TS-CKHI02

Attachment #11

Health Effects Data on the New Chemical Substance

Acute Oral Toxicity Test in Rats

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301



### FINAL REPORT

ACUTE ORAL TOXICITY STUDY

OF TEST ITEM

Polymer

IN RATS

Date of Final Report: 07 March 2011 STUDY CODE: 10/339-001P

### STUDY CODE: 10/339-001P Page 5 of 26



## LAB GENERAL INFORMATION

STUDY TITLE: Acute Oral Toxicity Study of Test Item

Polymer in Rats

SPONSOR:

STUDY PERFORMED BY: LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

Phone: +36 (88) 545-300

Fax: +36 (88) 545-301

STUDY DIRECTOR: István Buda, M.Sc.

RESPONSIBLE PERSONS: Judit Tavaszi, M.Sc. - assistant scientist

István Pásztor DVM - veterinary control

Peter Maslej, DVM, PhD – Head of Pathology Unit

Tamás Mészáros , PhD – Technical Team Leader of

Central Dispensary

### STUDY CODE: 10/339-001P Page 6 of 26



## LAB CONTENTS

1. SUMMARY 7

2. INTRODUCTION 9

2.1. STUDY SCHEDULE 9

3. MATERIALS AND METHOD S 10

3.1. TEST ITEM 10

3.2. EXPERIMENTAL A NIMALS 11

3.3. ADMINISTRATION OF TH E TEST ITEM 12

3.4. OBSERVATIONS 13

3.5. NECROPSY 13

3.6. EVALUATION OF THE RE SULTS 13

3.7. DEVIATION FROM THE STUDY PLAN 14

3.8. ARCHIVES 14

3.9. THE PERMISSION OF THE IACUC 14

3.10. DISTRIBUTION OF THE FINAL REPORT 14

4. REFERENCES 15

5. RESULTS 15

6. CONCLUSION 15

APPENDICES 16

APPENDIX 1: CLINICAL OBSERVATIONS 17

APPENDIX 2: BODY WEIGHT DATA 18

APPENDIX 3: NECROPSY FINDINGS 19

APPENDIX 4: COPY OF THE PATHOLOGY REPORT 20

APPENDIX 5: COPY OF THE TEST REPORTS AND CONTENTS OF THE DIETS 21

APPENDIX 6: COPY OF THE TEST ITEM DATA SHEET 23

APPENDIX 7: COPY OF THE GLP CERTIFICATE 26

APPENDIX 8: COPY OF THE STUDY PLAN 1-17

### STUDY CODE: 10/339-001P Page 7 of 26



## LAB 1. SUMMARY

The s ingle

- dose oral toxicity of Polymer was performed according to the

acute toxic class method (OECD 423 and Directive 2004/73/EC B.1.tris) in RjHan:WI

rats.

Two groups of three female RjHan Wistar rats were treated with

Polymer at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. As no

mortality was observed, a confirmatory group (Group 2) was treated at the same dose

level. No mortality was o bserved in the confirmatory group, therefore no further

testing was required according to OECD 423 and Directive 2004/73/EC B.1. tris.

A single oral treatment was carried out by gavage for each animal after an overnight

food withdrawal. Food was made avai lable again 3 hours after the treatment.

Polymer was administered as a suspension prepared in Poly ethylene

glycol (PEG 400) at a concentration of 200 m g/mL with a dosing volume of 10

mL /kg bw.

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing

and daily for 14 days thereafter. Body weight was measured on Day s - 1, 0 and 7 and

before necropsy. All animals were subjected to a necropsy and a macroscopic

examination.

Results

Mortality

Polymer did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical observations

Treatment with Polymer cause d faeces liquid (6/6).

Body weight and body weight gain

Body weight gains of Polymer treated animals during the study

showed no indicatio n of a treatment

- related effect

.

### STUDY CODE: 10/339-001P Page 8 of 26



## LAB Macroscopic Findings

There was no evidence of the test item

- related macroscopic observations at a dose

level of 2000 mg/kg bw.

U terus in estrus seen in two female s (4076 and 4079) and dark/red discoloration of the

lungs (4074) were regarded as incidental.

Conclusion:

Under the conditions of this study, t he acute oral LD 50 value of the test item

Polymer was found to be above 2000 mg/kg bw in female

RjHan:WI rats.

Accord ing the GHS criteria, Polymer should be ranked as

"Category 5" or "Unclassified".

### STUDY CODE: 10/339-001P Page 9 of 26



## LAB 2. INTRODUCTION

The objective of the study was to assess the toxicity of test item

Polymer when administered as a single oral gavage dose to rats. The results of the

study allow the test item to be ranked according to most classification systems

currently used

.

2.1. STUDY SCHEDULE

Experimental Starting Date 01 February 2011

Experimental Completion Date 16 February 2011

Reception of An imals 13 January 2011

Treatment 01 February 2011 (female s no. 4072, 4074, 4075)

02 February 2011 (female s no. 4071, 4076, 4079)

Observation 01 – 15 February 2011

(female s no. 4072, 4074, 4075)

02 – 16 February 2011

(female s no. 4071, 4076, 4079)

Necropsy: 15 February 2011 (female s no. 4072, 4074, 4075)

16 February 2011 (female s no. 4071, 4076, 4079)

### STUDY CODE: 10/339-001P Page 10 of 26



## LAB 3. MATERIALS AND METHOD S

3.1. TEST ITEM

Name: Polymer

Batch/Lot Number:

Chemical Name:

Description: White waxy solid

Purity: 98.6 %

Manufacturer: See Sponsor

Manufacture date: 24 August 2010

Expiry Date: 23 August 2012

Storage conditions: Room temperature (15 -30 °C), humidity (40

- 70%) and

protect from light

Safety Precautions: Routine safety precautions (gloves, goggles, face mask

, lab coat ) for unknown materials were applied to assure

personnel health and safety.

Vehicle: Polyethylene glycol (PEG 400)

Batch number: 1435799

Expiry Date: February 2011

Produced by: Fluka

Dose volume: 10 mL/kg bw

3.1.1. Identification, Receipt

The test item of a suitable chemical purity together with all precautions required in the

handling and disposal of the test item were supplied by the Sponsor. The identification

of the test item was made in the Central Dispensary Unit of LAB Research Ltd. on the

basis of the information provided by Sponsor.

3.1.2. Formulation

Test item was freshly form ulated at a concentration of 200 mg/mL in the vehicle, in

the Central Dispensary Unit of LAB Research Ltd. on the day of administration. The

formulation container was stirred continuously during administration with the

objective of ensuring that the syring e was filled from a homogenous suspension.

### STUDY CODE: 10/339-001P Page 11 of 26



## LAB 3.1.3. Details of Other Materials:

Name: Euthasol ® 40 %

Lot No.: 10C25 7

Expiry Date: February 2013

Produced by: AST Beheer B.V. Oudewater Netherlands

(Produlab Pharma, Raamsdonksveer)

Storage: Room tempera ture

3.2. EXPERIMENTAL ANIMALS

Species and strain: RjHan:WI rats

Source: Laboratoire Elevage Janvier, B.P. 4105, Route des

Chênes Secs, 53940 Le Genest

- St

- Isle CEDEX

FRANCE

Hygienic level at arrival: SPF

Hygienic level during

the study: Standard housing conditions

Number of animals: 6 animals, 3 animals/group

Sex: Female, nulliparous and non-pregnant.

Age of animals at dosing: Young healthy adult rats, 10-11 weeks old

Date of receipt: 13 January 2011

Body weight at treatment: 233 – 256 g

Acclimatio n period: At least 19 days

3.2.1. Husbandry

Animal health: Only healthy animals were used for the test. The

veterinarian certified health status.

Number of animal room: 244/5

Housing: 3 animals / cage

Cage type: Type II polypropylene/polycarbonate

Beddi ng: Lignocel Bedding for Laboratory Animals was

available to animals during the study.

A copy of the Certificate of Analysis is retained in the

archive at LAB Research Ltd.

Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Temperature: 22 ± 3 °C

Relative humidity: 24 - 63 %

Ventilation: 15-20 air exchanges/hour

Enrichment: Animals were housed by group to allow social

interaction and with deep wood sawdust bedding to

allow digging and other normal rodent activities.

The temperature and relativ e humidity were recorded twice daily during the study.

### STUDY CODE: 10/339-001P Page 12 of 26



## LAB 3.2.2. Food and Water Supply

Animals received ssniff ® SM R/M

- Z+H "Autoclavable complete feed for rats and mice

– breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D

- 59494 Soest

Ger many ad libitum , and tap water from the municipal supply, as for human

consumption from 500 ml bottle ad libitum

. The food is considered not to contain any

contaminants that could reasonably be expected to affect the purpose or integrity of the

study.

For contents and test report of the diet see Appendix 5 . The supplier provided an

analytical certificate for the batch used.

Water quality control analysis is performed once every three months and

microbiological assessment is performed monthly, by Veszprém C ounty Institute of State Public Health and Medical Officer Service (ÁNTSZ, H

- 8201 Veszprém, József

A.u.36., Hungary). The quality control results are retained in the archives at LAB

Research Ltd.

3.2.3. Animal Identification

Animals were individually i dentified using numbers written on the tail with an indelible

marker pen . The numbers were given on the basis of LAB Research Ltd.' s Master File, for

each animal allocated to the treatment groups. The cages were identified by cards, with

information about study code, sex, dose group, cage number and individual animal

numbers.

3.3. ADMINISTRATION OF TH E TEST ITEM

3.3.1. Doses

Justification of the dose:

The initial dose level was selected by the study director to be that which is most likely

to produc e mortality in some of the dosed animals. In the lack of any preliminary

toxicological information, 2000 mg/kg bw was selected to be the starting dose.

Initially, t hree female animals were treated with 2000 mg/kg bw of

Polymer

. No mortality was observed , therefore further 3 animals were treated at the

dose level of 2000 mg/kg bw. As no mortality was observed in this second dose group,

f urther testing was not r e quired according to the test guidelines (OECD 423, Directive

2004/73/EC B.1. tris).

### STUDY CODE: 10/339-001P Page 13 of 26



## LAB 3.3.2. Procedure

A single oral gavage administration was followed by a fourteen

- day observation period. On

the day before treatment

, the animals were fasted. The food but not water was

withheld during an overnight period. Animals were weighed just be fore treatment. The

test item was administered by oral gavage in the morning. The food was returned 3

hours after the treatment.

3.4. OBSERVATIONS

3.4.1. Clinical Observations

Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6

hours after dosing and daily for 14 days thereafter. Individual observations were

performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory,

autonomic and central nervous system, somatomotor activity and behaviour pattern

. Particular a ttention was directed to observation of tremors, convulsions, salivation,

diarrhoea, lethargy, sleep and coma.

3.4.2. Body Weight Measurement

The body weight was recorded on the day before treatment (Day -1) , on the day of the

treatment (D ay 0) and weekly after.

3.5. NECROPSY

Macroscopic examination was performed on all animals

. The animals were sacrificed

by exsanguination under pentobarbital anaesthesia ( Euthasol ® 40 %; details are

presented in 3.1. 3 .) . After examination of the external appearance

, the cranial, thoracic

and the abdominal cavities were opened and the organs and the tissues were observed.

Macroscopic abnormalities were recorded

.

3.6. EVALUATION OF THE RE SULTS

The method used wa s not intended to allow the calculation of a precise LD 50 value.

The test item wa s ranked into categories of Globally Harmonized Classification

System (GHS) described in the OECD Guideline No. 423.

Clinical signs, body weight, body weight gain and gross macroscopic data were

tabulated.

### STUDY CODE: 10/339-001P Page 14 of 26



## LAB 3.7. DEVIATION FR OM THE STUDY PLAN

During the acclimatization period , the humidity value was in range 25 – 50%, from

24 to 31 January 2011.

During the observation period, the humidity value was in range 24 – 34%, from

15 to 16 February 2011.

Th ese are a deviation s from the range stated in the guidelines (30 – 70%).

Th is deviation is considered not to have had an impact on the objectives of the study

3.8. ARCHIVES

The study documents and samples:

- study plan,

- all raw data,

- s ample of test item,

- one original of Final Report,

- correspondence

are stored according to the Hungarian GLP and to the applicable SOP ’ s in the archives

of LAB Research Ltd. , 8200 Veszprém, Szabadságpuszta Hungary.

After the retention time agreed with the Sponsor has elapsed

, all the archived materials

listed above w ill be offered to the Sponsor or retained for a further period if agreed by

a contract. Otherwise the materials will be discarded.

3.9. THE PERMISSION OF TH E IACUC

The Institutional Animal Care and Use Committee (IACUC ) of LA B Research Ltd.

reviewed the study plan and authorised the conduct of the study.

3.10. DISTRIBUTION OF THE FINAL REPORT

Sponsor: A PDF document sent by email, and 1 bound certified copy,

1 unbound certified copy sent by courier.

Archive: 1 original

### STUDY CODE: 10/339-001P Page 15 of 26



## LAB 4. REFERENCES

OECD Guidelin es for Testing of Chemicals No. 423. Acute Oral Toxicity

– Acute Toxic

Class Method. Adopted: 17 December 2001

Hungarian GLP Regulations: 9/2001. (III.30.) EüM

- FVM joint decree of the Minister

of Health and the Minister of Agr iculture and Regional Development, which

corresponds to the OECD GLP, ENV/MC/CHEM (98) 17.

EEC Directive 2004/73 B.1.tris (O.J. L152)

5. RESULTS

Mortality

Polymer did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical obse rvations

Treatment with Polymer caused faeces liquid (6/6).

Body weight and body weight gain

Body weight gains of Polymer treated animals during the study

showed no indication of a treatment- related effect

.

Macroscopic Fi ndings

There was no evidence of the test item

- related macroscopic observations at a dose

level of 2000 mg/kg bw.

Uterus in estrus seen in two females (4076 and 4079) and dark/red discoloration of the

lungs (4074) were regarded as incidental.

6. C ONCLU SION

Under the conditions of this study, t he acute oral LD 50 value of the test item

Polymer was found to be above 2000 mg/kg bw in female

RjHan:WI rats

.

According the GHS criteria, Polymer should be ranked as

"Category 5 " or "Unclassified".

### STUDY CODE: 10/339-001P Page 16 of 26



## LAB 

## APPENDICES

### STUDY CODE: 10/339-001P Page 17 of 26



## LAB APPENDIX 1 :

CLINICAL OBSE R VATION S

DOSE LEVEL: 2000 mg/kg bw SEX: FEMALE

30' 1h 2h 3h 4h 6h

Symptom Free + + + + + - + 19/20

Faeces liquid - - - - - 2 - 1/20

Symptom Free + + + + + - + 19/20

Faeces liquid - - - - - 2 - 1/20

Symptom Free + + + + + - + 19/20

Faeces liquid - - - - - 2 - 1/20

Symptom Free + + + + - - + 18/20

Faeces liquid - - - - 2 2 - 2/20

Symptom Free + + + + - - + 18/20

Faeces liquid - - - - 2 2 - 2/20

Symptom Free + + + + - - + 18/20

Faeces liquid - - - - 2 2 - 2/20

+: present

h=hour (s) 4075

4071

4076

4079

Treatment day= Day 0

Frequency of observation = number of occurence of observation / total number of

observations Frequency 0 1-14

Remarks: -: absent Observations Observation days

Cage No. Animal

Number

4072

4074 1

2

### STUDY CODE: 10/339-001P Page 18 of 26



## LAB APPENDIX 2 :

BODY WEIGHT DATA

DOSE LEVEL: 2000 mg/kg bw SEX: FEMALE

-1 0 7 14 -1-0 0-7 7- 14 -1 - 14

4072 276 256 302 317 -20 46 15 41

4074 253 235 271 275 -18 36 4 22

4075 256 240 276 281 -16 36 5 25

4071 247 234 263 280 -13 29 17 33

4076 255 240 277 284 -15 37 7 29

4079 240 233 260 279 -7 27 19 39

254.5 239.7 274.8 286.0 -14.8 35.2 11.2 31.5

12.1 8.5 15.0 15.5 4.5 6.7 6.6 7.6

Remark: 1

2

Treatment day= Day 0 Mean:

Standard deviation: Body Weight Gain (g) Days Cage

No. Animal No. Body weight (g)

### STUDY CODE: 10/339-001P Page 19 of 26



## LAB APPENDIX 3:

NECROPSY FINDINGS

DOSE LEVEL: 2000 mg/kg bw SEX: FEMALE

Cage

No. Animal

ID Necropsy Date External Observations Internal Observations Organ/Tissue

4072 15 February 2011 No external observations recorded No internal observations recorded Not applicable

4074 15 February 2011 No external observations recorded Dark discoloration, red, diffuse, all lobes Lungs

4075 15 February 2011 No external observations recorded No internal observations recorded Not applicable

4071 16 February 2011 No external observations recorded No internal observations recorded Not applicable

4076 16 February 2011 No external observations recorded In estrus Uterus

4079 16 February 2011 No external observations recorded In estrus Uterus 1

2

### STUDY CODE: 10/339-001P Page 20 of 26



## LAB APPENDIX 4:

COPY OF THE PATHOLOGY REPORT

### STUDY CODE: 10/339-001P Page 21 of 26



## LAB APPENDIX 5:

COPY OF THE TEST REP ORT S AND CONTENTS OF THE DIET S

### STUDY CODE: 10/339-001P Page 22 of 26



## LAB APPENDIX 5:

CONTENTS OF THE DIET S

SSNIFF ® SM R/M

- Z+H COMPLETE DIET FOR RATS AND MICE

Crude protein 19.00 %

Crude fat 3.50 %

Crude fibre 3.60 %

Crude Ash 6.50 %

Lysine 1.10 %

Methionine 0.56 %

Calcium 1.00 %

Sodium 0.20 %

Magnesium 0.22 %

Phosphorus 0.70 %

Vitamin A 25000 IU

Vitamin D 3 1000 IU

Vitamin E 125 mg/kg

These data are standard and guaranteed values provided by the supplier.

### STUDY CODE: 10/339-001P Page 23 of 26



## LAB APPENDIX 6:

COPY OF THE TEST ITEM DATA SHEET

### STUDY CODE: 10/339-001P Page 24 of 26



## LAB APPENDIX 6:

COPY OF THE TEST ITEM DATA SHEET

(CONTINUED)

### STUDY CODE: 10/339-001P Page 25 of 26



## LAB APPENDIX 6:

COPY OF THE TEST ITEM DATA SHEET

(CONTINUED)

### STUDY CODE: 10/339-001P Page 26 of 26



## LAB A PPENDIX 7:

COPY OF THE GLP CERTIFICATE

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301 APPENDIX 8:

COPY O F THE STUDY PL AN



### STUDY PLAN

ACUTE ORAL TOXICITY STUDY OF

TEST ITEM Polymer

IN RATS



### Date of Study Plan: 2 7 January 201 1 STUDY CODE: 10/339

- 001P

### STUDY CODE: 10/339-001P Page 2 of 17



## L A B CONTENTS

Page

GENERAL INFORMATION ................................................................................................... 3

1. OBJECTIVE OF STUDY

................................ ................................ ................................ 5

2. MATERIALS AND METHOD S

................................ ................................ ..................... 5

2.1. TEST ITEM AND VEHICL E

................................ ................................ ............................. 5

2.2. EXPERIMENTAL ANIMALS

................................ ................................ ........................... 6

2.3. ADMINISTRATION OF TH E TEST ITEM

................................ ................................ ...... 8

2.4. OBSERVATIONS

................................ ................................ ................................ .............. 9

2.5. NECROPSY

................................ ................................ ................................ ........................ 9

2.6. EVALUATION

................................ ................................ ................................ ................. 10

3. GOOD LABORATORY PRAC TICE (GLP) AND QUALI TY ASSURANCE

........ 10

4. AMENDMENT AND DEVIAT ION PROCEDURES

................................ ................. 10

5. STUDY REPORT

................................ ................................ ................................ ........... 11

6. ARCHIVES

................................ ................................ ................................ ..................... 12

7. DISTRIBUTION OF THE STUDY PLAN

................................ ................................ .. 12

8. SIGNATURES

................................ ................................ ................................ ................ 12

9. ANIMAL WELFARE

................................ ................................ ................................ ..... 12

APPENDICES

................................ ................................ ................................ .......................... 13

APPENDIX 1: CONTENTS OF THE DIET

................................ ................................ ........... 14

APPENDIX 2: PROPOSED STUDY SCHEDULE

................................ ................................ . 15

APPENDIX 3: TEST PROCEDURE DIAGRAMS (FROM OECD 423)

............................... 16

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## L A B GENERAL INFORMATION

SPONSOR:

### STUDY CODE: 10/339-001P Page 4 of 17



## L A B RESPONSIBLE PERSONS * : Judit Tavaszi , M.Sc.

- assistant scientist

István Pásztor DVM

- veterinary control

Peter Maslej , DVM, PhD

– Head of Pathology Unit

Animal House Staff

Central Dispensary Staff

Pathology Staff

* Other trained, competent personnel may work on the study as required; to be

recorded and reported.

START OF EXPERIMENT: 01 February 201 1

END OF EXPE RIMENT: Not later than 01 March 201 1

DATE OF DRAFT REPORT: Not later than 29 March 201 1

BASIS OF STUDY: OECD GUIDELINES FOR TESTING OF

CHEMICALS (423, adopted at 17 th Dec. 2001)

EEC Directive 2004/73 B.1.tris (O.J. L152)

### STUDY CODE: 10/339-001P Page 5 of 17



## L A B 1. OBJECTIVE OF STUDY

The objective of the study is to assess the acute oral toxicity of the test item when

administered in a single dose to rats at one or more defined dose levels. The results of

the study allow the test item to be ranked according to most classification systems

currently in use.

2. MATERIALS AND METHODS

2.1. TEST ITEM AND VEHICLE

Name: Polymer

Batch/Lot Number:

Chemical Name:

De scription: White waxy solid

Purity: 98.6 %

Manufacturer: See Sponsor

Manufacture date: 2 4 August 2010

Expiry Date: 2 3 August 201 2

Storage conditions: Room temperature (15

- 30 °C) , humidity (40

- 70%) and

protect from light

Safet y Precautions : Routine safety precautions (gloves, goggles, face mask,

lab coat) for unknown materials will be applied to

assure personnel health and safety.

Vehicle: Poly ethylene glycol (PEG 400) *

Dose volume: 10 mL/kg bw

*Details of the vehicle will be given in the Report.

2.1.1. Identification, Receipt

The test item of a suitable chemical purity was provided by the Sponsor. All

precautions required in the handling and disposal of the test item were outlined by the

Sponsor. Identification of the test item was performed on the basis of information

provided by the Sponsor in the Central Dispensary of LAB Research Ltd.

### STUDY CODE: 10/339-001P Page 6 of 17



## L A B 2.1.2. Formulation

Test item will be freshly formulated at appropriate concentrations in the vehicle in the

Central Dispensary Unit of LAB Research Ltd. on the day of administration. The

formulation will be stirred with a magnetic stirrer up to finishing the treatment.

2.1.3. Other Materials

For euthanasia:

Name: Euthasol ® 40 %

Lot No.: 10C25 7 *

Expiry Date: February 201 3

Produced by: AST Beh eer B.V. Oudewater Netherlands

* should any other Lot be employed, it will not be considered as a deviation but

stated in the report

2.2. EXPERIMENTAL ANIMALS

Species and strain: RjHan:WI rats

Source: Laboratoire Elevage Janvier, B.P. 4105, Route de s

Chênes Secs, 53940 Le Genest

- St

- Isle CEDEX

FRANCE

Hygienic level at supplier: SPF

Hygienic level during

the study: Standard housing conditions

Justification of strain: The Wistar rat as a rodent is one of the standard species

of acute toxicity studies

N umber of animals: 3 animals/group

Sex: Female, nulliparous and non pregnant animals

Age of animals at dosing: Young adult rats, 8

- 12 weeks old

Planned body weight range

at dosing: 150

- 300 g

Acclimatisation period: At least 5 days

### STUDY CODE: 10/339-001P Page 7 of 17



## L A B 2.2.1. Husbandry

An imal health: Only healthy animals will be used for the test. The

health status will be certified by the veterinarian.

Housing: Group caging (3 animals/cage)

Cage type: Type II. polypropylene/polycarbonate

Bedding: Certified laboratory wood bedding

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Temperature: 22 ± 3 °C

Relative humidity: 30 - 70 %

Ventilation: 15 -20 air exchanges/hour

Enrichment: Rodents are housed with more than one animal per

cage to allow social interaction and with deep wood

sawdust bedding to allow digging and other normal

rodent activities.

The temperature and relative humidity will be recorded twice daily during the study.

2.2.2. Food and Water Supply

Animals will receive ssniff ® SM R/M-Z+H "Autoclavable complete feed for rats and

mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494

Soest Germany ad libitum , and tap water from the municipal supply, as for human

consumption from 500 ml bottle ad libitum . The food is considered not to contain any

contam inants that could reasonably be expected to affect the purpose or integrity of

the study.

For contents of the standard diet see Appendix 1. The supplier will provide an

analytical certificate for the batch used.

Water quality control analysis is performed once every three months and

microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József

A.u.36., Hungary). The quality control results are retained in the archives of LAB

Research Ltd.

### STUDY CODE: 10/339-001P Page 8 of 17



## L A B 2.2.3. Bedding

Lignocel Hygienic Animal Bedding will be available to animals during the study.

2.2.4. Identification

Animals will be individually identified by numbers written on the tail with an

indelible pen. The numbers were given on the basis of LAB Research Ltd.'s master

file, for each animal allocated to the study.

The boxes will be identified by cards holding information on the study code, the sex

of animals, the dose group, the cage number and the individual animal number.

2.3. ADMINISTRATION OF THE TEST ITEM

2.3.1. Dosages

Justification of the doses

A limit of 2000 mg/kg bw dose is normally used, unless the Sponsor has justified in

wr iting a specific regulatory need for an additional upper dose level of 5000 mg/kg

body weight as required by guideline OECD 423. The limit dose for this study is

2000 mg/kg bw.

The starting dose level (2000 mg/kg bw ) was selected by the Study Director in

absence of any toxicity data or from any other available information and was selected

from four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. The flow charts in

Appendix 3 describe the procedure that will be followed for each of the starting doses.

Three female animals will be used for each step.

In summary, if two or more animals die at a dose when the lower dose level has not

yet been tested, then the test will be continued at the next lower dose level on a further

3 rats. If zero or one animals die at a dose when the higher dose level has not yet been

tested, then the test will be continued at the next higher dose level on a further 3 rats.

If the lower or higher dose level mentioned above have already been tested, or if there

is no valid lower or higher dose to be tested, then the test will be continued at the

same dose level on a further 3 rats.

The test will be complete when the criteria for termination given in Annex 2d of OECD

Guideline No. 423. (Presented in Appendix 3) is met.

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## L A B 2.3.2. Procedure

The day before treatment the animals will be fasted. Food, but not water, will be

withheld overnight. Animals will be weighed before dosing. A single oral gavage

administration will be administered using a constant dose volume of 10 mL/kg bw. Food

will be replaced 3 hours after the treatment.

The observation period will be fourteen-days.

2.4. OBSERVATIONS

2.4.1. Clinical Observations

Animals will be observed individually after dosing at least once during the first

30 minutes, then 1, 2, 3, 4 and 6 hours after the treatment and once each day for

14 consecutive days thereafter. Individual observations will be performed on the skin

and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and

central nervous system, somatomotor activity and behaviour pattern. Particular

attention will be directed to observation of tremors, convulsions, salivation, diarrhoea,

lethargy, sleep and coma. The time of death will be recorded as precisely as possible.

2.4.2. Measurement of Body Weight

Body weight will be recorded on Day -1 (day prior to treatment), Day 0 (treatment

day) and on Days 7, 14.

2.5. NECROPSY

All animals (including those that die during the test or are removed from the study for

animal welfare reasons) will be subjected to a necropsy and a macroscopic examination.

All surviving animals will be exsanguinated after verification of narcosis following an

injection of Euthasol ® 40 %. After examination of the external appearance, the cranial,

thoracic and abdominal cavities will be opened and the appearance of the tissues and

organs will be observed. All gross macroscopic changes will be recorded for each

animal.

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## L A B 2.6. EVALUATION

The method used is not intended to allow the calculation of a precise LD 50 value.

The test item will be ranked into classes of Globally Harmonized Classification

System (GHS) described in the OECD Guideline No. 423.

The frequency of the clinical symptoms will be summarised in tabular form. The mean

of the body weight and body weight gain will be calculated by Excel spreadsheet

software. Necropsy findings will be described and summarised in tabular form.

3. GOOD LABORATORY PRACTICE (GLP) AND QUALITY ASSURANCE

In order to enable the results of this study to meet legal requirements including

regulatory submission and/or safety assessment, it will be performed in compliance with

t he principles of Good Laboratory Practice Regulations as requested by the Sponsor and

specified in national Hungarian GLP Regulations: 9/2001. (III.30.) EüM-FVM joint

decree of the Minister of Health and the Minister of Agriculture and Regional

Development that corresponds to the OECD GLP, ENV/MC/CHEM (98) 17.

The Quality Assurance Unit will conduct inspections of the study plan, various phases

of the study, certain repetitive operations and the report will be audited according to

applicable Standard Operating Procedures.

4. AMENDMENT AND DEVIATION PROCEDURES

Planned changes to the study plan can be made at the discretion of the Study Director

and will be agreed with the Sponsor. Detailed descriptions of all study plan

amendments will be effective at the time of Study Director's signature. All such

amendments will be sent to the Sponsor. The study plan amendment will be

distributed and added to all copies of the study plan.

Unplanned changes to the study plan will be documented in the raw data and

communicated to the Sponsor. The final report will reflect every study plan deviation,

the reason for the study plan deviation and its anticipated effect on the outcome of the

study.

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## L A B 5. STUDY REPORT

The results of the study will be reported in a detailed Final Report in line with

EPA 86-5 format requirements.

The original English final report will be sent to the Sponsor and will include but will

not be lim ited to:

- Name and address of the Sponsor, the test facility and the study schedule.

- The names of the Study Director and other scientists and supervisory

personnel involved in the study.

- The signatures of the personnel of test facility, who signed the Study

Protocol.

- The signature of the Sponsor (if requested by the Sponsor).

- The statement of compliance, signed by the Study Director.

- The signature and statement of the management.

- The quality assurance statement, signed by the QAU.

- Characterisation of test item: The identification of the test item, either by

name or code number. The concentration, purity, stability, composition and

other appropriate characteristics of the test item, if the Sponsor provides data.

- A copy of the analytical certificate of the test item.

- A description of the animals: strain, health status, sex, source, identification,

weight at commencement of the study, group size and animal husbandry.

- Details of food and water quality (including diet type/source, water source).

- Rationale for initial dose level selection.

- Details of the administration of the test item.

- Tabulation of individual and test group data, response data for each animal

and dose level (i.e., number of animals exposed, number of animals showing

signs of toxicity including nature, severity, duration of effects, and mortality,

number of animals that were sacrificed in extremis). Time course of onset of

signs of toxicity and whether these were reversible for each animal.

- Tabulation of body weight/body weight changes.

- Individual weights of animals at the day of dosing, in weekly intervals

thereafter, and at the time of death or sacrifice.

- Necropsy findings for each animal, if available.

- Evaluation of results (description of computer routine used and spreadsheet

tabulation of calculations).

- Discussion and interpretation of results.

- Conclusions.

- A copy of the GLP-Certificate of the test facility.

- Details of archiving - the storage location, list of archived data and samples.

The draft report will be sent to the Sponsor by e-mail for comment. Following receipt

of comments, the final report will be issued as a PDF document sent to the Sponsor by

e-mail, with one bound and one unbound hard copy sent by courier.

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## L A B 6. ARCHIVES

The study documents:

- study plan and any amendments,

- all raw data,

- sample of the test item,

- study report and any amendments,

- correspondence

will be archived according to the Hungarian GLP and to applicable SOP’s in the

archives of LAB Research Ltd. 8200 Veszprém, Szabadságpuszta.

After the retention time agreed with the Sponsor has elapsed, all the archived

materials listed above will be offered to the Sponsor or retained for a further period if

agreed by a contract. Otherwise the materials will be discarded.

7. DISTRIBUTION OF THE STUDY PLAN

Sponsor: 1 x PDF-file, 1x copy

Study Director: 1 copy

QAU : 1 copy

Archive: 1 original

Departments 1 : 1 copy, each

8. SIGNATURES

The Study Director, LAB Research Ltd.'s management, QAU and IACUC will sign

the Study Plan before the start of the study. The PDF version of the Study Plan will be

sent to the Sponsor after the content of the Study Plan has been agreed. If the Sponsor

does not sign the Study Plan within 5 working days (or as specified in the contract)

then the study may be started with the signature of the Sponsor being made after study

initiation.

9. ANIMAL WELFARE

Procedures involving the care and use of animals in this study will be reviewed and

approved by the Institutional Animal Care and Use Committee (IACUC). During the

study, the care and use of animals will be conducted in accordance with the relevant

principles currently used with regard to animal welfare.

1 Central Dispensary, Scientific Documentation and Statistics, Pathology

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## L A B 

## A P P E N D I C E S

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## L A B APPENDIX 1:

CONTENTS OF THE DIET

SSNIFF ® SM R/M-Z+H, AUTOCLAVABLE

Complete feed for rats and mice – breeding and maintenance

Crude protein 19.00 %

Crude fat 3.50 %

Crude fibre 4.00 %

Crude Ash 6.60 %

Lysine 1.10 %

Methionine 0.60 %

Calcium 1.00 %

Sodium 0.20 %

Magnesium 0.20 %

Phosphorus 0.80 %

Vitamin A 25000 IU

Vitamin D 3 1000 IU

Vitamin E 120 mg/kg

These data are standard and guaranteed values provided by the supplier.

These values can slightly differ in case of changing the Batch of the feed used under

the duration of the study. Should any other Batch will be used, the details will be

documented in draft report, but it will not be considered as a deviation to the Study

Plan.

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## L A B APPENDIX 2:

PROPOSED STUDY SCHEDULE

Relative Date Absolute Date

Animal receipt: Day [-1 9 ] 13 January 201 1

Veterinary control: According to applicable SOPs.

Animal identification: Day [-1] 31 January 201 1

TREATMENT PERIOD

The day of treatment

with the initial dose: Day 0 0 1 February 201 1

Body weight measurement

of initial dose: Day -1, 0, 7, 14 31 January 2011 and

01, 08, 15 February 2011

Clinical observation: At least once during the first 30 minutes, then 1, 2, 3, 4 and

6 hours after the treatment and once a day for 14 consecutive days

thereafter.

Necropsy of initial dose: Just after death (if possible) or on

Day 14 (surviving animals). 15 February 201 1

Draft Report: Not later than

29 March 2011

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## L A B APPENDIX 3:

TEST PROCEDURE DIAGRAMS (FROM OECD 423)

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## L A B