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 



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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 
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CONTENTS

1. SUMMARY 7

2. INTRODUCTION 9

2.1. STUDY SCHEDULE 9

3. MATERIALS AND METHODS 10

3.1. TEST ITEM 10

3.2. EXPERIMENTAL ANIMALS 11

3.3. ADMINISTRATION OF THE TEST ITEM 12

3.4. OBSERVATIONS 13

3.5. NECROPSY 13

3.6. EVALUATION OF THE RESULTS 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 
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1. SUMMARY

The single-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 observed 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 available again 3 hours after the treatment. 

 Polymer was administered as a suspension prepared in Polyethylene 

glycol (PEG 400) at a concentration of 200 mg/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 Days -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 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. 
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Macroscopic Findings

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. 

Conclusion:

Under the conditions of this study, the acute oral LD50 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".
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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 Animals 13 January 2011 

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

02 February 2011 (females no. 4071, 4076, 4079) 

 

Observation 01 – 15 February 2011 

(females no. 4072, 4074, 4075) 

02 – 16 February 2011 

(females no. 4071, 4076, 4079) 

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

16 February 2011 (females no. 4071, 4076, 4079) 
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3. MATERIALS AND METHODS 

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 formulated 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 syringe was filled from a homogenous suspension. 
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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 temperature

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 

Acclimation 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 

Bedding: 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 relative humidity were recorded twice daily during the study. 
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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 

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 

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 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 at LAB 

Research Ltd.

3.2.3. Animal Identification

Animals were individually identified 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 THE 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 produce 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, three 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, 

further testing was not required according to the test guidelines (OECD 423, Directive 

2004/73/EC B.1. tris). 
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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 before 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 attention 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 (Day 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 RESULTS

The method used was not intended to allow the calculation of a precise LD50 value.

The test item was 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.
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3.7. DEVIATION FROM 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. 

These are a deviations from the range stated in the guidelines (30 – 70%).

This 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,

- sample 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 will 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 THE IACUC 

The Institutional Animal Care and Use Committee (IACUC) of LAB 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 
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4. REFERENCES

OECD Guidelines 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 Agriculture 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 observations 

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 Findings

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. CONCLUSION

Under the conditions of this study, the acute oral LD50 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".
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APPENDICES
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APPENDIX 1: 

CLINICAL OBSERVATIONS 

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

0 Frequency 1-14

Remarks: -: absent

Observations

Observation days

Cage No. Animal 

Number

4072

1 4074

2
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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)
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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
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APPENDIX 4: 

COPY OF THE PATHOLOGY REPORT
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APPENDIX 5: 

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

CONTENTS OF THE DIETS

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 D3 1000 IU

Vitamin E 125 mg/kg

These data are standard and guaranteed values provided by the supplier. 
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APPENDIX 6: 

COPY OF THE TEST ITEM DATA SHEET
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APPENDIX 6: 

COPY OF THE TEST ITEM DATA SHEET

(CONTINUED)
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APPENDIX 6: 

COPY OF THE TEST ITEM DATA SHEET

(CONTINUED)
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APPENDIX 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 OF THE STUDY PLAN

STUDY PLAN 

ACUTE ORAL TOXICITY STUDY OF 

TEST ITEM Polymer

IN RATS

Date of Study Plan: 27 January 2011 STUDY CODE: 10/339-001P
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CONTENTS 

Page 

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

1. OBJECTIVE OF STUDY ................................................................................................ 5

2. MATERIALS AND METHODS..................................................................................... 5

2.1. TEST ITEM AND VEHICLE............................................................................................. 5

2.2. EXPERIMENTAL ANIMALS........................................................................................... 6

2.3. ADMINISTRATION OF THE TEST ITEM...................................................................... 8

2.4. OBSERVATIONS .............................................................................................................. 9

2.5. NECROPSY........................................................................................................................ 9

2.6. EVALUATION................................................................................................................. 10

3. GOOD LABORATORY PRACTICE (GLP) AND QUALITY ASSURANCE........ 10

4. AMENDMENT AND DEVIATION 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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 GENERAL INFORMATION 

SPONSOR: 
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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 2011

END OF EXPERIMENT: Not later than 01 March 2011

DATE OF DRAFT REPORT: Not later than 29 March 2011

BASIS OF STUDY: OECD GUIDELINES FOR TESTING OF 

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

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

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 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.
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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 2013

Produced by: AST Beheer 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 des 

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

Number 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
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2.2.1. Husbandry 

Animal 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 

contaminants 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. 
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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 

writing 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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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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2.6. EVALUATION 

The method used is not intended to allow the calculation of a precise LD50 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 

the 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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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 limited 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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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 

Departments1: 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. 

 

1Central Dispensary, Scientific Documentation and Statistics, Pathology 
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A P P E N D I C E S 
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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 D3 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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APPENDIX 2: 

PROPOSED STUDY SCHEDULE 

Relative Date Absolute Date 

Animal receipt: Day [-19] 13 January 2011 

Veterinary control: According to applicable SOPs. 

Animal identification: Day [-1] 31 January 2011 

TREATMENT PERIOD

The day of treatment 

with the initial dose: Day 0 01 February 2011 

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 2011 

Draft Report: Not later than 

 29 March 2011 
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APPENDIX 3: 

TEST PROCEDURE DIAGRAMS (FROM OECD 423) 
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