TS-CKHI01

Attachment #11

Health Effects Data on the New Chemical Substance

Skin Irritation Test in Rabbits

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301 0



### FINAL REPORT

ACUTE SKIN IRRITATION STUDY

IN RABBITS

Date of Final Report: 25 July 2011 

### STUDY CODE: 11/076-006N

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

SPONSOR :

STUDY PERFORMED BY : LAB Rese arch Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

HUNGARY

Phone: +36 88 545 -300

Fax: +36 88 545 -301

STUDY DIRECTOR : Viktória Zelenák, M.Sc.

VETERINARY CONTROL : István Pásztor, D. V. M.

Veterinarian

CENTRAL DISPENSARY : Tamás Mészáros, PhD

QUALITY ASSURANCE : Szabolcs Gáty, M.Sc. Head of QA

R ESPONSIBLE PERSONS : Judit Tavaszi M.Sc. – Assistant Scientist

Animal House staff

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## LAB CONTENTS

Page

1. SUMMARY

........................................................................................................... 7

2. INTRODUCTION

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

3. MATERIALS AND METHODS

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

3.1. TEST ITEM

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

3.1.1. Test Item

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

3.1.2. Other Materials

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

3.2. EXPERIMENTAL ANIMALS

............................................................................. 10 3.2.1. Husbandry ............................................................................................................. 10 3.2.2. Food and Feeding .................................................................................................. 11 3.2.3. Water Supply ......................................................................................................... 11

3.2.4. Water Analysis

...................................................................................................... 11 3.2.5. Study schedule....................................................................................................... 11

3.3. ADMINISTRATION OF TH E TEST ITEM

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

3.3.1. Dosage

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

3.3.2. Application of the Test Item

.................................................................................. 12 3.3.3. Duration of Exposure ............................................................................................ 13

3.4. OBSERVATIONS AND SCO RING

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

3.4.1. Clinical Observations

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

3.4.2. Scoring and Assessment of Local Reactions

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

3.4.3. Classification of Test Item

.................................................................................... 13 3.4.4. Measurement of Body Weight .............................................................................. 15

3.4.5. Termination

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

3.5. ARCHIVES

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

3.6. THE PERMISSION OF THE INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC ) .............................................................................. 16

3.7. DE VIATION TO THE STUDY PLAN

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

4. RESULTS

............................................................................................................ 17

4.1. MORTALITY

....................................................................................................... 17

4.2. BODY WEIGHTS

................................................................................................. 17

4.3. CLINICAL OBSERVATION

............................................................................... 17

4.3.1. General Daily Examination

................................................................................... 17

4.3.2. Examination of Skin- Irritancy

............................................................................... 17

APPE NDICES

.................................................................................................................... 21

APPENDIX 1: SCORING SYSTEM OF ERYTHEMA AND OEDEMA

FORMATION

....................................................................................................... 22

APPENDIX 2: CONTENTS OF PURINA BASE

- LAP GR. DIET FOR

RABBITS

.............................................................................................................. 23

APPENDIX 3: COPY OF THE CERTIFICATE O F ANALYSIS

..................................... 24

APPENDIX 4: COPY OF THE TEST ITEM DATA SHEET

........................................... 25

APPENDIX 5: COPY OF THE GLP CERTIFICATE

....................................................... 27

APPENDIX 6 : C OPY OF THE STUDY PLA N .............................................................. 1-19

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## LAB 1. SUMMARY

An acute skin irritation study was performed with in New Zealand White rabbits. Parameters monitored during this study included mortality, body weight measurements and

clinical observations. The irrit ancy of the test item w as evalu ated according to the Draize method (OECD No.: 404, 2002).

The test item was administered undiluted, a t a single dose of 0.5 ml. Gauze was placed onto the hairless skin of the rabbit, test item was applied to the gauze, additional gauze was placed over t he test item and an adhesive clear plastic patch applied. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin o f each animal served as control

.

After 4 hours, the remaining test item was removed with water at body temperature.

To assess skin irritation, animals were examined at 1, 24, 48 and 72 hours after the patch removal . Additional general examinations were performed daily.

There was no mortality or systemic clinical changes related to administration

.

There was no effect of treatment on body weight.

At observation one , 24 , 48 and 72 hour s after patch removal t here were no

observed clinical signs noted on the skin of the treated animals

.

As no clinical signs were observed up to 72 ho urs after patch removal, the study was

terminated after the 72 hours observation.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0. 00, 0.00 and 0.00 respectively

.

The Pri mary Irritation Index (considering readings at 24, 48 and 72 hours after patch removal) was calculated as 0.00.

According to D irective 2001/59/EC

, does not require classification as a

skin irritant.

According to the UN Globally Harmonised System of Classificat ion and Labelling of Chemicals, does not require classification as a skin irritant

.

According to the classification system based on the scheme devised by Draize (1959), is a "non irritant".

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## LAB 2. INTRODUCTION

The objective of the stud y was to determine the acute skin irritation effect of the test

item in New Zealand White Rabbits.

The test item was applied to intact skin of rabbits as a single dose. The degree of irritation was scored at specified intervals. Informati on derived f rom this test serve to indicate the existence of possible hazards likely to arise from exposure of the skin to the test item. The duration of the study was sufficient to evaluate fully the reversibility or irreversibility of any effects observed.

3. MATER IALS AND METHODS

Day of Treatment: 24 May 2011 End of Expe riment: 27 May 2011

3.1. TEST ITEM

3.1.1. Test Item

Name:

Chemical Name:

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

Production date: 10 May 2010

E xpiry date: 24 October 2012 Storage: Room temperature ( 15-30°C) Safety Precautions: Routine safety precautions (gloves, goggles, face mask

, lab coat ) for unknown materials we re applied to assure personnel health and safety.

The pH of Test Item was measure d as 4.5.

The test item of a suitable chemical purity, all precautions required in the handling and disposal of t he test item and t he Test Item Data Sheet were supplied by the Sponsor. The identification of test item was made by its appe arance and colour in the Central Dispensary Unit of LAB Research Ltd. on the basis of the information provided by Sponsor.

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## LAB 3.1.2. Other Materials

For treatment: Disposable Syringe (1 ml)

Batch No.: 08H04 Expiry Date: 08 2011 Produced by: Penta Ferte

Sterilux

Batch N o.: 005232390 Expiry Date: 12 2015 Produced by: Paul Hartmann AG, Germany

Betasilk

Batch No.: 10092334 Expiry Date: 09 2015 Produced by: GS Medical Ltd., Dublin

Ramofix

Batch No.: 3005905100 Expiry Date: 03 2015

Produced b y: Ramona Zrt.

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 D ate: February 2013 Produced by: AST Beheer B.V. Oudewater Netherlands

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## LAB 3.2. 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 al bino rabbit is one of the standard strains used for acute irritation toxicity

studies.

Animal health: Only animals in acceptable health condition were used

for the test.

Number of animals: 3 Age of animals at treatment : ~14 weeks old

Sex: M ale Body weight range at the beginning of the life phase : 3074-3547 g

at the end of the life phase : 3238-3671 g Date of receipt: 29 April 2011

Acclimation time: 25 days

Animal identification: The animals were identified by engraved ear tags. The cages were marked with in dividual identity cards with information about study code, sex, cage number, dose group and individual animal number.

3.2.1. Husbandry

Number of animal room: 610 Lighting periods: 12 hours daily, from 6.00 a.m. to 6.00 p.m. Temperature: 18.0-21.6 °C

R elative humidity: 30-70% Housing/Enrichment: Rabbits were individually housed in AAALC approved metal wire rabbit cages (65x65 cm with height of

45 cm). Cages we re of an open wire structure and cages

we re placed together to allow some social interaction

wi th rabbit(s) in adjoining cages.

Ventilation: 15-20 air exchanges/hour

The environmental parameters were recorded twice daily during the acclimatisation and experimental phases.

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## LAB 3.2.2. Food and Feeding

Animals received PURINA Base

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

- 5300 Karcag, Madarasi út , Hungary, ad

libitum . See Appendix 2. for contents of PURINA Base – Lap gr. diet for rabbits.

3.2.3. Water Supply

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

, from an automatic system

.

3.2.4. Water Analysis

The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The quality control analysis is performed once every three month s 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). Copies of the re levant Certificates of Analysis are retained in the archives at

LAB Research Ltd.

3.2.5. Study schedule

PRE

- EXPERIMENTAL PERIOD

Study Day Absolute date

Animal arrival Day [

- 25 ] 29 April 2011

Veterinary control Day [

- 25 ] 29 April 2011

Removing of hair Day [

- 1] 23 May 2011

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## LAB TREATMENT PERIOD

Day of treatment Day [0 ] 24 May 201 1

Animal skin examination and assignment to study Day 0 (pre

- treatment) 24 May 2011

Body weight measurement Day [0, 3] 24, 27 May 2011

Clinical observat ion Scoring at 1, 24, 48 and 72 hours after treatment (24, 25, 26 and 27 May 2011)

End of in life phase Day [ 3 ] 27 May 2011

3.3. ADMINISTRATION OF TH E TEST ITEM

3.3.1. Dosage

The test item was used as supplied , as a single dose of 0 .5 ml applied to the

test area. The untreated skin of each animal served as a co ntrol.

3.3.2. Application of the Test Item

Patch testing was used to detect primary irritating effects of the test item. Three male animals in acceptable health condi tion were selected for this test.

Approximately 24 hours prior to the test

, the hair was clipped from the back and

flanks of the animals. Remov al of hair was performed in two steps. T he majority of hair was clipped with an electronic hair clipper and the remaining hair was moistened

with water and shaved with a razor.

The test item was applied to an approximately 6 cm2 area of intact skin as follows:

**•** A single layer of a fine medical gauze (open

- weave with large holes) of approximately 5x5 cm was placed over the application area,

**•** The appropriate amount of test item was carefully spread over the application area (the gauze helped maintain the test item in place),

**•** Three more layers of gauze were placed over the test item,

**•** These gauze patches were kept in contact with the skin by a patch of clear plastic with a surrounding adhesive hypoallergenic plaster to ensure continued good contact between the moistened test item and the shaved skin.

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## LAB **•** The entire trunks of the animals were wrapped with plastic wrap for 4 hours.

**•** Medical elastic tubing was placed over the plastic to keep it in place.

A n initial test was performed using one animal. One hour after application of the test item, the application site was examined. No severe irritation or corrosive effect was

fo und in the initial test, therefore the bandage was replaced and the exposure continued for a further 3 hours (a total 4 hours exposure). Two additional animals were then included in the study.

3.3.3. Duration of Exposure

Duration of exposure: 4 hours. After the treatment period

, the test item was removed

with water at body temperature

3.4. OBSERVATIONS AND SCO RING

3.4.1. Clinical Observations

Animals were examined for signs of erythema and oedema, and the responses scored
at 60 minutes and then at 24, 48 and 72 hours after patch removal.

3.4.2. Scoring and Assessment of Local Reactions

The dermal irritation scores were evaluated according to the scoring system by Draiz e (1959) shown in Appendix 1 . The animals were observed for 72 hours and the duration of the study was sufficient to evaluate fully the reversibility or irreversibility

of the effects observed.

3.4.3. Classification of Test Item

Results wer e presented and interpreted according to (I) Directive 2001/59/EC, (II) Regulation (EC) No 1272/2008 of the 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 and the (III) UN Globally Harmonised System of Classification and Labelling of Chemicals as follows :

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## LAB (I) Directive 2001/59/EC:

R 35 Causes severe burns

If, when applied to healthy intact animal skin, full thickness destruction of skin tissue

occurs as a r esult of up to three minutes exposure, or if this result can be predicted.

R 34 Causes burns

If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to four hours exposure, or if this result can be predicted.

R38 Irritating to skin

Substances and preparations which cause significant inflammation of the skin which persists for at least 24 hours after an exposure period of up to four hours determined

on the rabbit.

Inflammation of the skin is significant if:

● the mean value of the scores for either erythema and eschar formation or oedema
formation, calculated over all the animals tested, is 2 or more: or

● in the case where the test has been completed using three animals, either erythema and eschar formation or oedema formation equivalent to a mean value of 2 or more calculated for each animal separately has been observed in two or more animals.

In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect are used in calculating the respective mean values.

( II) EC Regulation + (III) GHS :

CATEGORY 1 CORROSIVE

A corrosive is a test material that produces destruction of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least 1 of 3 tested animal s after an exposure of 4 hour s . Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of the observation at 14 days, by discoloration due to blanching of the skin, complete areas of alopecia and scars.

Corrosive subcategories Corrosive in > 1 of 3 animals

Exposure Observation

1A < 3 minutes ≤ 1 hour

1B > 3 minutes

- ≤ 1 hour ≤ 14 days

1C > 1 hour

- ≤ 4 hours ≤ 14 days

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## LAB CATEGORY 2 IRRITANT

1 Mean value of for erythema/eschar formation or oedema is greater or equal than 2.3, but less or equal than 4.0 in at least 2 of 3 tested animals from grades at 24, 48 and 72 hours after patch removal or, if reactions are delayed from grades on 3 consecutive days after the onset of skin reactions; or

2 Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

3 In some cases where there is a pronounced variability of response among animals,

wi th very definite positive effects related to chemical exposure in a single animal

but less than the criteria above

(III) GHS :

CATEGORY 3 MILD IRRITANT

Mean value for erythema/eschar formation or oedema is greater than 1.5, but less than

2.3 in at least 2 of 3 tested animals from grades at 24, 48 and 72 hours after patch removal, or if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions (when not included in the irritant category above).

PRIMARY IRRITATION INDEX

P rimary Irritation Index was calculated by added together the scores for erythema and oedema obtained at 24, 48 and 72 hours after completion of the 4 hours exposure for all animals ( 18 values) and divided by 9 (number of test sites x 3 scoring intervals).

This index was used to classify the test material according to the following scheme devised by Draize (1959):

Primary Irritation Index

Classification

0 - non- irritant

> 0 – 2 - mild irritant

> 2 – 5 - moderate irritant

> 5 – 8 - severe irritant

3.4.4. Measurement of Body Weight

Body weights were recorded at the beginning and at the end of experiment.

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## LAB 3.4.5. Termination

At the end of the observation period

, euthanasia of the animal was by intramuscular injections of CP

- Ketamin 10 % and CP

- Xylaz in 2 % followed by i.v. Euthasol ® 40%

anaesthesia (see details in 3.1.2.). Death was verified by checking pupil and cornea reflex, absence of respiration and pulse.

3.5. ARCHIVES

The study documents:

- study plan,

- all raw data,

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

are stored in the archives of LAB Research Ltd .'s , 8200 Veszprém, Szabadságpuszta, Hungary according to the Hungarian GLP and applica b le SOP ’ s.

After the retention time ha s elapsed

, all the arc hived materials listed above will be offered to the Sponsor for further retention, if agreed by contract, or disposal.

3.6. THE PERMISSION OF THE INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE ( IACUC )

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

3.7. DEVIATION TO THE STUDY PLAN

There was no deviation from the Study Plan.

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## LAB 4. RESULTS

4.1. MORTALITY

There was no mortality observed during the study.

4.2. BODY WEIGHTS

There was no effect of treatment on body weight.

4.3. CLINICAL OBSERVATION

4.3.1. General D aily E xamination

There were no treatment

- related clinical signs noted

.

4.3.2. Examination of Skin-I rritancy

At observation one, 24, 48 and 72 hours after patch removal t here were no

observed clinical signs noted on the skin of the treated animals

.

As no clinical signs were observed up to 72 hours after patch removal, the study was

terminated after the 72 hours observation.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively

.

The Primary Irritation Index (considering readings at 24, 48 and 72 hours after patch removal) was calculated as 0.00.

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## LAB CONCLUSION

According to D irective 2001/59/EC

, does not require classification as a

skin irritant.

According to the UN Globally Harmonised System of Classificat ion and Labelling of Chemicals, does not require classification as a sk in irritant

.

According to the classification system based on the scheme devised by Draize (1959), is a "non irritant".

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## LAB SCORING OF ERYTHEMA FORMATION

TABLE 1

Animal No./

Sex Body weight (g)

1 h 24 h 48 h 72 h at the beginning

of the study at the end of the study

00233 / M 3521 3662 0 0 0 0

00245 / M 3547 3671 0 0 0 0

00239 / M 3074 3238 0 0 0 0

TOTAL

- - 0 0 0 0

SCORING OF OEDEMA FORMATION

TABLE 2

Animal No./

Sex Body weight (g)

1 h 24 h 48 h 72 h at the beginning

of the study at the end of the study

00233 / M 3521 3662 0 0 0 0

00245 / M 3547 3671 0 0 0 0

00239 / M 3074 3238 0 0 0 0

TOTAL

- - 0 0 0 0

M = male

d = day

h = hour

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## LAB MEAN VALUES OF SKIN IRRITATION SCORES (24, 48, 72 hours reading)

TABLE 3

Animal Nu mber Sex Erythema Oedema

00233 male 0.00 0.00

00245 male 0.00 0.00

00239 male 0.00 0.00

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## LAB 

## APPENDICES

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## LAB APPENDIX 1:

SCORING SYSTEM OF ERYTHEMA AND OEDEMA FORMATION



### Erythema and eschar formation

No erythema 0

Very slight erythema (barely percep tible) 1

Well defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Maximum possible erythema score: 4



### Oedema formation

No oedema 0

Very slight oedema (barely perceptible) 1

Slight oedema (edges of it will be well defined by definite raising) 2

Moderate oedema (raised appr. 1 mm) 3

Severe oedema (raised more than 1 mm and extending beyond area of exp.) 4

Maximum possible oedema score: 4

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## LAB APPEN DIX 2:

C ONTENTS OF PURINA BASE - LAP GR. DIET FOR RABBITS

Batch number: 0530 04 9 003 0 0411 Expiry date: 10 July 2011 11 July 2011

NUTRIENTS

Wet 14.00% 11.2 0% Crude protein 16.2 0% 16.2 0%

Crude fat 2.50 % 2.70 %

C rude fibre 16.2 0% 15.7 0%

Ash 7.8 0% 7.6 0%

MINERALS

Calcium 1. 09 % 1. 10 % Phosphorus 0.55 % 0.58 %

Sodium 0.18 % 0.18 % Cu(CuSo 4 5H 2 0) 20.000 mg/kg 20.000 mg/kg

AMINO

ACIDS

Lysine 0.69 % 0.68%

Methionine 0.25% 0.25% Met.+Cyst. 0.53 % 0.54 %

VITAMINS

Vitamin A 15010 IU/kg 15010 IU/kg

Vitamin D 3 1250 IU/kg 1250 IU/kg

Vitamin E 50.00 mg/kg 50.00 mg/kg

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

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## LAB APPENDIX 3:

COPY OF THE CERTIFICATE OF ANALYSIS

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## LAB APPENDIX 4 :

COPY OF THE TEST ITE M DATA SHEET

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## LAB APPENDIX 4 :

COPY OF THE TEST ITEM DATA SHEET (continued)

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## LAB APPENDIX 5:

COPY OF THE GLP CERTIFICATE

## LAB Research Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301 APPENDIX 6:

COPY OF THE STUDY PL AN



### STUDY PLAN

ACUTE SKIN IRRITATION STUDY

IN RABBITS

Date of Study Plan: 20 May 2011 

### STUDY CODE: 1 1/076-006N

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## L A B GENERAL INFORMATION

SPONSOR :

TI TLE : Acute Skin Irritation Study in Rabbits

TEST ITEM :

STUDY PERFORMED BY : LAB Research Ltd.

Address: H

- 8200 Veszprém, Szabadságpuszta

HUNGARY

Phone: +36 88 5 45

- 300

Fax: +36 88 545

- 301

### STUDY CODE : 11/076-006N Page 3 of 19



## L A B RESPONSIBLE PERSONS* : Judit Tavaszi M.Sc. – Assistant Scientist Istvánné Balogh- technician Nóra Végh

- technician

Péter Palotás

- technician Károly Pataki

- technician

Lukács Gábor

- technician

István Pásztor DVM/ Peter Maslej DVM, PhD

- veterinary control

Tamás Mészáros PhD

– central dispensary *: Other trained, competent personnel may work on the study as required

QUALITY ASSURANCE: Szabolcs Gáty, M.Sc. Head of QA Unit

PROPOSED STUDY SCHEDULE:

ST ART OF EXPERIMENT i : 24 May 2011

END OF EXPERIMENT : Not later than 07 June 2011

DRAFT REPORT : Not later than 22 July 2011

FINAL REPORT: Within 90 days of issuance of the Draft Report; depending upon receipt of the Sponsor’s comments and additional documentation (if any required).

BASIS OF STUDY : OECD GUIDELINES FOR TESTING OF CHEMICALS 404 (24 th April 2002) OECD: Paris, Commission Regulation (EC) No 440/2008, B.4 (L 142, 30 May 2008 ) OPPTS 870.2500 (EPA 712

- C

- 98

- 196) August 1998

REPORTING FORMAT : Final Report to LAB Research Ltd.'s standard

format

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

i A detailed study timing (target) is given in Appendix 3

.

### STUDY CODE : 11/076-006N Page 4 of 19



## L A B CONTENTS

1. OBJECTIVE OF THE STUDY

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

2. MATERIALS AND METHODS

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

2.1. TEST ITEM

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

2.2. EXPERIMENTAL ANIMALS

................................ ................................ .............................. 7

2.3. TESTING PROCEDURE

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

2.4. ADMINISTRATION OF TEST ITEM

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

2.5. OBSERVATIONS AND SCORING

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

3. ANIMAL WELFARE

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

4. GOOD LABORATORY PRACTICE (GLP) AND QUALITY ASSURANCE

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

5. AMENDMENT AND DEVIATION PROCEDURES

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

6. STUDY REPORT

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

7. ARCHIVES

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

8. DISTRIBUTION OF THE STUDY PLAN

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

9. SIGNATURES

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

APPENDICES ............................................................................................................................. 16

APPENDIX 1: DETAILS OF MATERIALS TO BE USED FOR EUTHANASIA ................... 17

APPENDIX 2: CONTENTS OF PURINA BASE - LAP GR. DIET FOR RABBITS ............... 18

APPENDIX 3: PROPOSED STUDY SCHEDULE .................................................................... 19

### STUDY CODE : 11/076-006N Page 5 of 19



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

The main objective of this study is to evaluate the skin irritation/corrosion potential of the test item after a single application to rabbit skin and to evaluate the reversibility of dermal

effects. Untreated skin areas of the test animal serve as the control.

Before testing starts, according to the guideline OECD 404, 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 dermal irritancy. It is not possible to provide information to make a regulatory classification based on analysis of Structure Activity Relationship (SAR) for skin irritation/corrosion.(a) There is no evidence that the dermal dose of approximately 100 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 skin irritancy assay currently recognised internationally as a replacement for animal testing for regulatory classification.

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 skin, 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. In the case of potential corrosivity, the sentinel rabbit will be treated with 3 patches for examination at 3 minutes, 1 hour and 4 hours respectively.

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## 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.

*p H will 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 provided by the Sponsor in the Central Dispensary of LAB Research Ltd.

2.1.2. Other Materials

For treatment:
Dispo sable Syringe (1ml)

Bandage for application etc.:
Sterile gauze pads Adhesive hypoallergenic plaster (Clinipore

- silk) Plastic wrap Elastic Stocking

For euthanasia (details are given in Appendix 1 ):

CP

- Ketamin 10 %

CP

- Xylazine 2 %

Euthasol ® 40%

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## L A B 2.2. EXPERIMENTAL ANIMALS

Species and strain: New Zealand White rabbit

Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, HUNGARY

Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies. Number of animals: Up to 3 animals

Sex: Male Age of animals at treatment: Young adult rabbits Target body weight range: ~ 2000-4000 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 (65x65 cm with height of 45 cm). Cages are of an open wire structure and cages are placed together to allow some social interaction with rabbit(s) in adjoining cages. 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 temperature and relative humidity values will be checked and recorded twice daily during the acclimatisation and experimental phases.

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 . For contents of PURINA Base – Lap gr. diets for rabbit: see Appendix 2

.

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## L A B 2.2.3. Water Supply and Quality Control

The animals will receive tap water, as for human consumption, ad libitum , from the automatic system supplied by the communal water network.

The water is considered not to contain 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 of LAB Research Ltd.

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 numbe r.

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 corrosive to animal skin. Patch testing will be used to detect primary irritating effects of the test item. The dermal route is selected because it is a potential route of human exposure.

Three male animals in acceptable health condition will be selected for this test. Approximately 24 hours prior to the test the hair will be clipped from the back and flanks of the animals. Animals showing any dermal defects or pre- existing dermal injury will not be used. Should dense patches of more prominent hair be observed,

such areas will not be used as test sites.

For ethical reasons, an initial test will be performed using a single animal. One hour after application of the test item to the skin of the sentinel animal, the application site will be examined. If the skin shows signs of a corrosive effect, the application period will be terminated (as described below for termination of the 4 hour exposure period) and the study will be immediately terminated. If the effect at one hour is less than corrosive, the bandage will be replaced and the exposure will continue for a further

3 hours (a total 4 hours exposure).

If at one hour in one rabbit there is no corrosive effect, no significant systemic toxicity and no other severe local effects observed in the initial test, then two additional animals will be included in this study. If the severity of the effects in the single rabbit prevents the inclusion of further rabbits, then the report will reflect this.

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## L A B The test item will be applied to an area of approximately 6 cm2 of intact skin. Sterile gauze pads will be placed on the skin of rabbits. These gauze pads will be kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals will then be wrapped with plastic wrap held in place with an elastic stocking for 4 hours.

The degree of irritation will be observed and scored at specified intervals by

examination of the treated area. The untreated skin of each animal will serve as control skin. The duration of the study will be sufficient to evaluate the reversibility o r irreversibility of the effects. At termination, animals will be sacrificed.

2.4. ADMINISTRATION OF TEST ITEM

2.4.1. Dose Level

A volume of 0.5 ml of undiluted liquid test item will be applied to the skin of the experimental animals. The test item will be applied as a single dose.

2.4.2. Duration of Exposure

Four hours after application, the test item will be removed by gently washing with body temperature water. In the case of difficulty removing the test item, the mildest appropriate method will be used (and documented). In the case of materials found to be corrosive, exposure will be terminated at one hour or less.

2.5. OBSERVATIONS AND SCORING

2.5.1. Clinical Observations

Animals will be examined for signs or effects of the test item. The degree of irritation will be evaluated at 1, 24 (±5 min), 48 and 72 hours (±60 min), then if necessary, at one and two weeks following the patch removal. If no sign of irritation is observed at 72 hours, the study will be terminated. If the animals become symptom-free before 14 days, the experiment will be terminated at that time. If signs remain at 14 days, the study may be continued at the discretion of the Study Director, but normally if signs

remain at 14 days the effect will be considered to be irreversible.

At each observation time, erythema and oedema scores will be recorded according to the scheme given in section 2.5.2. Any observations of other local effects observed will also be recorded. Any clinical signs of toxicity, mortality or signs of ill- health during the study will be recorded.

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## L A B At study termination or in the case of early termination due to skin effects or for moribund animals, euthanasia will be by intramuscular injections of Ketamin (10%) and Xylazine (2%) followed by i.v. Euthasol ® 40% terminal anaesthesia (see Appendix 1 for details).

2.5.2. Scoring and Assessment of Local Reaction

The dermal irritation scores will be evaluated according to the scoring system by Draize (1959) and OECD 404 (24 April 2002) presented by the following table:

Erythema and eschar formation

No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedem a formati on

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite raising) 2
Moderate oedema (raised approximately 1 mm) 3
Severe oedema (raised more than 1 mm and extending beyond area of exp.) 4

2.5.3. Classification of Test Item

Results will be presented and interpreted according to (I) Directive 2001/59/EC, (II) Regulation (EC) No 1272/2008 of the European Parliament and of the Council of

16 December 2008 on classification, labelling and packaging of subst ances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 and the (III) UN Globally Harmonised System of Classification and Labelling of Chemicals as follows:

(I) Directive 2001/59/EC:

R 35 Causes severe burns If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to three minutes exposure, or if this result can be predicted.

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## L A B R 34 Causes burns If, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs as a result of up to four hours exposure, or if this result can be predicted.

R38 Irritating to skin Substances and preparations which cause significant inflammation of the skin which persists for at least 24 hours after an exposure period of up to four hours determined

on the rabbit.

Inflammation of the skin is considered significant if:

- the mean value of the scores for either erythema and eschar formation or oedema formation, calculated over all the animals tested, is 2 or more: or

- in the case where the test has been completed using three animals, either erythema and eschar formation or oedema formation equivalent to a mean value of 2 or more calculated for each animal separately has been observed in two or more animals.

I n both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect will be used in calculating the respective mean values.

( II) EC Regulation + (III) GHS:

CATEGORY 1 CORROSIVE

A corrosive is a test material that produces destruction of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least 1 of 3 tested animals after exposure up to a 4 hour duration. Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of the observation at 14 days, by discoloration due to blanching of the skin, complete areas of alopecia and scars.

Corrosive subcategories Corrosive in > 1 of 3 animals

Exposure Observation

1A < 3 minutes ≤ 1 hour

1B > 3 minutes

- ≤ 1 hou r ≤ 14 days

1C > 1 hour

- ≤ 4 hours ≤ 14 days

CATEGORY 2 IRRITANT

1 Mean value of ≥ 2.3 - ≤ 4.0 for erythema/eschar formation or for oedema in at

lea st 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed from grades on 3 consecutive days after the onset of skin reactions; or

2 Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

3 In some cases where there is a pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal

but less than the criteria above.

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## L A B (III) GHS:

CATEGORY 3 MI LD IRRITANT

Mean value for erythema/eschar formation or oedema is greater than 1.5, but less than 2.3 in at least 2 of 3 tested animals from grades at 24, 48 and 72 hours after patch removal, or if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions (when not included in the irritant category above).

PRIMARY IRRITATION INDEX

Primary Irritation Index was calculated by added together the scores for erythema and oedema obtained at 24, 48 and 72 hours after completion of the 4 hours exposure for all animals (18 values) and divided by 9 (number of test sites x 3 scoring intervals).

This index was used to classify the test material according to the following scheme devised by Draize (1959):

Primary Irritation Index

Clas sification

0

- non

- irritant

> 0

– 2

- mild irritant

> 2

– 5

- moderate irritant

> 5

– 8

- severe irritant

2.5.4. Measurement of Body Weight

The 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. To ensure a minimum of animal distress, only a single animal will be exposed to the test chemical in the first instance. The treated site 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, but the animal will be washed to remove remaining material and the single animal will be observed as per study plan. If the effect is less than severe, exposure will continue to 4 hours in the single rabbit as per study plan. Two further rabbits will be treated as per study plan if the response in the single rabbit

is less than severe.

The bandaging and other study procedures are designed to minimise animal distress.

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## L A B 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 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 phases of the inspections will be included into the final report.

The results of all the study audits will be reported to the Study Director. The data presented in the final report wil l 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 hou rs for quality assurance purposes and also copy any study

- related original 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.

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## L A B 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 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.

- Inclusive dates of the test.

- A description of the animals: strain, health status, sex, source, identification, individual body weights, group size and animal husbandry.

- A complete and accurate description of the test procedures.

- Individual results of each animal at times of scoring.

- Description of any serious lesions observed.

- Na rrative 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 copy of the GLP-Certificate of the test facility.

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

The complete 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 will be offered to the Sponsor or retained for a further period if agreed by

a contract. Otherwise the materials will be discarded.

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## L A B 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

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 its content 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.

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## L A B 

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

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## L A B APPENDIX 1:

DETAILS OF MATERIALS TO BE USED FOR EUTHANASIA

Na me: CP -Ketamin 10 %

Batch No.: 10B18 3* Expiry Date: January 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

* Other lots may be used as required; to be documented in the raw data and study

report

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## L A B APPENDIX 2:

CONTENTS OF PURINA BASE - LAP GR. DIET FOR RABBITS

NU TRIENTS

Wet 12.10 % Crude p rotein 15.40 %

Crude fat 3.40 %

Crude fibre 16.00 %

Ash 7.60 %

MINERALS

Calcium 1.19 % Phosphorus 0.60 %

Sodium 0.35 % Cu (CuSO 4 *5H 2 O) 20.000 mg/kg

AMINO ACIDS

Lysine 0.60 %

Methionine 0.23 % Met.+Cyst. 0.25 %

VITAMINS

Vitamin A 13400 IU/kg

Vitam in D 3 < 1000 IU/kg

Vitamin E 71.8 mg/kg

These data are guaranteed values provided by the supplier.

These values can slightly differ in case of changing the Batch of the feed used under the duration of the study. Should any other Batch will be used, the details will be documented in draft report, but it will not be considered as a deviation to the Study

Plan.

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## L A B APPENDIX 3:

PROPOSED STUDY SCHEDULE

PRE-EXPERIMENTAL PERIOD

Relative date Absolute date

Animal arrival Day [

- 25 ] 29 April 2011

Veterinary c ontrol,

acclimation According to the applicable SOP.

Removing of hair Day [

- 1] 23 May 2011

TREATMENT PERIOD

Day of treatment Day [0] 24 May 2011

Animal skin examination and assignment to study Day 0 (pre

- treatment) 24 May 2011

Body weight measurement Day [0, terminating day] 24 May 2011, To be determined, based upon

the results of observation

Clinical observation Scoring at 1, 24 (±5 min), 48 and 72 hours (±60 min), then at 1 and 2 weeks after treatment if required (s ee relevant sections in the study plan)

End of in life phase Not later than Day [14] ( 07 June 2011) if appropriate

Draft Report Not later than 22 July 2011