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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STUDY TITLE : Acute Skin Irritation Study in Rabbits 

SPONSOR :

STUDY PERFORMED BY : LAB Research 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

 

RESPONSIBLE PERSONS : Judit Tavaszi M.Sc. – Assistant Scientist 

Animal House staff

 
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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 THE 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 SCORING .................................................................... 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. DEVIATION 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

APPENDICES .................................................................................................................... 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 OF ANALYSIS ..................................... 24

APPENDIX 4: COPY OF THE TEST ITEM DATA SHEET ........................................... 25

APPENDIX 5: COPY OF THE GLP CERTIFICATE ....................................................... 27

APPENDIX 6: COPY OF THE STUDY PLAN .............................................................. 1-19 
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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 irritancy of the test item was evaluated according to the Draize 

method (OECD No.: 404, 2002).

The test item was administered undiluted, at 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 the 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 of 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 hours after patch removal there 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. 

According to Directive 2001/59/EC, does not require classification as a 

skin irritant.

According to the UN Globally Harmonised System of Classification 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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2. INTRODUCTION

The objective of the study 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. Information derived from 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. MATERIALS AND METHODS

Day of Treatment: 24 May 2011 

End of Experiment: 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 

Expiry date: 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, all precautions required in the handling and 

disposal of the test item and the Test Item Data Sheet were supplied by the Sponsor. 

The identification of test item was made by its appearance and colour in the Central 

Dispensary Unit of LAB Research Ltd. on the basis of the information provided by 

Sponsor. 
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3.1.2. Other Materials 

For treatment:

Disposable Syringe (1ml)

Batch No.: 08H04 

Expiry Date: 08 2011 

Produced by: Penta Ferte

Sterilux

Batch No.: 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 by: 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 Date: February 2013 

Produced by: AST Beheer B.V. Oudewater Netherlands
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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 albino 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: Male

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 individual 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 

Relative humidity: 30-70% 

Housing/Enrichment: Rabbits were individually housed in AAALC approved 

metal wire rabbit cages (65x65 cm with height of 

45 cm). 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 parameters were recorded twice daily during the acclimatisation and 

experimental phases.
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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 consumption, 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 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). Copies of the relevant 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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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, 3] 24, 27 May 2011

Clinical observation 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 THE 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 control. 

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 condition were selected for this test. 

Approximately 24 hours prior to the test, the hair was clipped from the back and 

flanks of the animals. Removal of hair was performed in two steps. The 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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• 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.

An 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 

found 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 SCORING

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 Draize 

(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 were 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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(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. 

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 animals 

after an exposure of 4 hours. 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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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, 

with 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

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

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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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-Xylazin 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 applicable SOP’s.

After the retention time has elapsed, all the archived 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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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 Daily Examination

There were no treatment-related clinical signs noted. 

4.3.2. Examination of Skin-Irritancy

At observation one, 24, 48 and 72 hours after patch removal there 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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CONCLUSION

According to Directive 2001/59/EC, does not require classification as a 

skin irritant.

According to the UN Globally Harmonised System of Classification 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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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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MEAN VALUES OF SKIN IRRITATION SCORES

(24, 48, 72 hours reading) 

TABLE 3

Animal Number 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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APPENDICES
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APPENDIX 1:

SCORING SYSTEM OF ERYTHEMA AND OEDEMA FORMATION

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 

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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APPENDIX 2:

CONTENTS OF PURINA BASE - LAP GR. DIET FOR RABBITS

Batch number: 0530 049 003 0 0411

Expiry date: 10 July 2011 11 July 2011

NUTRIENTS

Wet 14.00% 11.20%

Crude protein 16.20% 16.20%

Crude fat 2.50% 2.70%

Crude fibre 16.20% 15.70%

Ash 7.80% 7.60%

MINERALS

Calcium 1.09% 1.10%

Phosphorus 0.55% 0.58%

Sodium 0.18% 0.18%

Cu(CuSo45H20) 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 D3 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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APPENDIX 3:

COPY OF THE CERTIFICATE OF ANALYSIS
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APPENDIX 4: 

COPY OF THE TEST ITEM DATA SHEET
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APPENDIX 4: 

COPY OF THE TEST ITEM DATA SHEET

(continued)
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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 PLAN

STUDY PLAN 

ACUTE SKIN IRRITATION STUDY 

IN RABBITS 

Date of Study Plan: 20 May 2011 STUDY CODE: 11/076-006N
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L A B 

GENERAL INFORMATION

SPONSOR : 

TITLE : Acute Skin Irritation Study in Rabbits

TEST ITEM :

STUDY PERFORMED BY : LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

HUNGARY

Phone: +36 88 545 -300

Fax: +36 88 545 -301
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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:

START OF EXPERIMENTi : 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 (24th 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. 
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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
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1. OBJECTIVE OF THE STUDY

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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2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: *

Chemical Name: 

Batch No.: 

Appearance: Colourless viscous liquid 

Purity: 97.4% 

Production date: 10 May 2010

Expiry date: 24 October 2012 

Storage: Room temperature (15-30°C) 

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

lab coat) for unknown materials will be applied to 

assure personnel health and safety. 

*pH 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:

Disposable 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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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 % 

Ventilation: 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 

AGRIBRANDS 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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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 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 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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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 or 

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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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 

Oedema formation

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 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:

(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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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. 

In 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 hour ≤ 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 

least 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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(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 

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

Classification

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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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 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 

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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6. STUDY REPORT 

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

will include but will not be limited to: 

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

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

personnel involved in the study. 

- The 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. 

- 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 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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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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A P P E N D I C E S 
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APPENDIX 1: 

DETAILS OF MATERIALS TO BE USED FOR EUTHANASIA 

Name: 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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APPENDIX 2: 

CONTENTS OF PURINA BASE - LAP GR. DIET FOR RABBITS 

NUTRIENTS

Wet 12.10 %

Crude protein 15.40 %

Crude fat 3.40 %

Crude fibre 16.00 %

Ash 7.60 %

MINERALS

Calcium 1.19 %

Phosphorus 0.60 %

Sodium 0.35 %

Cu (CuSO4*5H2O) 20.000 mg/kg

AMINO ACIDS

Lysine 0.60 %

Methionine 0.23 %

Met.+Cyst. 0.25 %

VITAMINS

Vitamin A 13400 IU/kg

Vitamin D3 <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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APPENDIX 3: 

PROPOSED STUDY SCHEDULE 

PRE-EXPERIMENTAL PERIOD 

Relative date Absolute date 

Animal arrival Day [-25] 29 April 2011

Veterinary control, 

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 (see 

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