TS-CKHI01

Attachment #10

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

IN RATS

Date of Final Report: 07 Ju ly 2011 STUDY CODE: 11/076-001P

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## LAB GENERAL INFORMATION

STUDY TITLE: Acute Oral Toxicity Study of Tes t Item

in Rats

SPONSOR:

STUDY PERFORMED BY: LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

Ph one: +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 – Head of Central Dispensary

Animal House Staff

Central Dispensary Staff

Pathology Staff

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## 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 ANIMALS 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 14

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

APPEN DIX 5: COPY OF THE TEST REPORT AND CONT ENTS OF THE DIET 21

APPENDIX 6: COPY OF THE TEST IT EM DATA SHEET 23

APPENDIX 7: COPY OF THE GLP CERTIFICATE 24

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## LAB 1. SUMMARY

The single

- dose oral toxicity of was performed according to the acute toxic class

method (OECD 423 and C ommission Re gulation (EC) NO 440/2008 of 30 M ay 2008,

B.1.T ris) in RjHan:WI rats.

Two groups of three female RjHan Wistar rats were treated with 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 a ccording to OECD 423 and C ommission R egu lation (EC) NO

440/2008 of 30 M ay 2008, B.1.T ris

.

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.

was administered as a suspension prepar ed in corn oil at a concentration of 200

mg/mL at 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

be fore necropsy. All animals were subjected to a necropsy and a macroscopic

examination.

Results

Mortality

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

Clinical observations

Treatment with did not cause any test item related adver se effect during the 14

days observation period.

Body weight and body weight gain

Body weight gains of treated animals during the study showed no indication of

a treatment

- related effect

.

Macroscopic Findings

There was no evidence of the test ite m- related macroscopic observations at a dose

level of 2000 mg/kg bw.

U terus in estrus was seen in 1/6 female, but was considered incidental and unrelated to

treatment.

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## LAB Conclusion:

T reatment with did not cause any test item related adverse effect at a

dose level of 2000 mg/kg bw

.

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

was found to be above 2000 mg/kg bw in female RjHan:WI rats.

According the GHS criteria, should be ranked as "Category 5" or

"Un classified".

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## LAB 2. INTRODUCTION

The objective of the study was to assess the toxicity of test item 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 26 May 2011

Experimental Completion Date 10 June 2011

Reception of Animals 05 May 2011

Treatment 26 May 2011 (female s no. 6408, 6409, 6413)

27 May 2011 (female s no. 6406, 6411, 6414)

Observation 26 May 2011 to 09 June 2011

(female s no. 6408, 6409, 6413)

27 May 2011 to 10 June 2011

(females no. 6406, 6411, 6414)

Necropsy: 09 June 2011 (females no. 6408, 6409, 6413)

10 June 2011 (females no. 6406, 6411, 6414)

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## LAB 3. MATERIALS AND METHOD S

3.1. TEST ITEM

Name:

Synonym: Polymer

Chemical Name:

CAS Number:

Lot Number: TQA1067

Molecular weight: >2000 Da

Description: Colourless liquid*

Purity: 97.4 %

Manufacture Date: 10 May 2010

Expiry Date: 24 October 2012

Storage conditions: Room temperature (15-30 o C)

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

, lab coat ) for unknown materials were applied to assure

personnel health and safety.

*On the Test Item Data Sheet (dated 16 M ay 2011) colourless, viscous liquid was s t ated. However,

according to the Sponsor

, the observed appearance is acceptable.

Vehicle: Corn oil

Batch number: MKBF6012V

Expiry Date: 11 May 2012

Produced by: SIGMA

- ALDRICH

Dose volume: 10 mL/kg bw

3.1.1. Identificati on, 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 f illed from a homogeneous opalescent

suspension.

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## LAB 3.1.3. Details of Other Materials:

Name: Euthasol ® 40 %

Lot No.: 10C25 7

Expiry D ate: 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

C hênes 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, 11 weeks old

Date of receipt: 05 May 2011

Body weight at treatment: 230 – 246 g

Acclima tion period: At least 21 days

3.2.1. Husbandry

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

veterinarian certified hea lth status.

Number of animal room: 522/9 and 244/1

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: 30 - 70 %

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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## 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

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 quali ty 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 L td.' 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 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, t hree female animals were treated with 2000 mg/kg bw of 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 gro up, f urther

testing was not r e quired according to the test guidelines (OECD 423, C ommission

R egulation (EC) NO 440/2008 of 30 M ay 2008, B.1.T ris ).

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## 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 before treatment. The

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

3 hours a fter 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 sacrifice d

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) desc ribed in the OECD Guideline No. 423.

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

tabulated.

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## LAB 3.7. DEVIATION FROM THE STUDY PLAN

There was no deviation during 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.

Aft er 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 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

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 Agriculture and Regional Development, which

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

C ommission R egulation (EC) NO 440/2008 of 30 M ay 2008, B.1.T ris

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## LAB 5. RESULTS

Mortality

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

Clinical observations

Treatment with did not cause any test item related adverse effect during the 14

days observation period.

Body weight and body weight gain

Body weight gains of 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.

U terus in estrus was seen in 1/6 female, but was considered incidental and unrelated to

treatment.

6. C ONCLUSION

Treatment with did not cause any test item related adverse effect at a

dose level of 2000 mg/kg bw.

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

was found to be above 2000 mg/kg bw in female RjHan:WI rats

.

According the GHS criteria, should be ranked as "Category 5" or

"Unclassified".

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## LAB 

## APPENDICES

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## LAB APPENDIX 1 :

CLINICAL OBSE R VATION S

DOSE LEVEL: 2000 mg/kg bw SEX: FEMALE

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

6408 Symptom Free + + + + + + + + + + 20/20

6409 Symptom Free + + + + + + + + + + 20/20

6413 Symptom Free + + + + + + + + + + 20/20

6406 Symptom Free + + + + + + + + + + 20/20

6411 Symptom Free + + + + + + + + + + 20/20

6414 Symptom Free + + + + + + + + + + 20/20

+: present

h=hour (s) 4-14 2

Treatment day= Day 0

Frequency of observation = number of occurence of observation / total number of observations Frequency 0

'= minute (s) 1 3

Remarks: Observations Observation days

Cage No. Animal

Number

1

2

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## LAB APPENDIX 2 :

BODY WEIGHT DAT A

DOSE LEVEL: 2000 mg/kg bw SEX: FEMALE

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

6408 248 233 249 261 -15 16 12 13

6409 259 244 282 297 -15 38 15 38

6413 246 232 263 269 -14 31 6 23

6406 248 246 258 258 -2 12 0 10

6411 239 230 257 262 -9 27 5 23

6414 250 242 269 271 -8 27 2 21

248.3 237.8 263.0 269.7 -10.5 25.2 6.7 21.3

6.5 6.9 11.4 14.3 5.2 9.6 5.8 9.8

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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## 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

6408 09 June 2011 No external observations recorded In estrus Uterus

6409 09 June 2011 No external observations recorded No internal observations recorded Not applicable

6413 09 June 2011 No external observations recorded No internal observations recorded Not applicable

6406 10 June 2011 No external observations recorded No internal observations recorded Not applicable

6411 10 June 2011 No external observations recorded No internal observations recorded Not applicable

6414 10 June 2011 No external observations recorded No internal observations recorded Not applicable 2 1

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## LAB APPENDIX 4:

COPY OF THE PATHOLOGY REPORT

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## LAB APPENDIX 5:

COPY OF THE TEST REP ORT AND CONTENTS OF THE DIET

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## LAB APPENDIX 5:

CONTENTS OF THE DIET

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

Thes e data are standard and guaranteed values provided by the supplier.

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## LAB APPENDIX 6:

COPY OF THE CERTIFICATE OF ANALY SIS

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## LAB A PPENDIX 7:

COPY OF THE GLP CERTIFICATE