. RESULTS 

Throughout the chapter "results", the term "signifICant" implies that the inter-group differences 

have attained statistical significance (p .::: 0.05) when compared with the control group. 

4.1. ANALYSES 

4.1.1. Stability analyses 

The stability of the test substance in the vehicle was demonstrated over a period of up to 96 

hours at room temperature (see PART III). As the suspensions were stored no longer than 

this time period, the stability was guaranteed. 

4.1.2. Homogeneity control 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 of all concentrations revealed that the values were in the 

expected range of the target concentrations, i.e. were always in a range of about 95.5­

118.6% of the nominal concentrations. Although the concentration of the low dose was 

118.6% of the nominal concentration, the data are assessed to be valid. 

For details see PART III. 

4.1.4. Food analyses 

On the basis of duration of use and the analytical findings with respect to chemical and 

microbiological contaminants the diet was 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 of thel I 

I I 

4.1.5. Drinking water analyses 

On the basis of the 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 of the I 1...-----1 

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

Page 43 of 368 
4.1.6. Bedding and enrichment analyses 

On the basis of the analytical findings the 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 be found in the archives of the 

I I 


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

Page 44 of 368 

4.2. CLINICAL EXAMINATIONS 

Summary tables of the results are given in the Appendix of PART I; individual values are 

given in Part A of PART II. 

4.2.1. Mortal ity 

1 male and 1 female of group 3 (60 mg/kg bw'day) died prematurely on day 6 and on day 7, 

respectively; 1 female of group 3 (45 mg/kg bw/day) was sacrificed moribund on day 11. 

These cases of deaths were assessed as related to the test-substance. 

4.2.2. Clinical examinations 

(Tables IA- 1 - IA-16) 

High dose (60 mglkg body weight/day; from day 0 until day 7): 

• 	 Convulsions, clonic were observed moderate in 4 females, severe in 2 females, 

epileptoid slight in 1 male, moderate in 1 female, severe in 2 males and 1 female, tonic 

slight in 1 female 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 

• 	 Hyperexcitability was 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 1 female 

• 	 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 mglkg body weight/day; from day 8 until the end of the administration period): 

• 	 Anogenital region, smeared with urine was observed severe in 1 female 

• 	 Convulsions, tonic were observed slight, moderate and severe in each 1 female 

• 	 Hyperactivity was observed in 1 female 

• 	 Hyperesthesia was observed slight in 2 males and 1 female 

• 	 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 1 female 

• 	 Nose, discharge, red was observed slight in 1 female and severe in 2 females 

• 	 Piloerection was observed in 2 males and 2 females 

• 	 Reduced general condition was observed slight in 1 female and severe in 2 females 

• 	 Salivation was observed slight in 1 female, moderate in 2 females and severe in 1 female 

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

Page 45 of 368 
• 	 Twitching was observed slight in 4 males and 3 females, moderate in 1 male and 4 

females and severe in 2 females 

Mid dose (30 mg/kg body weight/day): 

• 	 Convulsions, tonic were observed moderate in 1 male 

• 	 Hyperactivity was observed in 1 female 

• 	 Hyperesthesia was observed slight in 1 male and 5 females 

• 	 Hyperexcitability was observed slight in 5 males and 5 females, moderate in 3 males and 

4 females and severe in 1 female 

• 	 Piloerection was observed in 1 male 

• 	 TWitching was observed slight in 5 males and females, moderate in 4 females and severe 

in 1 female 

Most of the above-mentioned findings were obseNed on different days of the study, starting 


on day 0 after treatment and were present most of the 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 to the 


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 of group 3 (60 mg/kg bw/day) on day 7; up to -11.9% in males 

and statistically significantly of -15.3% in females of group 3 (45 mg/kg bw/day) on day 14; 

up to -9.8% in males and statistically significanUy of -18.0% in females of group 2 (30 mg/kg 

bw/day) on day 7 and up to -6.1 % in males and stati&ically significantly of -12.3% in females 

of group 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. 	Water consumption 

(Tables IA-21 - IA-24) 

Water consumption was predominantly increased in all dosed animals, more pronounced in 

females; viz of +13.9% in males and statistically significantly of +36.9% in females of group 3 

(60 mg/kg bw/day) on day 7; up to +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 day 21, +26.4% in females 

on day 14 and statistically significantly of +24.6% in females of group 2 (30 mg/kg bw/day) on 

day 28 and up to +14.0% in males on day 21 and +27.3% in females of group 1 (10 mg/kg 

bw/day) on day 14. 

Because of the high toxic effect of the test-substance in all dosed animals, this was assessed 

as substance-related. 

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

Page 46 of 368 
The decreased values of -8.5% in males group 3 (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. 	Body weight data 

(Figures 1 - 2; Tables IA-25 - IA-32) 

Body weight was statistically Significantly decreased of -13.9% in males and not statistically 

significantly decreased of -6.5% in females of group 3 (60 mg/kg bw/day) on day 7; it was 

also decreased during the administration period, up to -8.6% in males and -4.4% in females 

of group 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 of group 2 (30 mg/kg bw/day) on day 7 and up to -4.8% in males on days 7 and 28 

and -4.3% in females of group 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 deviations to control groups were assessed to be 

substa nce-related. 

Body weight change was statistically Significantly decreased of -60.8% in males and -50.9% 

in females of group 3 (60 mg/kg bw/day) on day 7; it was also decreased during the 

administration period in all dosed animals, up to -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 of group 2 (30 mg/kg bw/day) on day 7 and -20.9% in males and 

statistically Significantly of -35.3 in females of group 1 (10 mg/kg bw/day) on day 7. 

All above-mentioned body weight change deviations to 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 of group 3 (60 mg/kg 

bw/day) on day 7, statistically Significantly increased in males of group 3 (45 mg/kg bw/day) 

on day 14 and not statistically increased in both sexes during the administration period; food 

efficiency was also statistically significantly decreased in both sexes of group 2 (30 mg/kg 

bw/day) and in females of group 1 (10 mg/kg bw/day) on day 7. 

Because of a clear relationship to other clinical findings, like body weight data and food 

consumption, this finding was assessed as substance-related. 

On several occasions, food efficiency differed (increased/decreased values) in treatment 

groups 2 and 1 from controls in males and females on some days of the study. 

These findings were spontaneous in nature and therefore not substance-related. 

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

Page 47 of 368 
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. 

Beside this, 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 of group 3 (45 mg/kg bw/day). 


This was assessed as substance-related, because of the systemic toxicity of the 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. 

Sensorimotor tests/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 2/3 females of group 3 (45 mg/kg bw/day) 


This was assessed as substance-related, because of the systemic toxicity of the test article. 


Quantitative parameters: 

(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 of group 1 (10 mg/kg bw/day). 


Due to a clear dose-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 hind limbs" 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 of group 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 of the test article. 


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

Page 48 of 368 
4.2.8. Motor activity measurement 

(Figures 3 - 4; Males see Tables lA-53 -lA-54 and Females see Tables lA-55 - lA-56) 

Regarding the overall motor activity statistically significantly increased deviations were 


measured in males and females of group 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 showed a statistically significantly increase. 


These findings were clearly associated to the treatment with the test article and the above­


mentioned systemic toxicity. 


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

Page 49 of 368 
4.3. CLINICAL PATHOLOGY 

Summary tables of the results are given in the Appendix 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 of the test substance for 4 weeks there was noticed a statistically weakly 

significant decrease of the haemoglobin concentration in the male 10 mg/kg bw/d as well as 

in the 45 mg/kg bw/d dose group. This decrease of the haemoglobin levels was not 

accompanied by a deviation of any other red blood cell marker. Compared to the historical 

control data (min value: 9.1 mmollL, max. value 9.8 mmollL), the median of the control rats in 

this study was slightly higher than the historical controls, whereas the values of the 10 mg/kg 

bw/d as well as the 45 mg/kg bw/d dose groups were within the historical controls. 

Therefore, this decrease of the 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 there was observed a decrease of the median of the total protein level 

beginning in the 30 mg/kg bw/d dose group. This decrease was reflected in the decrease of 

the globulin fraction in the 45 mg/kg bw/d dose group. 

Even in the female rats of the 45 mg/kg bw/d dose group the total protein as well as the 

albumin and globulin fraction were decreased even not statistically significant. This decrease 

is accompanied by a slight but not statistically significant decrease of the serum glucose and 

triglyceride values in the dosed animals of both sexes. 

4.3.3. Urinalyses 

(Tables IB-15 -IB-18) 

There were no treatment-related changes in the urinalyses parameters measured. 

4.3.4. Other deviations 

Additionally, there was found a decreased median of the potassium levels in the 10 mg/kg 


bw/d as well as 30 mg/kg bw/d dose group of the female rats (Table IB-13). 


This deviation was inconsistent, when compared with the other sex, and lack dose-response 


relationship. Accordingly, this finding is considered to be of no toxicological significance. 


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

Page 50 of 368 
4.4. PATHOLOGY 

Summary tables of the results are to be found in the Appendix of PART I; individual tables 

are to be found 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 dose animal (animal no. 38) 7 days after start of exposure. One female high 

dose animal (animal no. 36) has to be sacrificed in a moribund state 11 days after start of 

exposure. 

4.4.2. Weight parameters 

(Tables IC- 1 - IC- 4) 

4.4.2.1. Absolute organ weights 

There were no treatment-related weight deviations recorded. 

The significant weight decrease of the thymus in dose group 2 females is regarded to be 

incidental and of no biological relevance (no dose-response relationship, no substanceinduced histopathological lesions in the high dose). 

4.4.2.2. Relative organ weights 

There were no treatment-related weight deviations recorded. 

The significant weight increase of the brain in dose group 2 females is regarded to be 

incidental and of no biological relevance (no dose-response relationship, no substanceinduced histopathological lesions in the high dose). 

4.4.3. 	Gross lesions 

(Tables IC- 5) 

There were 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. 

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

Page 51 of 368 
5. DISCUSSION 

Regarding clinical examinations, the oral administration ofliby gavage for 4 weeks 

caused severe signs of general systemic toxicity. Reduced f50crana increased water 

consumption, impaired body weight and body weight change, food efficiency, increased 

rearing, reduced grip strength in fore- and hildlimbs and increased deviations in overall 

motor activity were obtained down to 10 mg/kg body weight/day in both sexes. 

Moreover, 1 high dose animal of each sex was found dead within the first week of the 

administration period, Additionally, 1 high dose female was prematurely sacrificed moribund 

on study day 11. 

Due to this exceeding of the maximum tolerated dose level, the highest dose was reduced 

from 60 to 45 rng/kg body weight from study day 8 onwards. 

Regarding the clinical pathology, the decrease of the protein levels in the dosed rats of both 

sexes combined with slightly lower glucose and triglyceride levels is regarded as a result of a 

dysbalance between food intake and metabolic activity of the rats. 

There is no hint in clinical pathology for a failure of the liver cells to synthesize proteins. Even 

pretended decreased protein levels because of decreased haematocrit values can be 

excluded in the rats of this study. 

Regarding pathology, there were no treatment-related findings noted. All findings recorded 

are considered to be incidental in nature and not related to treatment. 

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

Page 52 of 368 
6. CONCLUSION 

In conclusion, the oral administration o~ Iby gavage over a period of 4 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 

respect to the tissue and organs examined during necropsy, organ weight determinations and 

light microscopy. 

Therefore, the no observed adverse effect level (NOAEL) under the conditions of this study 

was below 10 mg/kg body weight/day for male as well as female Wistar rats. 

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

Page 53 of 368 
7. APPENDIX 

7.1. LIST OF ABBREVIATIONS USED IN TABLES IA (CLINICAL EXAMINATIONS) 

Food Cons. 

Water Cons. 

BW change 

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 

Water consumption 

body weight change 

food efficiency 

milligram 

kilogram 

body weight 

weight in gram 

mean value 

standard deviation 

number of animals for determining M and SD 

animal 

rearing 

grip strength forelimbs 

grip strength hind limbs 

landing foot-splay test 

summary 

deviation in percent 

centimetre 

interval 

beam Interrupts 

twitching 

animal dead 

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

Page 54 of 368 
7.2. LIST OF ABBREVIATIONS USED IN TABLES IB (CLINICAL PATHOLOGy) 

SD = standard deviation 

N = number of values 

HEMATOLOGY: 

WBC = white blood cells (leukocytes) 

RBC = red blood cells (erythrocytes) 

HGB = hemoglobin 

HCT = hematocrit 

MCV = mean corpuscular volume 

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 = polymorphonuclear neutrophils (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 

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

Page 55 of 368 
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 = 1.00 gil, 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 

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

Page 56 of 368 
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 101211iter 

giga/l gigalliter = 1 09 Iliter 

IJkatll = microkatai/liter 

nkatll = nanokatalliiter 

mg/kg bw/day = milligram/kilogram body weightlday 

IJI = microliter 

ML ml (milliliter) 

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

Page 57 of 368 
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 = number of values measured forthe determination of mean value and 

standard deviation 

NAD = number of animals without gross lesions 

ppm = parts per million under dose level 

SD = standard deviation 

s = suspect weight (not included in the mean weights) 

% = percentage related to the reference weight in relative organ weight 

calculations 

Codes for the 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 takes into consideration either the severity or 

the number or the size of a microscopic finding. 

Severity Number Size 

Grade 1 Minimal Very few 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 microscopic finding was indicated to be present (P). 

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

Page 58 of 368 
;! 

or 

g 

c: Project No. L-____---l Body weight, males Figure 1 ~ 3­

iii' 
 300 1l 

""0 

I 

o 

g 

1l 

~ :::l 

~ (i) 

~ "1J 0. 250 

Il> is' to c. (1) g 

CJ1""O 

tO~ 

a w ~I I/) c: E ./x 

0') ~ I!! CXl~ 

o 

5' 

al 

~ 

01 

200 

- """­

i: ~ -0-0 mg/kg bw/day 

~ c: -CI- 10mg/kg bw/day 

. ~. -& 30mg/kg bw/day 

i I-x- 60/45mg/kg bw/day I :::l 

""0 

a> 

3 

~. 

5" 

:::l 

~ 150 +-~"""""""""" 5' 

a> 

""0 

(/J o 7 14 21 28

:::l 

o 60 mg/kg bw/day from day 0 till day 7 days

Q 45 mg/kg bw/day from day 8 till day 28 (/J 
--

-- -- o 

<10 

o 

z 

ti 

II) 

"ell. 

SWBJ6 

T 00 N 

I 

<10 1"-1"- N 

:>. :>. 

Cll Cll UU 

:.;:::; 

o <10 

:>. :>. 

Cll Cll uu 

E E 

0 0 

L.. L.. 

:>. :>. 

Cll Cll U U 

~~ ..c ..c Ol Ol ..loI:: ..loI:: 

0l0l 

oE E 01.1') 0 <O-.:t ("') 

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

Page 60 of 368 
CD 

N 

>­ I'C 

"C 

C 

o 

.... 

c 

CIJ 

E 

~ ::I 

:Q 

CIJ 

E 

>­ := 

> 

~ I'C 

... 

o 

-
 o 

:iE 

0 

z 

-
f.) 

CIJ 'eD.. 

N

i r I I I I 

I 

O 

0) 

I '\ I \ /

\y4 

c\ /" \ / \ 

/ 

J 

I 

I 

I 

I 

I 

~ I 

o 

o 

co 

o 

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

Page 61 of 368 
---

, fl[~

',/ \ I' 

/ \ 

\ 

\ 

, " 

lo(J 

Ii 

~ Ii \ ; i '. 

1:1 0 

I // ! '/ / 1/

I 

I 

II 

~ 

! I 

I i 

I I

I J ~ \ 

\ 

N 

0 

>. 

i~ ~ >. C'(I C'(I ~ ~ -a 32 ~ 

CJ ~ ~ ~ ~i u::: C> C> i C> Sf ..loG ..loG ..loG C> Ol Ol 

I -C> / E E E E <l 0 0 I/) 

0 (") ~ \ 

I I 

~ I I 

0 + ~ 

I 

\ 

\ 

/ 

I

/ 

.;1 1/1

Q) /

iii I 

I 

E I 

I 

Q) I ! 

I I -..: ! 

N 

>. .p ! 


C'(I 
 I 

I "C 

c I 

-

C 

0 

I 

/

i 

Q) <&E CJ l"­

... '. 

\\ 

' 

II I 

E ,\ Q) ! 

I iii ~ I c: 1/1 \ \ I Q) C'(I I

\\ Q) / \\ -.5 E \\ / 

-'S; 

\\ I 

>. (0 ,:¥ ... 

U 

/' /

I 

I III ,:// ... 

0 /7

- '/

0 *<i I/) 

2 II

1\ 

ci 

z 

-

u 

Q) 

'C)

... 0 0 0 o o o o o o o c... 0 Cf> to (0 I/) ~ ("') N 

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

Page 62 of 368 
-I 

:T 

(ii' 

a. 

0 

n 

c: 

3 

(1) 

a 

(ii' 

S' 

(1) 

10 

a 

10 

(1) 

~ 

a 

S' 

(1) 

10'" 

0 

::J 

'"0 

ii1 

U) 

-0 

(1) 

co "U ::i 

Dl ~ r.o 0 ('\) ::J 

-0 Q') ll> 

W <0 

0

...... -

(1) 

~ 

w c:

Q') '"(1) 

co 0" 

'< 

~ ~ 

-0 

ll> 

~ 

'" 

~ c: 

;' 

'" 

it 

~ 

::J 

'0 

(1) 

3 

(ii' 

'"0' 

::J 

0" 

'< 

S' 

(1) 

10 '" 0 

::J 

'"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 

severe 

Incidence 1 ( 20%) 


Observed 1 


Mean onset (Days) 2 

-l

::r 

(ii' 

0. 

?l 

c 

3 

<l> 

a 

(ii' 

SC1) 

"0 

a 

al 

;:I. 

'< 

Sl. 

S­

<l> 

(j) 

"0 

0 

:::I 

(j) 

0 

iil 

fi) 

"0 

C1) 

Q. '"'C :::!l 

C1) Q.) 0. CO 0 CD :::I 

0)"2 

~(QC1) 

fa,,,.... 

w c 

m 0) C1) 

00 0­ '< 

0 

S­

'!l 

"0 

OJ 

;:I.

iii' 

m 

til 

.Q 

C 

~. 

(fl 

~ ~ 

<l> 

:::I 

"0 

C1) 

3 

(ii" 

(fl ci" 

:::I 

0­

'< 

SC1) 

"0 '" 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 

slight 

Incidence 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 

severe 

Incidence 1 ( 20%) 


Observed 1 


Mean onset (Days) 2 


slight 

Incidence 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 


severe 

Incidence 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 

i3 

-0 

<D 

~ 

'< 

sa. 

5' 

<D 

Ol 

"0 

0 

::l 

U) 

0 

in 

~ -0 

<D 

n -0 9i 

<D ru Co (Q 0 CD ::l 

mal 

01""<D 

8,::­<: W Ol m <D 

(Xl c:r 

'< 

~ !!; 

-0 

til 

a 

<D 

U) 

<il 

.0 

<: 

ai" 

U) 

~" 

1if 

::l 

-0 

~ 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 


severe 

Incidence 1 ( 20'1;) 


Ol::>served 3 


Mean onset (Days) 2 


slight 

Inoidenoe 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 o 1 


Mean onset (Days) 6 

-i

:r 

in' 

0. 

0 

(') 

c: 

3 

<D 

2­

iii' 

go 

<D 

'tJ 

(3 

'tJ 

<D 

::l. 

'< 

a 

go 

<D 

(Jl 

'tJ 

0 

::l 

(Jl 

0 

iil 

(jl 

'tJ 

<D 

(') 

'"tl ~ 

<D t'l) 0. (Q 0 CD ::l 

O)al

0)<0<D 

o ... -., ~ 

c: 

0) <D 

CO cr 

(..V (Jl 

'< 

~ ~ 

'tJ 

ru 

::l. 

(5' 

(Jl 

til 

'" c: 

~. 

(Jl 

~ 

a>'"' 

::l 

iil 

3 

(ii' 

(Jl 

0' 

::l 

C" 

'< 

9' 

<D 

(Jl 

'tJ 

0 

:::l 

(Jl 

0 

til 

study: 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 

slight 

Incidence 

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 

II

c:: 

3 

(I) 

a 

(ii' 

9' 

(I) 

'C 

a 

'C 

(I) 

::l. 

'< 

sa. 

9' 

(I) 

(I) 

'C 

0 

::J 

(I) 

~ (i) 

'C 

(I) 

Q, 1J ::!\ 

(I) III Co (.Q 0 (1) ::J 

mal 

...... <0 (I) 

Q..,.... 

VJ c 

til m (I) 

ex> 0­ '< 

S? 

::r 

Ql 

'0 

Q) 

::l. 

Iii' 

til 

m 

.Q 

c:: 

~. 

til 

~ m 

::J 

16 

3 

(ii' 

til 

0' 

::J 

0­

'< 

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 

severe 

Incidence 1 ( 20%) 


Observed 3 


Mean onset (Days) 11 


slight 

Incidence 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 


slight 

Incidence 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) 

::liii· 

g. 

(l) 

""0 

a 

""0 

(l) 

~ 

9. 

g. 

(l) 

""0 '" 0 

:::l 

~ 

iil 

W 

""0 

(1) 

Q. -U ::!l

(1) D.) c. co 0 (I) :::l 

0') ""0 

W 

00 <0 -.::= 

. 

(l) 

o 

W c:

0') '" (l) 

00 0­

'< 

0 

g. 

g; 

""0 

ID 

e:l-. 

(l) 

(J) 

iil 

.0 

50. 

iil 

i '" 

:::l 

""0 3'" 

iii· 

'" o· 

:::l 

0­

'< 

g. 

(l) 

-g 

(Il 

:::l 0'" 

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 29 
BASF -'I....-____...J Print Date: 06-Nov-2006 

-1

:::r 

or 

a. 

0 

(") 

c: 

3 

<I> 

;:t 

ur 

5' 

<I> 

"0 

a 

"0 

<I> 

~ 

g 

5' 

<I> 

(J) 

"0 

0 

::J 

(J) 

0 

;;: 

(j) 

"0 

<I> 

Q. -U =" 

Q) <I>

a. co 0 m ::J 

0) "0 

<D <0

<I> '" 

0

_'::­

W c:

(J)

0) <I> 

co cr 

'< 

S!. 

:::r 

~ 

"0 

:J. '" iir 

(J) 

iil 

.n 

c: 

~. 

(J) 

~ 

~' 

::J 

"lil 

3

00· 

'" o· 

::J 

~ 

5' 

<I> 

"0 '" 0 

::J 

0 '" ;;: 

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 o 15 


Mean onset (Days) 3 

-; 

:::T u;" 

a. 

0 

() 

c 

3 

(J) 

;a. 

u;" 

5' 

(J) 

"0 

a 

"0 

(J) 

q 

Q, 

5' 

(J) 

fA 

"0 

0 

:> 

fA 

0 

ill 

(i;" 

"0 

(J) 

() 

:;t -U (J) tl) a. CO 0 CD :> 

"0 -."J OJ 

0 <0

(J) 

9,::-­

W c 

fA Ol (J) 

(X) C" 

'< 

£ 

:::T 

:J; 

al 

::l. 

iii 

fA 

iil 

.0 

C 

~. 

fA 

:Ea' (J) 

:> 

"0 

(J) 

3 

~. 

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 


severe 

Incidence 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 


severe 

Incidence 2 ( 40%) 


Observed 
 3 


Mean onset (Days) 5 

--l 

::l" 

iii' 

c. 

0 

(') 

c 

3 

(I) 

;a. 

<n' 

i 

'0 

0 

'0 

(I) 

;:l. 

'< 

Q. 

5' 

(I) 

(J) 

'0 

0 

:l 

(J) 

~ (i) 

'0 

(I) 

(') 

BASF - 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 

slight 

Incidence 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 


severe 

Incidence 1 ( 20%) 


Observeci 
 3 


Mean onset (Days) 3 


-U 

OJ 

<0 

(J) 

--.J 

..... 

o 

..... 

W 

(j) 

CD 

5i 

(I) 

c. 

0 

:l 

'0 

III 

<0 

(I) 

..... 

".... 

c 

CI) 

(1) 

a­

'< 

$l. 

::l" 

~ 

'0 

III 

;:l. 

iii' 

CI) 

(il 

.c 

c 

~. 

CI) 

~ 

(1) 
 = 

:l 


'0 

(I) 

'3 

(ii' 

CI) 

cr 

:l 

a­

'< 

5' 

(I) 

CI) 

'0 

0 

:l 

CI) 

0 

Ul 
-l 

:::T 

iir 

c0 

(") 

c: 

3 

<l>

;a 

In" 

S' 

<l> 

'0 

a 

'0 

<l> 

::l­

'< 

g, 

S' 

<l> 

(J) 

"0 

0 

(j) " 0 

U! 

(j) 

"0 

(1) 

Q. "'C 	 (1) Dl 	 "" C­ eo 0 (1) 	

"0 " ~ !l> 1\.)<0(1) 

9.0­

W 	 c:

(J) 

(j) 	 <l> 

CO~ 

~ ~ 

"0 

!l> 

::l­ roO 

(J) 

CD 

.0 

S:. 

iil 

'" 

li 

~ 

"0 " 

<l> 

3 

In" 

(J) 

0' 

=> 

CT 

'< 

S' 

(1) 

(J) 

"0 

0 

" '"0 

til 

BASF 	-1 Print Date: 06-Nov-2006

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 


severe 

Incidence 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 

or 

0. 

0 

(") 

c 

3 

<1l 

;:l. 

(ii' 

:;: 

<1l "a 

" <1l 

::I. 

'" 9­

:;: 

(1) 

(j) 

'8 

::1 

i!l 

Ul 

(i) 

(1) " C'l 

"1J 3i 

<1l OJ 0. (0 0 CD ::1 

""-I <0" W '" <1l 

.....

0-., 

W (j) 

c 

0') <1l 

CXl C' 

'" 

~ g; 

" a'" 

(1) 

(j) 

@ 

D 

c 

,if

(j) 

~,::::: 

<1l 

::1 

(1) " 3 

iii' 

S!), 

0 

:l 

C' 

'" :;: 

<1l 

(j) 

" 0 

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%) o 0%) o 0%) 1 ( 20%) 

Observed o o o 1 

Mean onset (Days) 7 

06-Nov-2006 

10:24:50 

IA 

11 
--I 

:::T 

00' 

a. 

0 

c 

3 " 

<1> 

:=. 

00' 

5' 

<1> 

u 

a 

u 

<1> 

;+ 

'< 

Q. 

5' 

<1> 

u'" 

0 

:::J 

'"0 

Ul 

V> 

u 

<1> 

-U 5" 

0> <1> 

a. to 0 (1) :::J 

OJ " u 

~(O<1> 

!a,::­

W c 

(j) '"<1> 

ex> 0­ '< 

0 

5' 

~ 

u 

OJ 

do 

<1> 

'" 

CD 

.0 

c 

~. 

'" 

~ 

a 

<1> 

:::J 

u 

<1> 

3 

00' 

'" o· 

:::J 

0­

'< 

5' 

<1> 

u'" 

0 

:::J 

'"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 

0 

"0 

<I> 

;:+ 

'< 

Q. 

g: 

<I> 

VI 

"0 

0 

::J 

VI 

Q 

VI 

(jj' 

"0 

<I> 

(") 

-U =0;

iii" Q) 0. 

to 0 <D ::J 

"0 -...J OJ 

(J1 co 

<I> 

9..::-­

W c 

VI 

(j) <I> 

CO 0­ '< 

Q.

::T 

~ 

"0 

OJ 

;:+

iii" 

VI 

CD 

.0 

C 

~. 

VI 

~ 

~ ::J 

"0 

<I> 

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 

slight 

Incidence 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 


severe 

Incidence 1 ( 20%) 2 ( 40%) 


Observed 1 11 


Mean onset (Days) 15 10 


slight 

Incidence 5 (100%) 3 ( 60%) 


Observed 18 28 


Mean onset (Days) 12 10 

-i

:::r 

iii' 

c. 

0 n

c:: 

3 

(!) 

~ 

iii' 

:T 

(!) "0 

" 

~ '< 

!2, 

:T 

(!) 

rn 

"0 

0 

:::J 

w 

0 

iil 

'VI 

"0 

(!)

n, '"tI (!) Q) "" c. CO 0 (1) :::J 

...,-g 

0)<0(!) 

Q..,..... c:: W w 0) (!) 

(Xl t:r 

'< 

~ !!; 

"0 

ru 

::lffi' 

w 

iii 

.Q 

c:: 

~. 

rn 

~ tii 

:::J 

(!) " 3 

iii' 

'" c)"

:::J 

t:r 

'< 

: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" 

5 

m 

"0 

a 

"0 

m 

~ 

'< 

g, 

5 

<1> 

VI 

"0 

0 

'"VI 

0 

en 

(i) 

"0 

<1> 

n 

"'U ~ 

Q) <1> 

0. (C 0 <D '" -...j~ -...j1O<1> 

9,.,­<: W III m m (X) 0­

'< 

0 

5 

!!l 

~ ~ 

or 

VI 

a; 

J:l 

<: 

~. 

III 

~ 

:= 

m 

"0'" 

<1> 3

.i" 

III 

15" 

'"0­

'< 

5 

<1> 

III 

"0 

0 

'" 113 

en 

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 


slight 

Incidence 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 

:::. '"

in" 

s: 

"0'" 

0 

"0 

::l. '" '< 

Q, 

s: 

Cl> 

"0'" 

0 

::J 

'"0 

en 

(i) 

"0 

Cl> 

0 

iJ ~ 

!l) Cl> 

a. CO 0 (1) :J 

"""-I'iil 

CD'"Cl> 


Q.::­

W c 

O'l '" CD~ '" 

0

s: 

!!; 

"0 

Q) 

::l.

iii" 

'" iil 

.a 

c 

~. 

i '" 

::J 

"0 

3 '" 

in 

l!j"

:J 

0" 

'< 

s: 

Cl> 

"0 '" 0 

:J 

[g 

en 

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 

severe 

Incidence 1 ( 20%) 2 ( 40%) 

Observed 1 8 

Mean onset (Days) 26 10 

slight 

Incidence 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 29 

Print Date: 06-Nov-2006 

Print Time: 11:30:30 

-I 

:;;; 

(ii" 

0. 

0 

n 

c 

3 

<1l

:;;. 

(ii" 

5' 

<1l 

"0 

i3 

"0 

<1l 

:+ 

'< 

Q. 

5' 

<1l 

<Jl 

"0 

0 

::> 

<Jl 

0 

i.il 

en 

~ n 

1J ~ 

<1l Q) 0. (Q 0 CD :::J 

"0 

(\) ....... 
 <0 <0 <1l 0-::­

W c (/) 

0> (!) 

00 a­

'< 

g. 

:;;; 

~ 

-g 

~ (/) 

iii 

.0 

C 

~. 

(/) 

:; 

a 

(!) 

::J 

al 

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 - Animal 
-l

::r 

en" 

a. 

0 

n 

c 

3 

C!> 

3­

en" 

s: 

C!> 

"0 

a 

"0 

~ '" 

a 

s: 

C!> 

II> 

"0 

0 

::l 

:g 

(il 

'iii" 

"0 

'" 0" '"'0 ::!! 

C!> t\) a. <.C 0 co ::l 

(XI "0 

OJ 

0 <C 

C!> 

..... 0 

w 

...... 
 II> 

c 


0') C!> 

(XI c:r 

'< 

a 

:;:,­

!!l 

"0 

OJ 

ilII> 

E 

c 

@" 

II> 

~" (if 

:> 

"0 

<l> 

;3 

en" 

CJ) 

0" 

::l 

~ 

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, * p <= 0.050, ** P <= 0.010 

Experimental Unit = Animal 
-I 

::T 

or 

Q. 

0 

C'l

c: 

3 

(l> 

~ 

<ii' 

5' 

(l> 

"0 

a 

"0 

(l> 

~ 

So 

5' 

(l> 

til 

"0 

0 

:J 

til 

0 

en 

<n 

"0 

(l> 

Q, ""0 ffi' OJ Q. 

(Q 

0 (]) :::l 

"0 CO Q) 

..... <0 

(l> 

0 ~ -
 w c: 

til 

en 
 (l> 


CO C" 


'< 

!? 

::T 

~ 

1il 

;:l 

(1)' 

til 

ro 

.c.

c:

=;. 

(l> 

til 

~ 

~ :J 

"0 

(l> 

3 

<ii' 

til 

0' 

::l 

0­

'< 

5' 

(l> 

til 

"0 

0 

:::l 

!/) 

0 

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 <= 0.010 

Experimental Unit = Animal 
-;

::r 

(i)" 

Q. 

0 n

c: 

3 

<l> 

:a 

(i)" 

5' 

<l> 

a 

16 

-Z 

g, 

5' 

<l> 

{Jl 

-g 

:::! 

<Jl 

0 

iil 

~ 

{Jl 

"0 

1II 

n \J ~ 

Q) 1II 

Q. 

to 

0 CD :::! 

(Xl "2 

I\) <0 

<l> 

9, "::-' 

W 

C

<Jl 


0) <l> 


(Xl c­

'< 

0 

5' 

~ 

"0 

OJ 

d-" 1II 

{Jl 

iil 

.0 

c: 

~. 

{Jl 

~ 

~ :::! 

"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, ** P <= 0.010 
;!

iE' 

0. 

0 

('I 

c: 

3 

<1> 

i2. 

ur 

5' 

<1> 

"0 

a 

"0 

<1> 

::I. 

"" g, 

:::r 

<1> 

Ul 

"0 

0 

:J 

rn 

Q 

Ul 

"0 

'"<1> 

Q. -U :::!l 

!l) <1> 

0. <.0 0 (1) 	 :J 

"0 OJ 	 III 

W (Q

<1> 

0 ::­ -W 	 c: 

rn 0'> 	 <1> 

OJ 	 cr

"" St

::r 

!!l 

"0 

III 

do 

<1> 

rn 

@ 

.c 

S;. 

@ 

rn 

~.::; 

<1> 

:J 

"0 

<1> 

3 

ur 

s!!. 

0 

:J 

cr 

"" 9' 

<1> 

rn 

"0 

0 

::l Q'" 

'" 

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 = Animal 
Print Date: 06-Nov-2006 

Print Time: 11:02:45 

-I

::.r 

iii' 

0­

0 

(") 

c: 

3 

(t> 

~ 

iii' 

s: 

(t> 

"0 

a 

"0 

(t> 

::l. 

'< 

Q. 

s: 

(t> 

fIl 

"0 

C 

:::> 

fIl 

C 

Ul 

<n 

16 

C'l 

-0 :5 

(t> Ol 0­ (Q C CD :::> 

"0 co III 

.,!:.. <0 

(t> 

8,::­

W fIl 

c 

C) (t> 

co c:r 

'< 

fa

::.r 

!ll 

"0 

Q) 

~ (t> 

fIl 

ill 

..0 

C 

~. 

~ 

~ :::> 

"0 

(t> 3

oj'

fIl 

0' 

:::> 

c:r 

'< 

s: 

(t> 

fIl 

"0 

0 

::J 

0 '" 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 <= 0.050, ** P <= 0.010 

Experimental Unit Animal 
:;J 

0:;' 

0­

0 

c: 

3 " 

CD 

a 

<ii' 

:;. 

CD 

'0 

a 

'0 

~ '< 

!2., 

:;. 

CD 

(/) 

'0 

0 

:J 

0 '" iil 

'0 

"' CD12, '1J ffi! 0) 0­ (0 0 m :J 

CD '0 

(J'I co'" 

CD

-

0 .,­

W c:

en CD'" 

CD ~ a::r 

~ 

'0 

::l­ '"

(ii' 

(/) 

co 

.D C

ai' 

(J) 

~ := 

CD 

:J 

'0 

CD 

3 

<ii' 

(J) 

5' 

:J 

~ 

~ (J) 

'0 

0 

:J 

(J) 

Q 

(/) 

Study: [ ~ 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 = Animal 

* p <= 0.050, ** P <= 0.010 
-i 

::r 

iii' 

a. 

0 

() 

c 

3 

;z. '"

iii' 

5' 

'" "0 

a 

"0 q'" 

Q. 

5' 

'" "0 '" 0 

:::> 

[!l 

iii 

(j) 

"0 

Q, '" \J ::!l 

Q) a. '" (0 0 CD :::> -

Q)a\ 

(j)'a; 

0 :­

eN c 

(j) '" 

Q) '"

C" 

'< 

0 

5' 

~ 

"0 

III 

a 

II) '" ii1 

J:J 

C 

~. 

en 

~, 

~ :::> 

i 

3 

iii' 

fIl, 

0 

:::> 

C" 

'< 

5' 

'" 

"0 '" 0 

:::> 

0 '" U\ 

Study: 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 -

(0 tn'" In 

0 ::­

W c:

en '" ro 

0.:> 0­ '< 

0 

5' 

!!l 

-0 

'":lro' 

'" 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 

c:o t\)

'0 

..... <0 

(1) 

.... 

W c:

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> 

1:1 l1ean

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 	 ::> 

1:1 SD co 	 III 

<0

N N 	 CD -

W 

%dev 0 	 .,.... 


c 
 l1ale, 0'> 	 CD'" 

co cr

'< 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/day 

69.9 

10.6 

5 

-10.3 

30 mg/kg hw/day 

60 4 

13.4 

5 

-22.6 

45 mg/kg hw!day 

59.0 

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 Q'"

(J) 

(j) 

"0 

<I> n, "1J :;, 

<I> ru a. ee 0 CD :J 

t01il 

W'al 

a~ 

W c::

fJ) 

0) <I> 

co c:r 

'< 

~ !!; 

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) 

;::Lco' 

'" 

~ 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 

CD :J

(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 

~ 

Q) 

"'C ~ 0.

CO 

0 CD :::l 

<0 (Q ...... "0 

'" (1) 

0 "::-' -

W c 

(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' 

c. 

0 

Project No. C ~ (') 

c: 

3 

(» male animals 

a 

~I HOME CAGE OBSERVATION -0 

a 

-0 posture (» 

:::I. • animal is sitting or lying '< • animal is staying or moving !2. • squatting posture 9' 

(» • abdominal position IJ> 

-0 abdominal position with splayed limbs C 

::! lateral position IJ> 

0 • oblique head posture (ij • opisthotonus Vi" 

"0 

(» tremors lJ a · no tremors 

(0 c. OJ iii' 

• slight tremors 

(J) g • moderate tremors (,O-g severe tremors (,Of!:>(» 

Q.::-­ convulsions 

VJ • no convulsions en 

c: 

0') (» • slight convulsions (Xl 0­

'< - moderate convulsions 0 · severe convulsions 


!!l 


9' 

"0 abnormal movements :::I. '" no abnormalities iii' 

en • manege movements ~ • head shaking .0 

c: • excessive cleaning ~. 

• frequent chewing 


!!j 


IJ> 

a impairment of gait (» 

::! • animal is not walking during observation "0 

3 

(» • no impairment of gait 


stiff gait 
 (ii' 


U> o slight impairment of coordination, unsteady gait 
 0' 


:J o moderate impairment of coordination, shuffling gait 

0' • severe impairment of coordination, dragging of the hindlimbs '< 

g: o severe impairment of coordination, with splayed limbs (» - animal is unable to walk (abdominal or lateral position) IJ> 

"0 

0 

:J other findings 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 behaviour when removed from cage 

~ - animal is tense, but it shows no resistance against handling 0 5 5 5 4
 '6 -animal shows a slight resistance against the handling 1 0 0 0 0 


:: - animal shows no resistance against the handling but appears indifferent 2 0 0 0 0 


ar -animal is difficult to handle, it shows aggressiveness 3 0 0 0 0 


~ animal is very difficult to handle, it shows severe aggressiveness 4 0 0 0 0 


::> 

~ fur 


~ - nothing abnormal detected 0 5 5 5 4 


{ij - discolored fur 1 0 0 0 0 


'1J ~< urine staining of anogenital 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 of pupils 

- 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 is staying or moving 

squatting postu re 

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) half closure 

- 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 10 mg/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 - slight tremors 1 0 0 0 0 


'"O~. - moderate tremors 2 0 0 0 0 


Ol 15' - severe tremors 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 

~ ~. Impairment of galt 

m - animal is not walking during observation 0 0 0 0 0 


:;: - no impairment of gait 1 5 5 5 4 


a stiff gait 2 0 0 0 0 


!g - slight impairment of coordination, unsteady gait 3 0 0 0 0 


16 - moderate impairment of coordination, shuffling gait 4 0 0 0 0 


~. - severe impairment of coordination, dragging of the 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 

W (1)

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 FielD OBSERVATIONS 

activity/arousal level 

- normal exploration of the area 

- reduced exploration of the area 

- severe reduced exploration of the area. animal apathetic 

- increased exploration of the area, sudden or jerky movements 

- hyperactivity 

feces 

- no defecation during observation period 

- feces without abnormalities 

- discolored feces 

- crumbly feces 

- soft feces 

- muicid feces 

- diarrhea 

urine 

- no urination during observation period 

- urine without abnormalities (some wet areas on the filter paper) 

- discoloration of urine 

- polyuria (great wet areas on the filter 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> "" CO 0.

0 ro :J 

..... "'0 

o OJ 

~~ 

0 

-+\ ;;: 

w '" <1> 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 10 mgfkg 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 of object 

touch response 

- no reaction 

- orientation to the stimulus 

- escape after touch 

- aggressive reaction and attacking of object 

- 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 of the pupil to light 

- retarded adaptation of the pupil to light 

- no adaptation of the pupil to light. pupils permanently contracted 

- no adaptation of the 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 of movements 

- nothing abnormal detected, immediate righting response 

- retarded righting response 

- fails to turn 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 

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) 

<0 :t

CD 0

::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, easy to handle, animal is tense, but it shows no resistance 

easy to handle, animal is limply hanging in the hand 


difficult to handle, animal shows a slight resistance 


to handle, animal shows a severe resistance 


vocalization 

- no or only sporadic vocalizations when touched 

- very frequent vocalizations when touched 

- vocalizations always when toUChed 

- vocalization without touching 

pain perception 

- nothing abnormal detected, immediate response to the stimulus 

- weak or retarded reaction to the stimulus 

no response to the stimulus 

- hyperreaction to the stimulus 

other findings 

- 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 

W -~ v;

<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 FUNCTIONAL OBSERVATIONAL 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 or lying 

- animal is staying or moving 

- squatting posture 

- abdominal position 

- abdominal position with splayed limbs 

- lateral position 

- oblique head posture 

- opisthotonus 

tremors 

- no tremors 

- slight tremors 

- moderate tremors 

- severe tremors 

convulsions 

- no convulsions 

- slight convulsions 

- moderate convulsions 

- severe convulsions 

abnormal movements 

- no abnormalities 

- manege movements 

- head shaking 

- excessive cleaning 

- frequent chewing 

impairment of gait 

- animal is not walking during observation 

- no impairment of gait 

- stiff gait 

slight impairment of coordination, unsteady gait 

- moderate impairment of coordination, shuffling gait 

- severe impairment of coordination, dragging of the hindlimbs 

- severe impairment of coordination, with splayed limbs 

animal is unable to walk (abdominal or lateral position) 

other findings 

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 it shows no resistance against handling 0 5 5 5 3 

- animal shows a slight resistance against the handling 1 0 0 0 0 

- animal shows no resistance against the handling but appears indifferent 2 0 0 0 0 

animal is difficult to handle, it shows aggressiveness 3 0 0 0 0 

- animal is very difficult to handle. it shows severe aggressiveness 4 0 0 0 0 

fur 

- nothing abnormal detected 0 5 5 5 3 - discolored fur 1 0 0 0 0 

- urine staining of anogenital 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 is cold 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 

- 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 

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 of pupils 

- oblique eye posture 

- opacity 

- cataract 

posture 

- animal is sitting or lying 

animal is staying 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. 

CD 0

::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 

res piratlon 

- nothing abnormal detected 

- respiration labored 

- gasping/respiratory sounds 

- respiration accelerated 

- respiration irregular 

tremors 

- no tremors 

- slight tremors 

- moderate tremors 

- severe tremors 

convulsions 

- no convulsions 

- slight convulsions 

- moderate convulsions 

- severe convulsions 

abnormal movements/stereotypies 

- no abnormalities 

- manege movements 

- head shaking 

- excessive cleaning 

- frequent chewing 

Impairment of galt 

- animal is not walking during observation 

- no impairment of gait 

- stiff gait 

- slight impairment of coordination, unsteady gait 

- moderate impairment of coordination, shuffling gait 

- severe impairment of coordination, dragging of the hindlimbs 

- severe impairment of coordination, with splayed limbs 

- animal is unable to 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 

0 (1)

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 of the area 

- reduced exploration of the area 

- severe reduced exploration of the area, animal apathetic 

- increased exploration of the area, sudden or jerky movements 

- hyperactivity 

feces 

- no defecation during observation period 

- feces without abnormalities 

- discolored feces 

- crumbly feces 

soft feces 

- muicid feces 

- diarrhea 

urine 

- no urination during observation period 

- urine without abnormalities (some wet areas on the filter paper) 

- discoloration of urine 

polyuria (great wet areas on the filter 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 

- approaching to object 

- escape reaction 

- aggressive reaction and attacking of object 

touch response 

- no reaction 

- orientation to the stimulus 

- escape after touch 

- aggressive reaction and attacking of object 

- 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 of the pupil to light 

- retarded adaptation of the pupil to light 

- no adaptation of the pupil to light, pupils permanently contracted 

- no adaptation of the 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 of movements 

nothing abnormal detected, immediate righting response 

- retarded righting response 

- fails to turn 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 w (l>

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 

SENSORIMOTOR TESTS/REFLEXES Rank N=5 N=5 N=5 N=3 

behaviour during handling 

- normal behavior, easy to handle, animal is tense, but it shows no resistance 0 5 

- very easy to handle, animal is limply hanging in the hand 1 0 

- slightly difficult to handle, animal shows a slight resistance against handling 2 0 

- difficult to 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 to the stimulus 0 5 

- weak or retarded reaction to the stimUlus 1 0 no response to the stimulUS 2 0 

- hyperreaction to the stimulus 3 0 

other findings 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 

2 
Study: 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 '" Ul SD

'iii' 

N '0 ,"dev ,.,

en 

'"0 

!l) ~ Male, (Q a. 0 ro :;J 

..... '0 Mean ..... Q) SO w'al -

w 

....

N

0 %dev 

5i 

0') en Male, 

CO '< 

cr 

0 

5' Mean

~ SO '0 

III 

N ::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 51 
Study: 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> 

a. 
 Female,

0 
 co 

CD ::l 

..... -0 Mean Q) ..... co SO

.f;l.. (I) N 


0 ~ 

%dev -. c: 

W fJ) 

<1> O'l Female,

CXl ~ 

::r 

~ Mean

!ll SO -0 

Q) 

N ::I. %dev iiifJ) 

~ Key: NC

FECES 

N 

Day 27 

k 

0 mg/kg bw/day 

0.0 

0.0 

5 

10 mg/kg bw/day 

0.0 

0.0 

5 

NC 

30 mg/kg bw/day 

0.0 

0.0 

5 

NC 

45 mg/kg bw/day 

0.0 

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 -W en 

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 

-W II> 

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 4 

Print 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) " QIJ) 

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 

4 
Print 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:: W (J)

(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 5 
study: 

:r 

-i NEUTA LYMPHA MONOA

(ii' giga/l giga/l giga/l 0. 

0 Day 29 Day 29 Day 29 (') 

c: 

3 

<D Female, 0 mg/kg bw/day

~ 

iii' Mean 0.58 4.18 0.07 

s: SD 0.27 0.98 0.01 <D 

'0 N 5 5 

5

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 (') 

=t N 5 5 

5

!l) <b 

co C. 

CD 0 Female, 45 mg/kg bw/day ::J 

..... '0 

I\.) tl> 

<0 Mean 0,50 3.76 0.06

,f;. <b SD 0.08 0.13 0.02 

0 ~ N 3 3 3 co -

W f/) 

<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 

co II

CD 

0

::> 

~ "0 

I\.) ru 

<0 

U1 (!) 0-::-

c W til

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> = co a.

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> Qtf> 

<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 = Animal 
Study: I -l

::r 

(ii' 

a. 

0 

c:'"' 3 

(1) 

a Female, 

(ii' 

S' Mean (1) 

"0 SD

a 

N "0 

~ '< Female, 

a 

S' Mean <!) 

(fj SD "0 

0 N ::I 

~ 

Ul Female,

Vi" 

"0 

(1) Mean

12, ""'"Cl :::!l SD

Q) (1) 

(.Q a. 

N

CD 

0

::I 

Female, 

I\) '" ..... "0 

<0 co <1> Mean 

a 

- SD c: w N ~ 0) 0" 

ALl' 

Jlkat/l 

Day 29 

k 

0 mg/kg bw/day 

0.43 

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 ~ ::r Experimental Unit = Animal

~ 

"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. 

CD 0

:::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. 

CD 	 0

::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 

:Tw" 

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. 

CI.l 0

::> 

...,\, "0 

!\) W co N <1> 

0 ­

c: 

-W (Jl 

<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/day 

2.18 

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 

W rn

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 -

W (J) 

<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 ':-' 

W - c:'"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 of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors 

Page 137 of 368 
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 is the property of the sponsors (specified on page 1). use by other parties requires written permission by the sponsors 

Page 138 of 368 
D PATHOLOGY REPORT 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 property of the sponsors (specified on page 1), use by other parties requires written permission by the 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 property of the sponsors (specified on page 1). use by other parties 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 

HypereIlll a 

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 

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

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 


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

Page 142 of 368 
D PATHOLOGY REPORT IC- 7/8


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 


Hema topoiesi s, extramedullar 1 


Necrosis, (IT.ul ti) 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 


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

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 

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

Page 144 of 368