TS-CKHI01

Attachment #12

Health Effects Data on the New Chemical Substance

Eye Irritation Test in Rabbits

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301 14

FINAL REPORT

Acute Eye Irritation Study in Rabbits

Date of Final Report: 24 June 2011 

### STUDY CODE: 11/076-005N

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## LAB STUDY TITLE : Acute Eye Irritation Study in Rabbits

S PONSOR

STUDY PERFORMED BY : LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta
Hungary

Phone: +36 88 545-300

Fax: +36 88 545-301

STUDY DIRECTO R : Magdolna Török- Bathó , M.Sc.

ASSISTANT SCIENTIST : Judit Tavaszi, M.Sc.

VETERINARY CONTROL : István Pásztor , DVM

CENTRAL DISPENSARY : Tamás Mészáros, PhD.

### STUDY CODE: 11/076-005N Page 6 of 22



## LAB CONTENTS

Page

1. SUMMARY 7

2. INTRODUCTION 8

3. MATERIALS AND METHOD S 8

3.1. TEST ITEM 8

3.2. OTHER MATERIALS 9

3.3. EXPERIMENTAL AN IMALS 9

3.4. HUSBANDRY 10

3.5. FOOD AND FEEDING 10

3.6. WATER SUPPLY AND QUALITY CONTROL OF WATE R 11

3.7. STUDY SCHEDULE 11

3.8. ADMINISTRATION OF TH E TEST ITEM 12

3.8.1. Dosage 12

3.8.2. Application of the Test Item 12

3.9. OBSERVATIONS AND SCO RING 12

3.9.1. Clinical Observations 12

3.9.2. Scoring and Assessment of Local Reaction 12

3.9.3. Classification of the Test Items 13

3.9.4. Measurement of Body We ight 14

4. ARCHIVES 14

5. THE PERMISSION OF TH E INSTITUTIONAL IACU C 14

6. DEVIATION TO THE STU DY PLAN 14

7. RESULTS 15

7.1. MORTALITY 15

7.2. BODY WEIGHTS 15

7.3. CLINICAL OBSERVATION 15

7.3.1. General daily examination 15

7.3.2. Examination of eye

- irritancy 15

8. CONCLUSION 16

INDIVIDUAL SCORES FOR OCULAR IRRITATION 17

MEAN VALUES OF EYE IRRITATION 18

BODY WEIGHT DATA 18

APPENDICES 19

APPENDIX 1: Scoring and Assessment o f Local Reaction 1 – 2 of Appendix 1

APPENDIX 2: Scoring o f Pain Reaction 1 of Appendix 2

APPENDIX 3: Contents o f Purina Base

- Lap Gr. Diet for Rabbits 1 of Appendix 3

APPENDIX 4: Copy o f t he Certificate of Analysis 1 of Appendix 4

APPENDIX 5: Copy o f t he GLP Certificate 1 of Appendix 5

APPENDIX 6 : Copy of the Study Plan 1 – 18 of Appendix 6

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## LAB 1. SUMMARY

An acute eye irritation study of the test item was performed in New Zealand

White rabbits. The irritation effects of the test item were evaluated according to the
Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal . The

untreated right eye served as control. A volume of 0.1 ml of the test item was administered as a single dose.

The eyes were examined at 1, 24, 48 and 72 hours after the application.

Initial Pain R eaction (IPR) was not observed in animal s.

One hour af ter the application , conjunctival redness (score 1) was observed in all

animals and discharge (score 3 ) was observed in all rabbits.

At 24, 48 and 72 hours after treatment there were no clinical signs observed

. As there were no clinical signs observed, t he study was terminated after the 72 hours

observation.

During the study, the control eye of all animals was symptom

- free.

The general state and behavior of animals were normal throughout the study period.

There were no notable body weight changes during the study period.

The animal’s individual mean scores (considering readings at 24, 48 and 72 hours
after the treatment) were as follows:

redness : 0.00, 0.00, 0.00

chemosis : 0.00, 0.00, 0.00 discharge : 0.00, 0.00, 0.00 cornea opacity : 0.00, 0.00, 0.00

iris : 0.00, 0.00, 0.00

The test item ( Batch No .: TQA1067) , applied to rabbits’ eye mucosa, caused conjunctival irritant effects at one hour which were fully reversible within

24 hours.

According to Regulation (EC) No 1272/2008, does not require classification as an eye irritant.

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## LAB 2. INTRODUCTION

The objective of the study was to determine the acute eye irritation effect of the test

item in the New Zealand White Rabbit. The test item was applied as a single dose to the left eye of treated animals. The degree of irritation was scored at specified

time intervals. Information derived from this test was used to determine the existence of possible hazards likely to arise from exposure of the eyes and adjacent mucous

membranes to the test item . The duration of the study was sufficient to evaluate fully the reversibility or irreversibility of the effects observed

.

3. MATERIALS AND METHOD S

Start of Treatment: 31 May 2011 End of Experiment: 03 June 2011

3.1. TEST ITEM

Name:

Chemical Name:

Batch No.:
Appearance: Colourless viscous liquid Purity: 97.4%

Production date: 10 May 2010 Expiry dat e: 24 October 2012
Storage: Room temperature (15-30°C) Safety Precautions: Routine safety precautions (gloves, goggles, face mask,
lab coat) for unknown materials were applied to assure personnel health and safety.

The pH of Test Item was measured as 4.5.

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 in the Central Dispensary Unit

of LAB Research Ltd. on the basis of name, batch number, appearance and colour.

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## LAB 3.2. OTHER MATERIALS

For application :

Name: Disposable syringe, Penta Ferte (1 mL)

Batch No.: 08H04 Expiry Date: August 2011

For euthanasia:

Name: CP -Ketamin 10 %

Batch No.: 10H12 4 Expiry Date: July 2012 Produced by: CP -Pharma, 31303 Burgdorf

Name: CP -Xylazin 2 %

Batch No.: 10G06 2 Expiry Date: June 2013 Produced by: CP -Pharma, 31303 Burgdorf

Name: Euthasol ® 40 %

Lot No.: 10C25 7 Expiry Date: February 2013 Produced by: AST Beheer B.V. Oudewater Netherlands

3.3. EXPERIMENTAL ANIMALS

Species and strain : New Zealand White rabbits

Source: S&K

- LAP Kft. 2173 Kartal, Császár út 135, Hungary

Justification of strain: The New Zealand White rabbit is one of the standard

strai ns used for acute irritation toxicity studies.

Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined approximately one hour before starting the study. An imals showing eye irritation, ocular defects or pre

- existing corneal injury

were not used.

Number of animals: 3 animal s

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## LAB Age of animals at treatment : ~ 11 weeks old (young adult)

Sex: M ale Body weight range at the beginning of the life phase : 2771 – 2841 g

end of the life phase : 2858 – 2946 g Date of receipt: 26 May 2011

Acclimatization time: 5 days

Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose group, cage number and individual animal number.

3.4. HUSBANDRY

Number of animal room: 619 Light : 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3 °C Relative humidity: 34 – 80 % Housing/Enrichment: Rabbits were individ ually housed in AAAL A C approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to
allow some social interaction with rabbit(s) in adjoining cages

Ventilation: 15-20 air exchanges/hour

The environmental param eters were recorded twice daily during the study. Minor

v ariations from the target humidity range were observed during the study. These deviations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results due to their low magnitude.

3.5. FOOD AND FEEDING

Animal received PURINA Base

– Lap gr. diet for rabbits produced by AGRIBRANDS
Europe Hungary PLC, H

- 5300 Karcag, Madarasi road, Hungary, ad libitum . Contents

of PURINA Base

- Lap gr. di et for rabbits, which were used during the experimental phase see in Appendix 3.

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## LAB 3.6. WATER SUPPLY AND QUALITY CONTROL OF WATE R

The animals received municipal tap water, as for human consumption, ad libitum

, from an automatic system.

The 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.

3.7. STUDY SCHEDULE

PRE

- EXPERIMENTAL PERIOD

Study Day Absolute date

Animal received Day [ -5] 26 May 2011

Veterinary c ontrol 2 6 May 2011

Acclimation Day [ -5] - Day [ -1] 26 May – 30 May 2011

TREATMENT PERIOD

Day of treatment Day [0 ] 31 May 2011

Body weight

measurement Day [0 ; 4] 31 May 2011

03 June 2011

Clinical observation Scoring 1, 24, 48

and 72 hours 31 May 2011

01 June 2011

02 June 2011

03 June 2011

End of in life phase 03 June 2011

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## LAB 3.8. ADMINISTRATION OF TH E TEST ITEM

3.8.1. Dosage

A single dose of 0.1 ml of the liquid test item was administered to each animal.

3.8.2. Application of the Test Item

Three mal e animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

An initial test was performed using one animal. The test item was instilled into the conjunctival sac of the left eye. The eyelids were held closed for several seconds to prevent the loss of the test item. The contralateral eye served as the control. Immediately after the administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 2.

After consideration of the ocular responses produced in the first animal, two additional

animals were treated.

3.9. OBSERVATIONS AND SCO RING

3.9.1. Clinical Obser vations

The eyes were examined at 1, 24, 48 and 72 hours after the treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill

- health during the study were

recorded.

At the end of the observation period, each animal was sacrificed by intramuscular injections of CP

- Ketamin 10% and CP

- Xylazin 2% followed by iv. Euthasol ® 40%
anaesthesia. (see details in 3.2.)

. Death was verified by checking pupil a nd cornea reflex, absence of respiration and pulse.

3.9.2. Scoring and Assessment of Local Reaction

The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002) shown in Appendix 1.

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## LAB 3.9.3. C lassification of the Test Items

Individual reactions of the animal were recorded at each observation time. The n ature, severity and duration of all lesions observed were described. Results were presented and interpreted according to Regulation (EC) No 1272/2008, as follows:

Irreversible effects on the eye/serious damage to eyes (Category 1) Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). These observations include animals wit h grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight.

Category for irreversible eye effects If, when applied to the eye of an animal, a substance produces:

— at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;

and/or

— at least in 2 of 3 tested animals, a positive response of:

o corneal opacity ≥ 3 and/or

o iritis > 1 .5 calculated as the mean scores following grading at 24, 48 and 72 hours after

installation of the test material.

Reversible effects on the eye /irritating to eyes (Category 2) Substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes).

Category for reversible eye effects If, when applied to the eye of an animal, a substance produces:

— at least in 2 of 3 tested animals, a positive response of:

o corneal opacity ≥ 1 and/or

o iri tis ≥ 1, and/or

o conjunctival redness ≥ 2 and/or

o conjunctival oedema (chemosis) ≥ 2

— calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 d ays

.

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## LAB 3.9.4. Measurement of Body Weight

Individual body weight w as recorded at the beginning and end of the experiment.
(Table 3)

4. ARCHIVES

The study documents:

- study plan,

- all raw data,

- sample of the test item, - study report and any amendme nt(s), - correspondence

are stored in the archives of LAB Research Ltd., 8200 Veszprém, Szabadságpuszta, Hungary according to the Hungarian GLP and applicable SOPs.

After the retention time has elapsed, all the archived materials listed above will be

offe red to the Sponsor for further storage or disposal.

5. THE PERMISSION OF TH E INSTITUTIONAL IACU C

The Institutional Animal Care and Use Committee (IACUC) of LAB Research Ltd. reviewed the study plan and authorised the conduct of the study.

6. DEVIATION TO THE STUDY PLAN

The relative humidity was out of the target range during part of the test (34 – 80%). Floor washing can cause transient high humidity in the animal room.

The animals were received on 26 May 2011 instead of 25 May 2011.

Th ese deviati on s were considered not to adversely affect the results or integrity of the study.

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## LAB 7. RESULTS

7.1. MORTALITY

There was no mortality observed during the study.

7.2. BODY WEIGHTS

The body w eight and the body weight change were considered to be normal with no

indication of treatment related effect.

7.3. CLINICAL OBSERVATION

7.3.1. General daily examination

There were no clinical signs observed that could be related to treatment.

7.3.2. Examination of eye- irritancy

An acute eye irritation study of the test item was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the
Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each an imal. The

untreated right eye served as control. A volume of 0.1 ml of the test item was administered as a single dose.

The eyes were examined at 1, 24, 48 and 72 hours after the application.

Th is deviation was considered not to adversely affect the res ults or integrity of the study

As there were no clinical signs observed, the study was terminated after the 72 hours

observation.

During the study, the control eye of all animals was symptom- free.

The general state and behavio u r of animals were normal throughout the study period.

There were no notable body weight changes during the study period.

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## LAB The animal’s individual mean scores (considering readings at 24, 48 and 72 hours
after the treatment) were as follows:

redness : 0.00, 0.00, 0.00

chemosi s : 0.00, 0.00, 0.00 discharge : 0.00, 0.00, 0.00 cornea opacity : 0.00, 0.00, 0.00

iris : 0.00, 0.00, 0.00

8. CONCLUSION

The test item ( Batch No .: ) , applied to rabbits’ eye mucosa, caused conjunctival irritant effects at one hour which were fully reversible within

24 hours.

According to Regulation (EC) No 1272/2008, does not require classification as an eye irritant.

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## LAB INDIVIDUAL SCORES FOR OCULAR IRRITATION

TABLE 1

Study Code: 11/076-005N Species: NZW Rabbit

Dose: 0.1 ml Sex: Male

Start of Exposure: 31 May 2011 – Day 0 Test Item:

Time Animal No. Score of irritation

IPR Conjunctivae Opacity of

cornea Iris Control

eye Other sign R CH D OD OE R

1 hour 00533 1 0 3 0 0 0 0

- 0

00550 1 0 3 0 0 0 0

- 0

00547 1 0 3 0 0 0 0

- 0

Time Animal No. Score of irritation

Conjunctivae Opacity of

cornea Iris Control

eye Other sign R CH D OD OE R

24 hours 00533 0 0 0 0 0 0 0

-
00550 0 0 0 0 0 0 0

-
00547 0 0 0 0 0 0 0

-

Time Animal No. Score of irritation

Conjunctivae Opacity of

cornea Iris Control

eye Other sign R CH D OD OE R

48 hours 00533 0 0 0 0 0 0 0

-
00550 0 0 0 0 0 0 0

-
00547 0 0 0 0 0 0 0

-

Time Animal No. Score of irritation

Conjunctivae Opacity of

cornea Iris Control

eye Other

si gn R CH D OD OE R

72 hours 00533 0 0 0 0 0 0 0

-
00550 0 0 0 0 0 0 0

-
00547 0 0 0 0 0 0 0

-

Abbreviations:

R = Redness ; OD = Opacity degree of density ; CH = Chemosis ; OE = Extension of opaque area ; D = Discharge ; IPR = Initial pain reaction ; - = No data

### STUDY CODE: 11/076-005N Page 18 of 22



## LAB MEAN VALUES OF EYE IRRITATION (24, 48, 72 hour reading)

TABLE 2

Study Code: 11/076-005N Species: NZW Rabbit

Dose: 0.1 ml Sex: Male Start of Exposure: 31 May 2011 – Day 0 Test Item:

Animal

Number Sex Cornea Opacity Iris Conjunct ivae

Redness Chemosis Discharge

00533 male 0.00 0.00 0.00 0.00 0.00

00550 male 0.00 0.00 0.00 0.00 0.00

00547 male 0.00 0.00 0.00 0.00 0.00

BODY WEIGHT DATA

TABLE 3

Animal

Number Beginning of study
(g) At the end of study
(g) Body weight gain
(g)

00533 2824 2946 122

00550 2771 2858 87

00547 2841 2904 63

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## LAB 

# APPENDICES

### STUDY CODE: 11/076-005N Page 1 of Appendix 1



## LAB APPENDIX 1:

SCORING AND ASSESSMENT OF LOCAL REACTION

Cornea

a. Opacity

- degree of density (Area most dense taken for reading)

No ulceration or opacity 0

Scatter ed or diffuse areas of opacity (other than slight

dulling or normal lustre): details of iris clearly visible 1

Easily discernible translucent area: details of iris slightly obscured 2

Nacreous area: no details of iris visible: size of pupil barely disc ernible 3

Opaque cornea: iris not discernible through the opacity 4

b. Area of cornea involved

One quarter (or less), but not zero 1

Greater than one quarter, but less than half 2

Greater than half, but less than three quarters 3

Greater than three qua rters, up to whole area 4

Iris

a. Values

Normal 0

Markedly deepened rugae, congestion, swelling, moderate

circumcorneal hyperaemia: or injection: iris reactive to light

(a sluggish reaction is considered to be an effect) 1

Haemorrhage, gross destructio n, or no reaction to light. 2

### STUDY CODE: 11/076-005N Page 2 of Appendix 1



## LAB APPENDIX 1:

SCORING AND ASSESMENT OF LOCAL REACTION
(c ontinued)

Conjunctivae

a. Redness (Palpebral and bulbar)

Normal 0

Some blood vessels hyperaemic (injected) 1

Diffuse, crimson colour, individual vessels not easily discernible 2

Diffuse beefy red 3

b. Chemosis

Normal 0

Some swelling above normal (includes nictating membrane) 1

Obvious swelling with partial eversion of lids 2

Swelling with lids about half closed 3

Swelling with lids more than half closed 4

c. Di scharge

No discharge 0

Any amount different from normal (does not include small amounts
observed in inner canthus of normal animals) 1

Discharge with moistening of the lids and hairs just adjacent to lids 2

Discharge with moistening of the lids and hairs, on considerable area around the eye 3

### STUDY CODE: 11/076-005N Page 1 of Appendix 2



## LAB APPENDIX 2:

SCORING OF PAIN REACTION

Class Reaction by Animal Descriptive Rating

0 No response No initial pain

1 A few blinks only, normal within one or two minutes Practically no initial pain

2 Rabbit blin ks and tries to open eye, but reflex closes it Slight initial pain

3 Rabbit holds eye shut and puts pressure on lids, may rub eye with paw Moderate initial pain

4 Rabbit holds eye shut vigorously, may squeal Severe initial pain

5 Rabbit holds eye shut v igorously, may squeal, claw at eye, jump and try to escape Very severe initial pain

The class 0, 1 and class 2 are not anaesthetised.

There is no correlation between the initial pain and the subsequent eye irritation.

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## LAB APPENDIX 3:

CONTENTS OF PURINA BASE
- LAP GR. DIET FOR RABBITS

Batch number: 003 004 11 Expiry date: 11 July 2011

NUTRIENTS

Wet 11.2 % Crude protein 16.2 %

Total fat 2

. 7 %

Crude fibre 15.7 %

Crude Ash 7.6 %

MINERALS

Calcium 1. 10 % Phosphorus 0. 58 %

Sodium 0. 18 %
Cu (CuSO 4 * 5H 2 O) 20.000 mg/kg

AMINO ACIDS

Lysine 0.6 8 %

Methionine 0.25% Met + Cystine 0. 54 %

VITAMINS

Vitamin A 1 501 0 IU/kg

Vitamin D 3 1 25 0 IU/kg

Vitamin E 50 mg/kg

These data are standard and guaranteed values which were pro vided by the supplier.

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## LAB APPENDIX 4:

COPY OF THE CERTIFICATE OF ANALYSIS

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## LAB APPENDIX 5:

COPY OF THE GLP CERTIFICATE

### STUDY CODE: 11/076-005N Page 1 of Appendix 6



## L A B 

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301 APPENDIX 6:

COPY OF THE STUDY PLAN

STUDY PLAN

Acute Eye Irritation Study in Rabbits

Date of Study Plan: 23 May 2011 

### STUDY C ODE: 11/076-005N

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## L A B GENERAL INFORMATION

TITLE OF THE STUDY : Acute Eye Irritation Study in Rabbits

TEST ITEM :

SPONSOR

S TUDY PERFORMED BY : LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta
Hungary

Phone: +36 88 545 300

Fax: +36 88 545 301

### STUDY CODE: 11/076-005N Page 3 of Appendix 6



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

. – Assistant Scientist István Pásztor DVM/ Peter Maslej DVM, PhD - veterinary control Szabolcs Gáty M.Sc. – Head of QAU

Animal House Staff Central Dispensary Staff

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

PROPOSED STUDY SCHEDULE i :

START OF EXPERIMENT : 31 May 2011

END OF EXPERIMENT : Not later than 21 June 2011

DRAFT REPORT : Not later than 31 July 2011

BASIS OF STUDY : OECD Guidelines for Testing of Chemicals 405 (24 th April 2002)

C ommission Regulation (EC) No 440/2008, B.5 ( L 142, 30 May 2008 ) OPPTS 870.2400 (EPA 712

- C

- 98

- 195) August 1998

REPORTING FORMAT : Final Report to LAB Research Ltd 's sta ndard

format

REPORT COPIES : Original report to be archived One PDF copy will be sent by email.

One bound and one unbound hard copy will be sent by courier.

i A detailed study timing (target) is given in Append ix 1

.

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## L A B CONTENTS

1. OBJECTIVE OF THE STUDY 5

2. MATERIALS AND METHOD S 6

2.1. TEST ITEM 6

2.1.1. Identification, Receipt 6

2.1.2. Other Materials 6

2.2. EXPERIMENTAL ANIMALS 6

2.2.1. Husbandry 7

2.2.2. Food and Feeding 7

2.2.3. Water Supply and Quality Control 7

2.2.4. Identifica tion 8

2.3. TESTING PROCEDURE 8

2.4. ADMINISTRATION OF TE ST ITEM 9

2.4.1. Dose Level 9

2.4.2. Application of the Test Item 9

2.4.3. Duration of Exposure 9

2.5. OBSERVATIONS AND SCO RING 10

2.5.1. Clinical Observations and Evaluation of Ocular Irritation 10

2.5.2. Scoring a nd Assessment of Local Reaction 10

2.5.3. Classification of the Test Item 12

2.5.4. Measurement of Body Weight 13

3. ANIMAL WELFARE 13

4

. GOOD LABORATORY PRAC TICE (GLP) AND QUALI TY ASSURANCE 13

5. AMENDMENT AND DEVIAT ION PROCEDURES 14

6. STUDY REPORT 14

7. ARCHIVES 15

8. DISTRIBUTION OF THE STUDY PLAN 15

9. SIGNATURES 16

10. REFERENCES 16

APPENDICES 17

APPENDIX 1: 18

PROPOSED STUDY SCHEDULE 18

### STUDY CODE: 11/076-005N Page 5 of Appendix 6



## L A B 1. OBJECTIVE OF THE STU DY

The objective of this study is to determine the degree of ocular irritation resulting from the test item when administered in a single dose into the conjunctival sac of one eye of up to three New Zealand White rabbits. The other eye, which will remain untreated, serves as the

control.

Before testing starts, according to the guideline OECD 405, it is the responsibility of the Sponsor to ensure that the following considerations have been addressed:

There is not adequate human or animal data to allow regulatory classification of this test item for eye irritancy.
It is not possible to provide information to make a regulatory classification based

on Structure Activity Relationship (SAR) for eye irritation/corrosion. (a)
There is no evidence that the dose of less than approximately 20 mg of test item per kg of animal body weight will cause severe systemic toxicity.
The test item is not considered to be corrosive to skin. (b).
It is noted that there is no in vitro eye irritancy assay currently recognised internationally as a replacement for animal testing for regulatory classification.

Following the above, as part of the initial evaluation, the pH of the test item will be measured by LAB Research Ltd. (pH target range 2-11.5). Should it fall outside the range required by the guideline, the test item will be reported as corrosive to eyes, unless the Sponsor requests and can justify the performance of an animal test.

Notes: (a) SAR is only applicable to pure test items, not mixtures or materials with any impurities. (b) Corrosivity data from any previous studies should be considered.

### STUDY CODE: 11/076-005N Page 6 of Appendix 6



## L A B 2. MATERIALS AND METHODS

2.1. TEST ITEM

Name:

Chemical Name:

B atch No.:
Appe arance: Colourless viscous liquid

Pur ity: 97.4%

Production date: 10 May 2010 Expiry date: 24 October 2012 Storage: Room temperature (15-30°C)

S afety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety.

pH wil l be measured prior to the start of treatment.

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

prov ided by the Sponsor in the Central Dispensary of LAB Research Ltd.

2.1.2. Other Materials

The details of the materials to be used for euthanasia will be given in the Study
Report

.

2.2. EXPERIMENTAL ANIMALS

Species and strain: New Zealand White rabbit

Source: S&K-LAP Kft.
2173 Kartal, Császár út 135, H ungary Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.

### STUDY CODE: 11/076-005N Page 7 of Appendix 6



## L A B Number of animals: Up to 3 animals

Sex: Male or female (if female, nulliparous and non pregnant) Age of animals at treatment: Young adult rabbits Target body weight range: 20 00 -40 00 g at dosing Acclimation period: At least 5 days

2.2.1. Husbandry

Animal health: Only healthy animals will be used for the study, as certified by the veterinarian. Housing/Enrichment: Rabbits will be individually housed in AAALAC approved metal wire rabbit cages. Cages are of an open wire structure and cages are placed together to allow some social interaction with rabbit(s) in adjoining

ca ges. Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C Relative humidity: 30-70 %

Ve ntilation: 15 -20 air exchanges/hour.

The room temperature and relative humidity values will be checked and recorded twice daily during the study (i ncluding acclimatization phase)

.

2.2.2. Food and Feeding

The animals will receive PURINA Base – Lap gr. diet for rabbits produced by

AG RIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi út, Hungary, ad libitum. The contents of PURINA Base – Lap gr. diet for rabbits will be given in the

re port.

2.2.3. Water Supply and Quality Control

The animals will receive tap water, as for human consumption, ad libitum , from an automatic system from the communal water network. The water is considered not to

co ntain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The quality control analysis is performed once every 3 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.

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## L A B 2.2.4. Identification

The individual animal identification number will be marked with indelible ink on the ear, or will be on an ear tag. The cages will be marked with individual identity cards with information about study code, sex, dose group, cage number and the individual

animal number.

2.3. TESTING PROCEDURE

The pH of the test item will be determined. If the pH is 2 or less or 11.5 or greater, the study will not be conducted, unless there is evidence that the test item is not irritating or corrosive to the eye.

Three animals in acceptable health condition will be selected for the test. Both the control eyes and the eyes to be treated will be carefully examined before treatment. Any animal with ocular lesions will be rejected and not assigned to the study.

F or ethical reasons, an initial test will be performed using a single animal. If the results of this test indicate the test item to be severely irritating or corrosive to the eye,

further testing will not be performed.

Immediately after administration of the test item, an assessment of the initial local pain reaction (IPR ) will be made according to the IPR scale detailed below:

Score Reaction by Animal Descriptive Rating

0 No response No initial pain

1 A few blinks only, normal within one or two minutes Practically no initial pain

2 Rabbit blinks and tries to open eye , but reflex closes it Slight initial pain

3 Rabbit holds eye shut and puts pressure on lids, may rub eye with paw Moderate initial pain

4 Rabbit holds eye shut vigorously, may squeal Severe initial pain

5 Rabbit holds eye shut vigorously, may squeal, c law at

eye, jump and try to escape Very severe initial pain

Class es 0, 1 and 2 will not be anaesthetised as it is considered that usually there is no correlation between the initial pain and the subsequent eye irritation.

Providing severe effects are not observed in the first treated animal, two additional

a nimals will subsequently be treated in an identical manner. Both eyes of these animals may be anaesthetised prior to treatment, at the discretion of the Study

Director.

### STUDY CODE: 11/076-005N Page 9 of Appendix 6



## L A B The test item will be applied in a single dose to the left eye of one or three animals. The degree of irritation will be observed and scored at specified intervals. Information derived from this test serves to indicate the existence of possible hazards likely to arise from exposure of the eyes and associated mucous membranes, to the test item. The duration of the study will be sufficient to evaluate the reversibility or irreversibility of
the effects. At termination, animals will be sacrificed.

2.4. ADMINISTRATION OF TEST ITEM

2.4 .1. Dose Level

A single dose of 0.1 ml of the liquid test item will be introduced into the eye.

2.4.2. Application of the Test Item

The test item will be administered into the conjunctival sac of the left eye of each animal. The eye lids will be gently held closed for several seconds to prevent the los s of the test item.

The untreated contralateral eye will serve as the control.

2.4.3. Duration of Exposure

Animals showing continuing severe pain or distress or any of the following irreversible changes, will be humanely euthanized without delay: corneal perforation or significant ulceration of the cornea or conjunctival membrane; blood in the anterior chamber of the eye; grade 4 corneal opacity which persists for 48 hours; absence of a light reflex (iridial response grade 2) which persists for 72 hours; necrosis of the conjunctivae or nictitating membrane; or sloughing.

In the case of corrosive effects, severe irritation, or at 24 hours after instillation,

wa shing of the eye with physiological saline may be performed at the discretion of the Study Director.

### STUDY CODE: 11/076-005N Page 10 of Appendix 6



## L A B 2.5. OBSERVATIONS AND SCORING

2.5.1. Clinical Observations and Evaluation of Ocular Irritation

Pre-treatment:

Both eyes of each experimental animal(s) provisionally selected for testing will be examined within 24 hours prior to testing. Animals showing eye irritation, ocular

defects or pre-existing corneal injury will not be used.

Post-treatment:

Evaluations will be made by comparing the treated eye to the control eye. The readings

will be made at 1, 24, 48, and 72 hours, then at 1, 2, and 3 weeks post-treatment.

If there is no evidence of irritation at 72 hours, the study will be terminated.

I f the animal(s) become symptom-free before 21 days, the experiment may be terminated at that time. As deemed necessary, after recording the observations at

24 hours, the eyes of the rabbit(s) may be further examined with the aid of fluorescein liquid. Examination will be performed by means of a hand slit-lamp.

Any clinical signs of toxicity or signs of ill-health during the study will be recorded daily.

At study termination, or in the case of early termination due to eye effects or for moribund animals, euthanasia will be performed by intramuscular injections of Ketamin (10%) and Xylazin (2%) followed by i.v. Euthasol® 40% terminal anaesthesia (details will be given in the report). Death will be verified by checking pupil and cornea reflex, absence of respiration and pulse.

2.5.2. Scoring and Assessment of Local Reaction

The eye irritation scores will be evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002) presented in the following table.

1. Conjunctivae

A. Redness (Palpebral and bulbar)

Normal 0

Some blood vessels hyperaemic (injected) 1

Diffuse, crimson colour, individual vessels not easily discernible 2

Diff use beefy red 3

### STUDY CODE: 11/076-005N Page 11 of Appendix 6



## L A B B. Chemosis

Normal 0

Some swelling above normal (includes nictating membrane) 1

Obvious swelling with partial eversion of lids 2

Swelling with lids about half closed 3

Swelling with lids more than half closed 4

C. Discharge

No discharge 0

Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) 1

Discharge with moistening of the lids and hairs just adjacent to lids 2

Disch arge with moistening of the lids and hairs, on considerable area around the eye 3

2. Iris

D. Values

Normal 0

Markedly deepened rugae, congestion, swelling, moderate

circumcorneal hyperaemia: or injection: iris reactive to light

(a sluggish reaction is considered to be an effect) 1

Haemorrhage, gross destruction, or no reaction to light. 2

3. Cornea

E. Opacity -degree of density (Area most dense taken for reading)

No ulceration or opacity 0

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre): details of iris clearly visible 1

Easily discernible translucent area: details of iris slightly obscured 2

Na crous area: no details of iris visible: size of pupil barely discernible 3

Opa que cornea: iris not discernible through the opacity 4

F. Area of cornea involved

One quarter (or less), but not zero 1

Greater than one quarter, but less than half 2

Greater than half, but less than three quarters 3

Greater than three quarters, up to whole area 4

### STUDY CODE: 11/076-005N Page 12 of Appendix 6



## L A B 2.5.3. Classification of the Test Item

The individual reactions of each animal will be recorded at each observation time. The

n ature, severity and duration of all lesions observed will be described. Results will be presented and interpreted according to Regulation (EC) No 1272/2008 of th e European

Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing

Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC)

No 1907/2006 , as follows:

Irrev ersible effects on the eye/serious damage to eyes (Category 1) Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). These observations include animals with grade 4 cornea lesions an d other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the

iris or other ef

fects that impair sight.

Category for irreversible eye effects

If, when applied to the eye of an animal, a substance produces:

— at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;

and/or

— at least in 2 of 3 tested animals, a positive response of:

o corneal opacity ≥ 3 and/or

o iritis > 1.5 calculated as the mean scores following grading at 24, 48 and 72 hours after

installation of the test material.

Reversible effects on the eye/irritating to eyes (Category 2) Substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes).

Category for reversible eye effects If, when applied to the eye of an animal, a substance produces:

— at least in 2 of 3 tested animals, a positive response of:

o corneal opacity ≥ 1 and/or

o iritis ≥ 1, and/or

o conjunctival redness ≥ 2 and/or

o conjunctival oedema (chemosis) ≥ 2

— calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 d ays

### STUDY CODE: 11/076-005N Page 13 of Appendix 6



## L A B 2.5.4. Measurement of Body Weight

Body weights will be recorded at the beginning and at the end of the experiment.

3. 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.

Chemicals likely to be corrosive will not be tested. To ensure a minimum of animal distress, initially, only a single animal will be exposed to the test chemical. The treated eye will be examined at 1 hour for signs of serious effects.

In the case of a clear corrosive effect, the animal will be sacrificed and the study
terminated. In the case of a severe irritant effect, less than a clear corrosive effect, no further animals will be treated and the single animal will be observed as per study plan. Otherwise, 2 additional rabbits will be used to evaluate the ocular effects of the

test item.

4. 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 which corresponds to the OECD GLP, ENV/MC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments.

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 our Standard Operating Procedures.

This study will be assessed in compliance with this Study Plan and the LAB Research Ltd. Standard Operating Procedures. The critical phases of this study as well as the test facility will be inspected by the Quality Assurance Unit (QAU) and the dates and the

p hases of the inspections will be included into the final report.

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## L A B The results of all the study audits will be reported to the Study Director. The data presented in the final report will be audited in comparison to the raw data. A quality assurance statement, signed by the Quality Assurance Unit, will be included in the Final
Report.

The Sponsor’s Study Monitor or authorized designate(s) may inspect this study during regular working hours for quality assurance purposes and also copy any study-related

or iginal data.

5. 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.

6. STUDY REPORT

A Report in English in LAB Research Ltd.’s format will be sent to the Sponsor and

will in clude 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 study director and the management.

- 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.

- C haracterisation 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 description of the animals: strain, health status, sex, source, identification, weight at commencement of the study, group size and animal husbandry.

- Details of the administration of the test item.

- Individual results of each animal at times of scoring.

- Description of any lesions observed.

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## L A B - Narrative description of the degree and nature of irritation observed.

- Description of any toxic effects.

- Interpretation of results.

- Conclusions.

- A copy of Study Plan including amendments to and deviations from the protocol. A list of all deviations to the Study Plan and a statement on their possible impact on quality and integrity of the study results.

- A copy of the Certificate of Analysis (if available)

- A c opy of the GLP-Certificate of the test facility.

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

The draft report will be sent to the Sponsor by e-mail for comment. The final report will be issued following comments by the Sponsor as a PDF document and a Word document sent to the Sponsor by e-mail. One bound and one unbound copy will be sent by courier.

7. ARCHIVES

The study documents (study plan and any amendments, all raw data, study report and any amendments, correspondence) and a sample of the test item will be archived according to the Hungarian GLP and 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.

8. DISTRIBUTION OF THE STUDY PLAN

Sponsor: 1 copy or 1x PDF-file

Study Director: 1 copy

QAU: 1 copy

Archive: 1 original

Central Dispensary: 1 copy

### STUDY CODE: 11/076-005N Page 16 of Appendix 6



## L A B 9. SIGNATURES

The Study Director, LAB Research Ltd.'s management, QAU and IACUC will sign the Study Plan before the start of the study. The signature page with 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.

10. REFERENCES

1. OECD Guidelines for Testing of Chemicals 405 (24 th April 2002)

2. Commission Regulation (EC) No 440/2008, B.5 (L 142, 30 May 2008)

3. OPPTS 870.2400 (EPA 712-C- 98 -195) August 1998

4. 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.

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## L A B 

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

### STUDY CODE: 11/076-005N Page 18 of Appendix 6



## L A B APPENDIX 1:

PROPOSED STUDY SCHEDULE

PRE-EXPERIMENTAL PERIOD

Relative date Absolute date

Animal arrival Day [

- 6 ] 25 May 2011

Veterinary control Within 48 hours of arrival

Acclimatization 6 days 25 May

– 3 0 May 2011

TREATMENT PERIOD

Day of treatment Day 0 31 May 2011

Animal ocular examination and assignment to study Day 0 (pre

- treatment) 31 May 2011

Body weight measurement D ay 0

T erminating day 31 May 2011
To be determined, based upon the results of

observation

Evaluation of pain reaction

Clinical observation Shortly after treatment

Scoring at 1, 24, 48 and 72 hours then at 1, 2 and 3 weeks after treatment if required (see relevant sections in the study plan)

End of in life phase Not later than Day [21] 21 June 2011

Draft Report Not later than 31 Ju ly 2011 ( Within 6 weeks after completion of the in

- life phase )