- 

FINAL REPORT 

STUDY TITLE 

ACUTE DERMAL IRRITATION STUDY IN 

RABBITS 

STUDY No.: 

DATA REQUIREMENT 

U.S. EPA Health Effects Test Guidelines, OPPTS 870.2500 (1998) 

OECD Guidelines for the Testing of Chemicals, Test No 404 (2002) 

JMAFF 12-Nousan-8147 (2000) 

Official Journal of the European Communities, Methods for the Determination of 

Toxicity and Other Health Effects, Part B.4 (Skin Irritation), Council Regulation (EC) 

No. 440/2004 

Copy No. 2/2 

AUTHOR 

STUDY COMPLETED ON 

24 May 2011 

SPONSOR 

DOW EUROPE GMBH 

BACHTOBELSTRASSE 3 

CH-8810 HORGEN 

SWITZERLAND 

TEST FACILITY 

DEPARTMENT OF SAFETY ASSESSMENT 

ADVINUS THERAPEUTICS LIMITED 

POST BOX No. 58 13, PLOT Nos. 2 1 & 22 

PEENY A II PHASE, BANGALORE - 560 058 

INDIA 

Page 1146 
- 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS 

Compound: (

Study Title: Acute Dermal Irritati on Study in Rabbits 

Except as noted below, the stud y described in this report was conducted in compl iance 

with the fo llowing Good Laboratory Practice Standards: 

Organ isation fo r Economic Co-operation and Development (OECD) 

ENV/MCICHEM (98) 17 

European Economi c Community (EEC) 

Commission Directive 200411 OIEC 

February 20, 2004 

Japanese Min istry of Agri culture, Forestry, and Fisheri es (JMAFF) 

Nousan Notifi cation No. 6283 

Agri cultural Production Bureau 

I October 1999 

Exception to GLP compliance is as listed below: 

• Testing homogeneity, concentration and stability of test substance in the 

vehicl e. 

• Archiving ofraw data at the test fac ility. 

• Exception to guideline 404, fo r not testing weight of evidence analysis by 

the test fac il ity. 

:)A- I""() 20 I \ 

Date 

Study Director 

SKI N 

Copy No. 212 

Page 2/46 
- 

QUALITY ASSURANCE STATEMENT 

The Study No.: entitled " Acute Dermal Irritation Study in 

Rabbits" has been inspected in accordance the OECD Principles of Good Laboratory 

Practice (as revised in 1997) ENV/MCICHEM (98)17, OECD Paris, 1998, JMAFF GLP 

standards: II -Nousan-No.6283, I October 1999, and EC Directive 200411 O/EC, Official 

Journal of the European Union, L50/44, Feb.20,2004. 

This study was inspected and findings reported to Management and to the Study Director 

on the dates shown below: 

INSPECTION REPORTING 

DATE PHASE DATE 

I I February 20 I I 

23 February 20 II 

23 March 20 II 

and 

24 March 20 I I 

23 May 201 1 

Initiation Phase 

Review of study plan 

In-Life Phase 

Test substance application 

Reporting Phase 

Review of draft report 

Review of final report 

I I February 20 I I 

28 February 20 I I 

24 March 20 I I 

23 May 2011 

Inspections were performed according to the Standard Operating Procedures of the test 

faci lity's Quality Assurance Unit. The fina l report was inspected against the approved 

study plan and pertinent raw data and accurately reflects the raw data. 

Date: 2~, t()I\Y , )1)\\ 

Copy No. 2/2 

Head, GLP Section 

Qual ity Assurance Unit 

Advinus Therapeutics Limited, 

Bangalore 

Page 3/4 6 
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1. STUDY PERSONNEL 

The following personnel participated in the conduct of the study. 

Na me, Responsibility, 

Section / Department 

Copy No. 2/2 

Function Signature with date 

Overall in-charge for the 

conduct of the study and 

report preparation 

Assistance in conduct of 

study and report 

preparation \

Veterinary services 

Assistance in the conduct 

of study 

Data entry and report 

compi lation 

Page 4/46 
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TABLE OF CONTENTS 

STUDY TITLE .............. .. ...................... ......... ..................................................................... 1 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ............... ... 2 

QUALITY ASSURANCE STATEMENT ... .. ............................ ................. ........... ............. 3 

I. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

8. 1 

8. 1. 1 

8.1.2 

8.2 

8.2. 1 

8.2.1. 1 

8.2.1.2 

8.2. 1.3 

8.2.1.4 

8.2. 1.5 

8.2. 1.6 

8.3 

8.3. I 

8.3.2 

8.3.3 

8.3.4 

STUDY PERSONNEL ......................... ................. .................. ........................... 4 

SUMMARY .............................................. .......... ........... .......... ........................... 7 

STUDY DETAILS ......................... .. ................... ........ ..... .. ......... .. ...................... 8 

OBJECTIVES .. ... ...... ........................ ...... .. .... ...................................................... 9 

JUSTIFICATION FOR SELECTION OF TEST SYSTEM .............................. 9 

JUSTIFICATION OF ROUTE OF ADMfNlSTRA TION ................................. 9 

TEST PROCEDURE GUIDELfNES .................................................. ........ .. ..... 9 

MATERIALS AND METHODS ...................................................................... I 0 

Materials ..................................................................... ... .............................. ..... 10 

Test substance Information ........ ...... ............... .................................................. 10 

Test System .............................. .. ......................... ........ .. .. ....... .. .. .. ........ ............. I I 

Methods ..... ........................................ .............................................................. .. 12 

Performance of the Test .............................................................. ...................... 12 

Husbandry ........ ....................................... ...... ... .. ................ .................... ........... 12 

Sample preparation ....... .. ...................................................... ........... ... .............. 13 

Preparation of Test Animals ...... ....................................................................... 13 

Testing Option .. ............................. ..... ... ... .. .... ................ .................................. 13 

Test substance Application ........ ...................................................................... . 13 

Exposure of An ima ls ............................. ........... .............................. ................... 13 

Observations ..................................... ..................... ... .. .......... ................. ........... I 3 

Duration of Observation ................................................................................... 13 

Toxic Signs and Pre-Terminal Deaths .............. .. .... .................... ....... ............... 14 

Body Weights ..... .... ...... ........................ .. ..................... ...................................... 14 

Skin Scorin g ................................................ ....... ............... ....... .. ....................... 14 

Copy No. 212 

Page 5/46 
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8.3.5 Grading of skin reactions .................................................................. ................ 14 

9. RESULTS ........................................... ........................................ ... ................... 15 

9. 1 Toxic signs and pre-terminal deaths ..................................... ............ .. .............. 15 

9.2 Body weights ................. ... .... .. ...................................................................... .... 15 

9.3 Skin reactions ............................................. .................................... ... ... ....... ...... 15 

10. CONCLUSION ...................................... .......................................... ................. 15 

II. ARCHIVING .......................... .. .... .................................................................... 15 

12. REPORT DISTRIBUTION .................................. ............................................ 16 

13. TABLES .......... .. ......... .... .. ......... ....................................................................... 17 

TABLE I. Body Weight and Pre-terminal Deaths ....................... ... .. .. .......... 18 

TABLE 2. Skin Reaction Scores ......... .......................................................... 19 

TABLE 3. Mean Skin Reaction Scores ................................ ....... .................. 20 

TABLE 4. Individual Toxic Signs ... ..... ................................. .... .. ................. 2 1 

14. APPEND ICES .................. ............. ....................................... .. ......................... . 22 

APPENDIX I. Certificate of Analysis .................................................. .. ... ..... 23 

APPENDIX 2. Study Plan ............................... .. ...... ............... ....... ....... .. ......... 24 

15. ANNEXURES ......................................................................... .......... ............... 40 

ANNEXURE I.Analysis Report - Anima l Diet Sample .............. .................. .41 

ANNEXURE 2. Analysis Report - Water Sample .. .. ............ ........................... .42 

ANNEXURE 3. GLP Certificate - Germany .................. .. .. ............................ . .43 

ANNEXURE 4. GLP Certificate - The Netherlands ... ............... ...... ......... ...... .44 

ANNEXURE 5. GLP Certificate - India ................................... .. ..................... 46 

Copy No. 212 

Page 6/46 
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2. SUMMARY 

The acute dermal irritation study in Rabbits was conducted to evaluate the skin 

irritation potential of

Five hundred mg (O.5g) of the test substance was made into a paste with 

sufficient volume of water and mixed with a glass rod and was aPflied to the 

dose site and covered with a gauze pad of size approximately 6 cm (2 x 3 cm6ply). The patches were secured to the body of the animal by a non-irritating 

semi-occlusive adhesive tape. After 4 hours contact period the bandage was 

removed and the dose sites were wiped with water. 

The degree of irritation was evaluated and scored by Draize's evaluation 

method at I, 24, 48 and 72 hours post removal of the test patch . The skin 

reactions are presented in the table below. 

There were no toxic signs, pre-terminal deaths and no skin reactions observed. 

The mean skin irritation scores were as follows: 

Copy No. 2/2 

Rabbit 

No. 

RB8764 

RB8765 

RB8766 

Sex 

M 

M 

M 

Treatment 

Period 

(hours) 

4 

4 

4 

Elapsed time Erythema (post removal of 

the test patch) score 

J hour 0 

24 hours 0 

48 hours 0 

72 hours 0 

I hour 0 

24 hours 0 

48 hours 0 

72 hours 0 

1 hour 0 

24 hours 0 

48 hours 0 

72 hours 0 

Edema Total 

Score score 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

0 0 

Page 7/46 
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3. STUDY DETAILS 

Study Title 

Test Substance 

Study No. 

Sponsor 

Monitoring Scientist 

Test Facility 

Study Initiation Date 

Experimental Startin g Date 

Accl imati zation 

Treatment 

Observations 

Experimental Comp letion Date 

Draft Report 

Study Completion Date 

Copy No. 212 

Acute Dermal 

Irritation study in Rabbits 

(

Dow Europe GmbH 

Bachtobelstrasse 3 

CH-8810 Horgen 

Switzerl and 

Donna Anderson 

The Dow Chemical Company 

Toxicology & Environmental Research & 

Consulting (TERC) 

Building 5 

727 Norristown Road, P.O. Box 904 

Spring House, PA 19477-0904 

Ema il: DonnaAnderson@dow.com 

Department of Safety Assessment 

Advinus Therapeutics Limited 

Post Box No. 5813, Plot Nos. 21 & 22 

Peenya II Phase, Bangalore 560 058, 

INDIA 

18 February 20 II 

18 February 20 II 

Start: 18 February 20 II 

End : 22 February 2011 (single anima l) 

and 23 February 20 II (additional two 

animals) 

Start: 23 February 20 II (single an imal) 

24 February 20 II (additional animals) 

Start: 23 February 20 II 

End: 27 February 20 II 

27 February 20 II 

30 March 20 II 

24 May 20 11 

Page 8/46 
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4. OBJECTIVES 

The objective of this acute dermal irritation study was to provide information 

on the potential for skin corrosivity / irritation to arise from a single topical 

exposure to the test substance. 

5. JUSTIFICATION FOR SELECTION OF TEST SYSTEM 

The rabbit is the system of choice because, historically, it has been the 

preferred and most commonly used species for primary skin irritation tests. 

6. JUSTIFICATION OF ROUTE OF ADMINISTRATION 

Topical appli cation was used because it is recommended in the referenced 

guidelines, and because human exposure may occur via this route. 

7. TEST PROCEDURE GUIDELINES 

The procedures as described in this study plan are based on the following 

testing guidelines: 

• OECD Gu ideline for Testing of Chemicals, Section 4, Number 404. "Acute 

Dermal Irritation / corrosion," adopted: 24th Apri l 2002. 

»> Exception to guideline 404, for not testing weight of evidence analysis 

by the test facility. 

• U.S. EPA Health Effects Test Guidelines, OPPTS 870.2500, Acute Dermal 

Irritation, August 1998 . 

• JMAFF 12-Nousan-8147, November 2000. 

• Official Journal of the European Communities, Methods for the 

Determination of Toxicity, Part B.4 (Skin Irritation), Directive 

2004/73/EC, 29 April 2004. 

Copy No. 212 

Page 9/46 
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8. MA TERIALS AND METHODS 

8.1 Materials 

8.1.1 Test substance Information 

(As furnished by the Sponsor) 

Test substance / Item 

Lot No. 

Manufactured by 

(Name and Address) 

Supplied by 

(Name and Address) 

Date of manufacture 

Date of expiry 

Purity as per 

Certificate of 

Analysis 

Physical appearance 

Storage conditions 

(

The Dow Chemical Company 

Midland, MI 48674 USA 

The Dow Chemical Company 

Mid land, MI 48674 USA 

Not Applicable 

06 March 2012 

Off-white solid 

Ambient (+ 18 to +36°C) 

The test substance, identified as was received on 03 

February 20 II and was further identified as 008-26 by test facility, Reference 

Number dated 07 February 20 I I. The test substance is a solid and is stored at 

ambient condition (+ 18 to + 36°C). Documentation of the methods of synthesis, 

fabrication, or derivation of the test substance is retained by the Sponsor. 

Determination of the stability, identity, strength, purity and composition of the 

test substance is the responsibi lity of the study Sponsor. The fo llowing 

information related to the characterization of the test substance was provided 

by the sponsor. 

Copy No. 2/2 

Pa ge 10/46 
- 

Information as provided by Sponsor are as fo llows: 

pH: 6.47 (Analysed in-house). 

Specifi c gravity / density: Not Applicable. Test substance is a solid 

8.1.2 Test System 

Animals 

Source 

Number and sex 

Age at treatment 

Date of birth 

Body weight range 

at treatment 

Identification 

Rabbit accession 

numbers 

Accl imatization 

Copy No. 2/2 

New Zealand White rabbits 

Inhouse Bred (Outbred) 

New Zealand White rabbits 

Toxicology 

Department of Safety Assessment 

Advinus Therapeutics Limited 

Bangalore 560 058, INDIA 

3 Ma le rabbits 

6-7 months 

30 July 20 10 

2.77 to 2.94 kg 

The an imals were ind ividual ly identifi ed by 

animal accession number, cage labe l and 

turmeric colour body marking. 

RB8764 to RB8766 

One rabbit (RB8764) was acclimatized for five 

days while the other two (RB8765 and RB8766) 

rabbits were accl imatized for six days under 

laboratory conditions after physical 

examination. 

Page 11146 
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8.2 Methods 

8.2.1 Performance of the Test 

8.2.1.1 Husbandry 

Conditions 

Animals were housed under standard laboratory conditions, air conditioned 

with adequate fresh air supply (12-15 air changes/hour). Environment: 

temperature 21 - 23°C, relative humidity 65 - 67%, with a 12 hour light and 12 

hour dark cycle. The maximum and minimum temperature and relative 

humidity in the experimental room were recorded once daily. The relative 

humidity in the experimental room was calculated from dry and wet bulb 

temperature recordings. 

Housing 

The rabbits were housed individually in rabbit cages (approx. size: L 65 x B 

65 x H 45 cm) with Noryl shallow cage body and facilities for pelleted food 

(Stainless steel feed hopper) and drinking water (750 mL markrolon bottle 

fitted with sipper tube). The litter collection tray (noryl waste tray) were 

washed daily with water (except on Sundays). Water was replenished daily. 

The water bottles and feed hoppers were changed once a week. 

Diet: ad libitum 

The animals were offered rabbit feed manufactured by Pranav agro Industries 

Ltd, Sangli. The diet ana lysis report is enclosed as Annexure I. 

Specific contaminants (heavy metals organochlorines and organophosphates) 

in the diet are ana lysed periodically and the reports are included in the raw data 

file. 

Water: ad libitum 

The animals were provided deep bore-well water passed through activated 

charcoal filter and exposed to UV rays in Aquaguard on-l ine water filter-cumpurifier manufactured by Eureka Forbes Ltd. , Mumbai - 400 00 I, Indi a. The 

water bottles were replen ished once dai Iy and the water bottles changed once a 

week. The water analysis report is enclosed as Annexure 2. 

Copy No. 2/2 

Page 12/46 
- 

8.2.1.2 

8.2.1.3 

8.2.1.4 

8.2.1.5 

8.2.1.6 

Sample preparation 

A quantity of 0.5 g of test substance as received was made into paste by adding 

sufficient volume of water and mixed with a glass rod. 

Preparation of Test Animals 

Approximately 24 hours prior to treatment, the fur on the left side of the 

dorsolateral trunk surface [about 14 x 19 cm] was clipped with an electric 

clipper -Aesculap® - Germany. Care was taken to avoid abrading the skin. 

After clipping and prior to application, the animal 's skin (test site) was 

checked for any abnormalities according to the "Primary Skin Irritation 

Scoring System", and the test site was found to be normal. 

Testing Option 

The study was initiated in a stepwise procedure ( I +2 Rabbits). 

Test substance Application 

Preparation of Dose Site 

Approximately 6 cm

2 intact test site was selected on each animal. The adjacent 

untreated skin of each animal served as the control site. 

Application of Test Substance 

A quantity of 0.5 g of test substance as received was made into paste by adding 

sufficient volume of water and mixed with a glass rod. The test substance was 

applied directly to the skin of the test site and covered with a gauze pad. The 

entire trunk of each animal was then wrapped with non-irritating semiocclusive adhesive tape to avoid dislocation of the patch. 

Exposure of Animals 

In a stepwise manner (1 +2 rabbits) all the three rabbits were exposed to the test 

substance each with one test patch only for a four hour exposure period. 

The rabbits were restrained using Eli zabethan collar for 72 hours post 

application of the test patch. 

8.3 Observations 

8.3.1 Duration of Observation 

The rabbits were observed up to 72 hours post removal of the test patch. 

Copy No. 212 

Page 13/46 
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8.3.2 Toxic Signs and Pre-Terminal Deaths 

Rabbits were observed for toxic signs and pre-terminal deaths for the first four 

hours (at hourl y intervals) on the day of test substance application and once 

daily thereafter ti ll the end of the observation period. 

8.3.3 Body Weights 

Individual body weights of the rabbits were recorded at the start of 

acclimatization, day I of treatment Gust before test substance application) and 

at the termination of observation. 

8.3.4 Skin Scoring 

Following the exposure period, test patches were removed and the application 

sites were washed with water and clean towels to remove any residual test 

substance. The 4 hour dose sites were examined for signs of erythema and 

edema within 60 minutes and at intervals of approximately 24, 48 and 72 hours 

after test patch removal accord ing to the "Primary Skin Irritation Scoring 

System" developed by Draize. 

Individual skin scores were recorded for each rabbit. 

8.3.5 Grading of skin reactions 

The skin reaction was assessed according to the numerical scoring system of 

Draize et.a l (1944). 

1. 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 SCORE - 4 

2. EDEMA FORMATION 

No edema 0 

Very sl ight edema (barely perceptible) .......................................... 1 

Slight edema (edges of area well defined by definite raising) ......... 2 

Moderate edema (raised approximately 1 millimetre)..................... 3 

Severe edema (raised more than 1 mill imetre and extending 

beyond area of expos ure)............................................................... 4 

MAXIMUM POSSIBLE SCORE - 4 

Copy No. 2/2 

Page 14/46 
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9. RESULTS 

The toxic signs, body weight and pre-terminal deaths are presented in Tables I 

and 4. The skin reaction scores are shown in Table 2 and mean skin reactions 

are presented in Table 3. 

9.1 Toxic signs and pre-terminal deaths 

Refer to Table I and Table 4 

There were no toxic signs and pre-terminal deaths during the course of the 

experiment. 

9.2 Body weights 

Refer to Table I 

The body we ights of all the rabbits increased through the observation period. 

9.3 Skin reactions 

Refer to Table 2 and Table 3 

There were no skin reactions observed III any of the rabbits through the 

observation period. 

10. CONCLUSION 

Under the testing cond itions of this study, the test substance 

did not show any evidence of dermal irritation. 

11. ARCHIVING 

The original signed final report wi ll be sent to the Sponsor and one signed final 

report wi ll be retained by Advinus. The original raw data, documentation, 

records, the original study plan, and final report generated as a result of this 

study will be sent to the Sponsor for retention fo llowing signing of the final 

report. Copies of the same (authorized by the Study Director) wi ll be retained 

by Advinus. Advinus wi ll maintain these records for a period of at least five 

years. After this time, the Sponsor of the study will be offered the opportunity 

to take possession of the records or will be charged an arch iving fee for 

continued archiving by Advinus or to dispose of the raw data in consultation 

with the Sponsor. A representative sample of approx imately one gram of the 

test substance was sent from the test substance stores to the archives in the test 

fac ility before the first dispensing of test substance to studies. This sample 

Copy No. 212 

Page 15/46 
- 

shall be stored for a period of 2 years from the date of this final report or ti ll 

next GLP inspection, whichever is later, however not beyond 5 years. 

In the event of any inspection by regulatory authorities, seeking original raw 

data for verification, the sponsor would be responsible either to send the 

original raw data to Advinus Therapeutics Limited or to the regulatory 

agenc ies for verification. 

12. REPORT DISTRIBUTION 

Copy No. 212 

The final report (original copies) wi ll be distributed as follows: 

Test Facility Archives: One signed final report in original (Copy No. 1/2). 

Sponsor: One signed final report in original (Copy No.2/2) and one Soft copy 

of the report in PDF format. 

Page 16/4 6 
- 

Copy No. 212 

13. TABLES 

Page 17/46 
- 

TABLE 1. Body Weight and Pre-terminal Deaths 

Body weight (kg) 

Rabbit At Start of At treatment At 

No. Sex Acc li matization (Day I) Termination 

RB8764 M 2.54 2.77 2.91 

RB8765 M 2.71 2.81 2.95 

RB8766 M 2.89 2.94 3.14 

M: Male, Note: There were no pre-terminal deaths. 

SKJN Page 18/46 

Copy No. 212 
- 

TABLE 2. Skin Reaction Scores 

Rabbit Treatment 

Sex period No. (hours) 

RB8764 M 4 

RB8765 M 4 

RB8766 M 4 

M:Male; 

0: No ErythemaINo Edema 

C opy No. 2/2 

Observation time 

(post remova l of 

the test patch) 

I hour 

24 hours 

48 hours 

72 hours 

I hour 

24 hours 

48 hours 

72 hours 

I hour 

24 hours 

48 hours 

72 hours 

Erythema Edema Tota l 

score Score score 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

0 0 0 

Page 19/46 
- 

TABLE 3. Mean Skin Reaction Scores 

Observation 

(post removal of Erythema Score Edema Score Tota l Mean Score 

the test patch) 

I hour 0 0 0 

24 hours 0 0 0 

48 hours 0 0 0 

72 hOllrs 0 0 0 

Page 20/46 

Copy No. 212 
- 

TABLE 4. Individual Toxic Signs 

Rabbit Day of Observation 

No. Sex Day I 2 3 

I hour 2 hours 3 hours 4 hours 24 hours 

RB8764 M N N N N N N N 

RB8765 M N N N N N N N 

RB8766 M N N N N N N N 

M: Male; N: Norma l 

Page 21/46 

Copy No. 2/2 
- 

·SKlN 

Copy No. 2/2 

14. APPENDICES 

Page 22/46 
- 

APPENDIX 1. Certificate of Analysis 

Page 3 of 19 

2oft8 

CERTIFlCATE OF ANALXSlS FOR TESTIREFERENCElCQNTROL SUBSTANCES 

TlTLE/OBJ£CTIVE: 

TF.ST/R[FERENCEICONTROL SUBSTANCE: 

DESCRIPTION: 

STRUCTURE: CAS No., "'"' 

MOLECULAR WEIGITT: 

STUDYTNTTIATION DATE: 15June2010 

METHODS USeD: 

PURITY: Gas chrontltography with name ioniutiOIl dettclion {(;C.FID) IUld Karl Fischer lilr.llion. 

IDENTIFICATION; Fourier Transfonn-Inframl (FT.IR) spectroscopy IJId gas chromatography.mass s~ctromctry 

(GC-MS) 

R£SULTS AND CONCLUSIONS: 

Structural confirmation ofLhc t~ 1 malerioJ was performed byFT.IR and GC·MS. 

x PURITY DETERMINATION 

Result: oonfidcooc level) after correcTing for the Karl Fischer witer esurL~. 

OTHER (Specify); 655 ppm water by Karl Fischer titration. No impurities were detected lit ~ O. J % by 

GC-FID analysis. 

CALCULATIONS: 

Arc. Percent: x (GC-FID) Internal SlandMd: N/A External StandMd; N/A 

Odler (e.'plain): Karl Fischer "trnllon 

AJI originall1lw data associated with thi, study were archived in the Sponsor facility archlv!:. 

Only desCliptive statistics were used unless otherwise noted in tile results. This study wu cunducted in accordAnce wilh the 

Good Laboratory Practice Standud, 40 CFR Part 792 unless ollierwist' nuted. Due to the shurt letm nature ofthc: study, the 

stnbilily of the lest substance under storAge conditions pcr 40 CFR Pnrt 792.1 05(e) lit Ihe lest site was 1I0t verified. 

Completion Date: 

7j"",ba ro 

_____ u.<kl. ;Lo.JQ_' __ _ 

jl",rSTUDY DIRECTOR AND TESTING J;ACILTTY ADDRESS: 

Impact Analytical, 1910 W. 51. Andrews Rd. Midland, Mr 4&640 

THIS IS A TRUE COpy OF 

THE ORrG1NA~ D,~ .. :·:.!\ 

CAe 12-2.3-- 10 

SK IN 

Copy No, 2/2 

Page 23/46 
- 

APPENDIX 2. Study Plan 

STUDY PLAN 

STU DY PLAN No. : SPTEST ITEM CODE I3Y 

TEST fACI LITY : 1)08-2 6 

STUDY CODE : SKIN 

ACUTE DEHMAL IRRITATION 

STUDY I RABmTS 

C op)' No. I I I 

SKJN 

Copy No. 2/2 

TEST ITEM: 

STUDY No.: 

SPONSOR 

Dow Europe GmbH 

Bachtobelstrasse 3 

e lI-88t 0 Horgen 

Switzerland 

TEST FACILITY 

DEI'AETM EN T OF SAFETY ASSESSMENT 

ADVINUS THERAPEUTICS LIMn-ED 

POST BOX No. 5813. PLOT Nos. 2 1 & 22 

PEENY A II PH.ASE, BANGALORE - 560 058 

I NDIA 

Page 1/16 

Page 24/46 
- 

APPENDIX 2 contd. Study plan 

CONTE TS 

I. STU DY D ETAI LS ........ " " ..... "". ". ".". " ... ,, " .,.". . . .• " .... " ...• " .,, " . "",4 

2. Q UALI TY ASSURANCE " .. . " ."." "". "" " ."" ....... " " ..... .. " " .......... " ",,5 

J . GJ.P COMPLTA NCE " .. . "" .... ". """""" "" " ". " .. ..... " .... .. " ".,,"" .... ". " ,,5 

4 . TEST PROCEDU RE G UIDE LI NES """.""""" ...• ,, . " " .. ""." " " .. """,,0 

5. AM EN D lENT PROCEDUR ES."."" ."" .... " ." " " .... "" .. ,, .• "".,,""" " " ,,0 

(, . SA r:ETY PREC A UTI O NS .". ""." " " " ". " ." ....•• "" ... ..•• ". " ..... . "",, ... "".6 

7. OBJECTI VE ".""". " """ .. ,, .,, . "" .. ""."" .. """."."""."",, .. ,, .,,"" " " " """ 7 

8. J USTIFICATION FOR S ELECTION OF TES T SYSTEM " " " .... ".".7 

9. JUST'IFICATION FOR RO UT E OF ADMIN ISTRATION" " ." .... ". " .7 

10. MA T ERTAT.S AN D M ETHODS. """ " """" " " " " .. " .. . " "". " ". " "" .. " .. ,,7 

10 .1 Materia ls.". " . .. " ".""" .. .. . "",,. " .. "" .. ,," " ". " ...... """ ...... .. " ...... " .. "" ,, . " .. 7 

1 0. 1.1 

10.1. 2 

10.2 

10 .2. 1 

10.2.2 

10.2.2. I 

10 .2 .2.2 

10 .2.2.3 

10. 2 .2.4 

10.2.2.5 

10.3 

10 .3. 1 

10 .3.2 

10.3.3 

10.3 .4 

I I. 

1 1. I 

12. 

T est ~(cl1l ". " ."" .. """ ...... " """ ." .. " .. ,, " ... "" " " """ ..... """ .. "".".7 

Test S ubstance Info rma tion ... ...... .... ..... .. .... ... ... ............. ,., .... ............ ... .. 8 

Methods ... .... " .. .... ..... .. ...... ... ...... ....•....... " ..... " ..... ........ ...... .. ... ....... .... .. ... 9 

Husbandry .... " . " .. " ..... " " ........... .. " ." "" " ." .... " " " .. " "" ...... " ".". ".9 

l")e rfo rtn ance o f te st ... .. ... _ ...... .. ...... ............... ... .... , ...... ... ...•... ........ ....... 10 

Sampl e Pre pa ratio n " """ " .",," .... " ..... " " "" " ... " " ... ". "." ..... ",," 1 ° 

Prep£tratio l1 o f test an imals ... .. .......... ... .... ........... .... ........ .. ...... ........... ... 10 

T esting o pt io n ................ ... ............... ...... ....... ........ .... 10 

Te sl substance applicatio n " ...... " ... " .... " .... ... " ..... .... " . " " " .. "" ... " " ..... 10 

Ex posu re or anima ls ... .. , ........ " ... .... ...... ... .. ... .. .. ... .. ..... ... .. ....... ..... 1 ] 

O bserva ti o ns " ".""" ".".",, ..... " " " ....... ". "."" "." .... .. " ... " ,, " ,, . "." .,, " ,, " I J 

C linical Ohservations .. ... ... ...... ... .. ... .. .••....•. ..... ... ... ..... ..... .... ...••... ...... .. . 1 ] 

Body we ig hts ." .. " ." .. . "" ... .... .. .. "."". " ".""." "" ..... ...... ... " .. " .... ,, ." I I 

Skill scoring .. ". ". "".". " .... " "" .. " " ... " ..... " " . " ..... "" " .. " .. " .... " ... " I I 

Grading or skin reac ti o n s ... .. ... ... .. ................. ...... .. .......... .............. .... .. 12 

R ESULTS " ... ".". " .. """ " .. "" .. .. ..... " " " " ". ,,"" '"'' ' ' ." .. " " ...... ". " " .. " " . 12 

/);\ '1'1\ C OMPTl .l\ T IO N .. " ...... " ... " ..... .. .. " .. ". " ." .. ". "" .. " .... " .. " .. " ... 12 

REPORTING ... " .. "" .... . " ... ,," .. . " .. ".". " ....... .. " ". " . " .. " .,, " " ... " """. "" 13 

SP-G7693_SI<IN 

Copy No. JII 

Pagl' 21 16 

Copy No. 2/2 

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APPENDIX 2 eoutd. Study piau 

13. ARCH IVING ... .......... ... ... .. .................................... .. 14 

14. STUD Y PLAN D ls TRIBlr nON .. ............... ............................ .. ....... 14 

15. REPO RT DISTRIB UTION .... ................. ............... ........ . .............. 15 

16. REFERENCES .. ............. .. ..... ... ....... .... ............. ................................. . 15 

17. AG REEMENT .................. ........ ......... ........................ ............. .... .. ..... . 16 

S PCOllY No . III 

Copy No. 2/2 

Page 3 / 1 (; 

Page 26/46 
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APPENDIX 2 contd. Study plan 

I. STUDY DETAILS 

Study T itle 

Test substance 

Study -Number 

Test Fac ility 

Sponsor 

l\1oni(oring Scientist 

Study Personne l 

Study DirccLOr 

Technical Co-ordinator 

Clinical Veterinarian 

SI uely Schedul e 

S tudy initiati o n date 

Experimenta l starting dute 

SP· SKtN 

Co py No. III 

·SKIN 

Copy No. 2/2 

Acute Dennni Irritat ion 

study in Rabbits 

(

Department o f Safety J\ssessment 

Advinlls T'herapeulics Limited 

Pas I Box 1 o. 58 13. Pl o t Nos. 21 & 22 

Pcenyn 1I Phas ~ Bangalorc 560 058, 

IND IA 

Dow Europe GmbH 

Bachtobclstrassc 3 

Cl 1-88 1 0 Borgen 

Switzerlcll1d 

Donna Anderson 

The Dow Chemical Company 

Toxicology & Environmenta l Research & 

Consulting (TERC) 

[3ui lding 5 

727 orristown Road. 1'.0. B ox 904 

Spring House, PA 19477-0904 

Email : DOl1r}(lAnder~on dow coll'l 

1'. J. Prakash, M.Se. 

S. B. Moh an Kumar. !" I.Sc. 

S. Rlllllesh , B.V.Se. & A.H. 

J 8 February 2011 

18 February 201 J 

PlI gl' 4/ 1 (i 

Page 27/46 
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APPENDIX 2 contd. Study plan 

Accl imatization 

Treatment 

Observations 

Experimental cOl llplction 

date 

Draft repo rt 

S tudy completion datc 

Start: 18 February 20 II 

End : 22 February 20 11 (single an imal) 

and 23 February 2011 (add iti onal two 

animalJs) * 

Start: 23 February 20 11 (si ngle animal) 

24 February 2011 (additional anima]!s)" 

Start: 23 February 20 I I 

Lat est by 15 March 20 II 

Latest by 31 March 20 I I 

Latest by JUlle 20 I I 

• . \Vill be included iflhc test substflnce proves to be non·corrosive and i or not 

a severe irritant depending on the single animal test obscrvaiiol1s. The 

confirmatory t~ l Illay be conducted in a seql1cntial manner using one 

animal p l.! 1' step or using l\.v(l additional animals. The details or 

confirmato ry test wiJl be included in the report. 

2. QUALITY ASSUUANCF. 

Tile Quality I\!=isurance U ni t o f Advinlls Therape llti cs Li mited. 

Dangalore - 560 058, Ind ia, w ill inspect the study, the r~l\:v dnta. the 

draft "nd fina l report. Findings of all inspections wi ll be reported to 

the ~1ana gc l11enl and to the Study Directo r. rnspecti ol1 dates and 

reporting dates wi ll be entered in the study re port. The Quality 

Assurance Unit has reviewed the final study plan and will receive a 

copy thereor. 

3. GLP COMPLIANCE 

The study w ill be perfo rmed in accordance with the Jollowing GLP 

rcguiatie)Jls: 

• OEe!) Pri nc iples ofGLP (as revised in 1997), ENVIMCICHEM 

(98) 17, OEeD, Pmis, 1998. 

• JMAFF GLP Standards fo r Agri cultura l Chemi ca ls: Il-NollsanNo. 6283, I October. 1999. 

• Ee Directive 20041101EC , Official Journal o f the Eu ropean 

Union, LSOI44, Feb, 20. 2004. 

• This study will also be c onducted as per the Illutually "greed 

study plan and the Standard Operating Procedures. 

SPCopy Nu. 1/ 1 

Pa,-!c 511" 

Copy No. 2/2 

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APPENDIX 2 coutd. Study plan 

EXt..:CpliollS (0 the G L P complia nce a rc Iistt:!d below: 

• Testing homogeneity. concc ntra t io n and slabi I it y of lest 

su bsta nce in the vehicle. 

• Archi vi n g of raw d ata at th e test fac ility . 

• Exceptio n to g u id e line 404, for n.ot testing weig ht of cvidcm.: c 

a na lysis by the test fac il ity. 

4. TEST PROC E D U R E GUI!)E LINES 

T he p rocedures as described in this study plan arc based o n the 

iollowing testing guide lines: 

• DECO G uideline rar Testi ng o f C hem icals, Sectio n 4 , N umber 

404. '"A.cute De rma l Irritati o n I corrof;ion," ad opted: 241h Apri l 

2002. 

• U.S. E PA H ea lth Effects Test Guide li nes, O PPTS g70.2500. 

ACLIte D eJ'm a llrritation, A ugust 1998. 

• JM AI'!' 12- o llsa n -S 147, November 2000. 

• O ffic ia l Jo urnal of the European Con11l1unitics Methods fo r 1h<..' 

D ctermination of Tuxic ity, Pa rt 13.4 (Sk in Irritatio n). Directive 

2004173!EC. 29 April 2004. 

5. A MENDMENT PROCEDURES 

A ll am~ ndll1 ls to uncllor cvifl ti ~ from th is study plan and the 

reasons the refore sha ll be documented, dated. sig ncd by the S tudy 

D irecto r, ap proved by management (as may be required) a nd 

mai ntai ned ,",vitb the sludy pla n . If lmme di ate action is ncccssnry, 

ve rba l agreem e nt \,,'ith lhe sponsor w il l b e con firmed ;:1$ soon a s 

possible by study plan amendme n t. Any Hlll encilllcnts to a nd/o r 

d evia ti o ns fro ln this study plan w ill be recorded in the raw d ata and 

docum e nted in t he fina l report. 

6. S AFETY J>HEC A UTIO NS 

Glove s, cap. nICe InHsk a nd goggles ( if re qui red ) w ill be used in 

add ition to pro tec tive body gn nnc nts a nd shoes to e nsure adeq uate 

pe rsona l he a lt h and safety a nd 1.0 avo id inha la tio n alld sk ill contact 

'with thc test s ubstance. In case of eye contne l, the l..'yc wi ll he 

\vashed thoro ug hl y ,vi th \vate r and me d ica l trc a tnH!llt \' ill be sought. 

111 case o f ski n contact, it ,:viI] be \:vashed \vith soap and wa tcr w ith 

s u bsequcnt Incdical a id. 

S P

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APPENDIX 2 contd. Study plan 

7. OB.JECTIVE 

The object.i ve of thi s acute dermal irritation study is to provide 

information on the potential for sk in corrosivity I irritation ari sing 

frol11 a si ng le topi cal exposure to the tcst substance. 

8. .JUSTIFICATION FOR SELECTION OF TEST SYSTEM 

The rabbit is the sySfcm of cho ice because ~ historicall y, it has been 

the preferred and lnost conllnon ly lIsed spec ie s for primary ~ki n 

irritation tests. 

9. JUSTIFICATION FOR ROUTE OF ADMINISTRATION 

to. 

10.1 

11l.l.! 

Topical application w ill be used because it is recommended it< the 

referenced guideli nes, and because h tl111an exposure 111ay occur via 

this route. 

MATERIALS AND METHOUS 

1\1atcrials 

'l 'cst SYS( CIn 

Anin1als 

Source 

No. of rabbits 

B ody \",eight range al 

treatlnent 

A ge at tn!atmenl 

ldentitication 

New Zealand White Rabbits 

Inho use Bred (Out b red) 

N e w Zealand White Rabbits 

Toxicology 

Department of Safety AsscssIl1 l.!nl 

AdvillUS T herap eutic s Limited 

Ba n ga lo re 560 058, IND IA 

U p to three m ak 

2.2 to 3.2 kg 

Young adult (the exact age w ill be 

menti o ned in thc report) 

Uy rabbit accession number. 

Idcntifi<.:alioll o r indi v idual nninutl is by 

cage label. turmeric colollr body 

111m"king. 

S I'· S I(J N 

C opy N o . t I l 

Pag e 7116 

Copy No. 212 

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APPENDIX 2 contd. Study plan 

10. 1.2 

Rabbit accession 

numbe rs 

A ce l i mali;(.ation 

RB8 764 to RJ3 8766 

After physical exmninati on fo r good 

health and suitability for experim ell(, 

the rabbits will be acc lima tized [or at 

least five days before treatment. 

T est S ubsta nce InforJllntiuli 

(As fUl11ished by rhe Spon sor) 

Test substance / J (e m 

Lot No. 

M anu fact.urcd b y 

(Name a nd Address) 

S upp lied by 

(Nam e a nd Add ress) 

Date of Jll anu faclure 

Date of expiry 

Puri ty as per 

Certi ficate of 

A nalysis 

Physical appearance 

Sto rage conditions 

The Dow Chem ica l Company 

Midland. M I 4 8674 USA 

The D o'\v Che luica i Com p uny 

M idland, M I 4 8674 US A 

N o t Appl icable 

06 Ma rch 20 12 

Off-white solid 

A m bient (+18 ( 0 + 36"C ) 

T he test s ubs(ancc . idcn ti ried a s B enzo y la (ed Ca tec hoL was 

rece ived o n 03 F e br uary 2 0 1 I a nd was furthe r identifie d as 0 08-26 

by test fa c ility. R cfercnce N umber d ated 07 Fehruary 20 11. T he 

test subs tance is a solid and is stored at al11bient condition 

(+ I 8 to +36°C). Docume ntali o n of the method s of synthesis. 

fabrication. or derivation of the test substance is rcta incd by the 

S ponsor. 

D Clerm inaUon o f the stability, icienti ty, strength. purity and 

composition of the lest substance is the responsibility of the study 

Spon::;or. ~ rol lo\oving information re lated to the chal'aclcri;:,ntion 

o r the test substance "vas provided by the sponsor. 

SPCo py No. III 

Copy No. 2/2 

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APPENDIX 2 contd. Study plan 

10.2 

10.2.1 

Informmi on as provided by Sponsor arc as fo llows: 

pH : 6.47 (Anal ysed ill-house). 

Specific gravity I density: Not Appli cable . Test substance is a 

solid , 

Methods 

Husbandry 

Conditio ns 

A nitnals \·vill be hOllsed under standard labora tory conditi ons, air 

conditi o ned v.dth adequate fresh air supply ( 12-15 air chang~s oll . 

Environment: temperature 17-23°C, re lative humidity 30-70%. with 

a J 2 ho ur lig ht and 12 hour dark cyck. The Inaximulll and minil1111111 

tell1peraturc and re lative Inlln idity in the expcrilnouta l f\.)om wi ll be 

recorded once dai ly. The relative hU111idity in the experimental 

room \Ni l! be ca lcul ated fro m d ry and wel bulb temperature 

recordings. 

Ho using 

l-lollsed indi viduall y in rabb it cages (approx. size : L 65 x B 65 x 

r r 45 em ) wi th naryl shallow cage body and faci lities [o r pcllctct! 

food (S tai n kss steel feed h opper) and drinking water (750 1111 

markrolon bOltle fitted w il'h sippe r lube). The liltcr collection tray 

(noryl \vas Le tray) w ill be chan ged daily (except o n Sundays), 

Diet: ad libifum 

R,abbit Ieed In(;tnufa<.:lurcd by Pranav agro Industries Ltd , Sangli, 

Maharashll"a , India , wil1 be provided ad libilurn to the rabbits. The 

procured feed w ill be subjected to analysis of composition and the 

analysis report included in the report. 

Specific contaminants (heavy me tals o rganoch lorillcs and 

organophosphHtes) in the diet arc analysed periodically and the 

report \vi lJ be incl uded in the study ti le. 

\Valer: ad libitum 

D ecp bo re-wel l water passed Uuough activated charcoal ti lter and 

exposed to UV rays ill Aquaguard in-Jine \vuter fihcr-{,; unl-purificr 

m a nufactured by Eureka rorb es Ltd .. MUlll ba i - 4 0 0 00 I, INDIA , 

w il l be provided. The \vater boule \vilJ be replenished OJH':C daily <-lIl ei 

J~

Co py ,,'0. III 

P ngc 9/ 1 (, 

Copy No. 212 

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APPENDIX 2 contd. Study plan 

the \vater bottl es changed on ce a vl cck. The \·vater analysis csu lt ~ 

w ill be f urnished in the re port. 

10.2.2 PCI"formancc of test 

J 0.2.2.1 Salnplc l)rcparation 

A quan tity o f 0.5 g of test itc I11 as received w ill be made into paste 

by adding su ffic ient voll1Jllc of "vater nnd m ixed w ith a g lass rod . 

10.2.2.2 PI~c plll'ntion of test aninlals 

Approximate ly 24 hours bcfol'e trcnllnent, the illr o n the len side of 

the dorsola tera l trun k s urfnee I about 12-20 x 18-20 e m 1 w i II be 

clipped vvilh a ll e lectri c clippe r c~cu la ® GCflnrln y. Care w ill be 

taken to avoid abra din g the skin. A fter clipping and prior to 

appli ca tion. the anhna l's skin \-vill be checked for any abno nlla li tics 

accord ing to {he "Primary S kin Irri tat io n Scori ng Systeln ". If any 

site scores a rc greate r than zero, that aninHl1 \vill be rem o ved fro m 

tcst a nd rep laced and ihis \'I.:i ll be recorded in rn\.v da t.a a nd \vill be 

reported. 

10.2.2.3 Testing option 

The study will be initi ute d with a ::;te pwisc procedure ( l + 2 R a bbi ts). 

10.2.2.4 Test s ubstance application 

P"cpnrntion of test S ite 

Appro:< in1ate ly () em 2 intact test site \vill be se lected on each aniJnal. 

The a dja<.:ell( untreated skin of each anima l \"i ll serve a s th e contro l 

site. 

Applicatiun of Test SlIbstllllCC 

/\ quantity of 0.5 g of te st itC Jll as received w ill be made in to paste 

by adding sufficient volume of \:vate r and mixed with a g Jass rod. 

Genera ll y, the tcst. su bstance wi ll be applied directly to tbe s kin of 

the test si te, however. in som e cases it may be n ecessary to apply the 

test s ubsta llce to the ga uze pad pri o r to a pp lica tio n to e nsure prupe r 

placcll1cIll <;tnd distribution. The e ntire trunk of each an ima l \vill then 

be \vrapped with nOIl-irrita t.ing semi-occlusive ad hesive tape to 

avoid dislocation of the patch. _Aninl nls will be exposed to the test' 

substance for a p eriod of 4 h o urs. El izabet han colla rs v.' HI be placed 

o n cach rabbit for the de sig n ated exposure period . 

S P

Co p y No. II I 

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APPENDIX 2 contd. Study plan 

10.2.2.5 EX(losli rc of anirnals 

10.3 

10.3.1 

10.3.2 

10.3.3 

In a step,"v isc m anne r ( I·t 2 R a b bits) a ll the t hree ra b bits wi ll be 

e xposed to the t(.;:<; t. sub lan c c[lch \,vith o ll e test patc h o nly fo r a t'01.lr 

h O ll!" expo!-iurc peri od . 

Obscn'ations 

Cl inical ObSCI"VH(ions 

H.a b bits \vi ll b e o bserved fo r rox ie sig n s and pre-te rmi nal d e aths fo r 

the first fo ur hours (at hourl y inte rva ls) o n the day of test s ubstance 

appl icatio n and o nce daily there a fter till the end o f the o b ser vati o n . 

O bservatio n will in c lude gross e v aluatio n o f skin and fur. eyes a nd 

JllUC()lIS ln embra n es. respira tory, c irc u lato ry, auto n Olll<:lti c a nd 

central n e rvo us SYStC1l1 S. SOJlH :l lo m o t o r activity a nd be hav io r patte rn . 

Particula r a ttentio n ,·vi1l be d irected to observ ation o f tren10 rs, 

convu lsion , saJi va1ion, dia rrhe a and coma. 

Jlod)' , veights 

Ind ividual bo d y ,-ve ig h(s o f the a nill1als w ill be re co rded a t the sta rt 

of acclitl1 ati zatio n , day I o f tre atll1ent Gust before tt:sl ub J1c c 

appJi calio n ) and a t termina ti o n o f ob servatio n ! death . 

Skin scoring 

Foll o ,ving the e xposure p e ri o d. the p a tches vw'ill be J"enH)vcd ,mel the 

app)j c at.io n site wiU be v:ash e d w ith w'ate r o r appro pri at e solvents 

using clean to\ve ls to r CI1l0Ve a n y residual test sub sta n ce. The 4 hour 

appli catio n s ites wi ll b e c x alnined fo r signs o f e ry thc nu t u nd edema 

\-vi thi n 3 0 -60 luinllte s a nd a t inte rvals of approx in1utc ly 2 4 . 4 8 and 

72 h o urs u n cr p a tc h re m o v a l a c cording to the.; "Prirn il ry S kin 

Irrita tion Scoring S yste m" dev elo p ed by Dnd£:c. 

If the re is n o e vide n ce o f d e rmal irrita ti o n in any lest rabbit at 

72 ho urs. the stud y ,~ ll be te rminated . If irrjtatio n persists in any 

ra bbit a t 72 h o u rs. scoring ,.v ill continue for all rabbits al 7 . 10 an.d 

14 d ays a fter patch rCllloval or until a ll sig n s o f irrita ti o n a rc 

c o mpletely r e versed (n sco r e o f :t..cro) in all n lbhi ts. \vhi c hcver 

occurs first. 

Ind iv id ual scores wi ll be recorde d fo r e aeh rabbit. A n a rra ti ve 

d escri p tio J1 or pertinent sk in o bse rva ti o n s Ul1d uny si gns of g ro ss 

lox ic ity \-v ill be re cord ed . In a d diti o n. evaluatio n o f th e reve rsib ili ty 

o r irreversihili ty o Cth e o bserv ed effects \·\lill b e n o te d . 

S P

co p~' r\·o. I I I 

Pn gc 11 116 

·SKlN 

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APPENDIX 2 contd. Study plan 

J f at nny time during the study. corrosion is noted. (h e St udy 

Di rector will notify the Sponsor that the study !-iho uld be 

terminated . fr damage is extensive find the Sponsor is 1I11a vai table 

to aut horize tenninalion or the study, the S tudy J)i rec tor may 

unilaterally decide to have the animals cUlhanizcd immed iately 

for hUl1UII1C reasons and the Sponsor will be n otified as suon as 

possible. 

10.3.4 Grad ing of skin l-cOlctions 

The skin reaction is assessed accord ing to the numerica l scoring 

system of Oraizc cut! (1944). 

1. ERYTHEMA AND ESCHAR FORMATION 

No erythema ... . .. ..... . o 

Very slight e rythema (bal'cly perceptible)... ........... I 

Well-de fined erythema ............................ . .. ........ . . ·· .. ·· .. · 2 

Moderate 1.0 severe erythema ....... ....... ...... .... ... . .......... 3 

Severe erythema (beet redness) to slight eschar formation 

(injuries in depth) ........ ..................... . . . . . ........... 4 

MAXIMUM l'OSSlULE SCORE - 4 

2. EDEMA FORMATION 

No edenl a . . ........ .......... . . .......... ....... ........ _. . .... ..... 0 

Very slight cdcl11u (barely perceptible) ................... 1 

S light edema (edges of area well defined by definit e raising)... 2 

rVlodcratc cdenla (raised approximately I rni llillll::lrc)... 3 

Severe CdCJl1.fl (raised morc than I millimetre and extend ing 

beyond area of exposlIre)............... 4 

MAXIMUM T' OSSrIlT,E SCORE - 4 

I I. RESULTS 

11.1 Dat" compilation 

Tile following data will be recorded on datu sheets and transcribed 

for compi latio n and a na lysis: irrita tion scores. desc ription of" the 

degree and nature of irritation. the presence of dermal lesions. und 

allY erfeets other than cle nn" i noted ciuring clinical observations and 

hody wei g hts. 

S PC opy No. I JI 

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APPENDIX 2 contd. Study plan 

All findings will be record ed in the st and a rd repo rling formu ls and 

prese nted in the repo rt. 

l2. REPORTING 

The final rep o rt w ill incl ude the ro llowin g infonnnrio n : 

y The name a nd ad d ress of the spo nsor a nd the lest faci lity a lo ng 

w ith the stud y schedule . 

>- The nrul1cs and signatures of a ll personneJ involved in the stuciy. 

includi ng t he Study D irect o r and other sc ie n tists. 

~ Test s ubsta nce: 

• Test s ubstance inro nnation as provided by the sponsor. 

>- D a ta derived a t e a c h s tag e of testing strategy, 

;;.. Descrip tion or r<::l evant data ava ilabl e fro m prior testing . 

J;.. T est anima ls: 

• species/strai n used; 

• l1L1111 ber ofcUlimals o f each sex; 

• indiv idua l a n inutl \·\lcig hts a t sta rt a nd concl usio n o f test; 

• age a t eat l11 t~ 

• source o f aninta ls. h o u sing conditions _ d jet. e t.c. 

r Test conditions: 

• lech niq ue of patch site prcpara n~ 

• d e ta ils of patch rn a te ria ls lIsed and patching tech niq ue: 

• detai ls of test s ubstance preparaUon, applicatio n and 

re ll1o val; 

>- Results: 

• tabulatio n o f irri tation/ con -osi,o n resp on se ~co cs for each 

a n inu li at t he rinle p o ints nlcas urcd; 

• d escripti o n or a ll lesions observed; 

,.. A d escri ptio n of a ll rcsui(s. 

r /\.11 study p lan deviatio ns, if nn)' . 

.I"'" l n di viduHllo xi<.- signs and necropsy find ings ir a n y. 

~ A d escript ion o f a ll c irclIIlls 1anccs if any that ma y 11:1\,(.' a lTcc k:d 

t he q uality o r int egrity Oflh c study. 

SI'-

C opy No. 111 

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APPENDIX 2 contd. Study plan 

)..- The Quality Assurance S tatclnenL sig ned by the Quality 

Ass Ufi:1l1CC U n i l. 

:;;... The GLP Compliance Statement, signed by the Study Direc tor. 

? The storage locations of aU ra v~' data. the report, a sample o r the 

test suhstance and the archiving pe ri od. 

» GLP c ertificates. 

13. A R CHIVING 

The original signed final report "..,ill be sen t to the Sponsor and o ne 

signed final report wil1 be retained by Advinus. The original raw 

data. dOClIll1Cntalion, records. the original study plan, and final 

report generated as a result of thjs stud y will be sent to the Sponsor 

fo r retention follo\.ving signing of the f:innl rep o rt. Copies of ('he 

same (authorized by the Study Director) will be retained by 

Advinus. Advinlls \.vill maintain these records fOI" u pe riod o f al least 

fi ve years. After this time. the S ponsor o f tbe study will be offered 

the opportunity to take possession of the re co rds o r ',"v iII be c harged 

an a rchiving fee for continued archiv ing by Advinlls or to dis pose of 

the ravl d ata in consultatio n w ith the Spo nsor, 1\ repre sentati ve 

s<i lnpie or approxi m ately o ne ,gram o f the tes t itenl \,,-'ill be: sent from 

the test iten1 sto re s to the archives in the te st facililY before; the firs! 

dis pensing o f test item to stud ies. This satnple sha ll be sto n:,' cI for a 

pe riod o f 2 yea rs fro l11 the date o f t bis final report 01' till next GLP 

inspe c ti o ll;> \"vh ichcve r is laler, however no t beyond 5 years. 

]n the e v e nt of any inspectio n by regulato ry aut tie s~ seeking 

o riginal nnv data for verification, the spo nsor \.vQu ld be e~po iblc 

either to send the o ri ginal raw dala to Adv inus Therape ulics Limited 

or to the regulatory agcncic!) for vc riJjcation. 

L4. STUOY l'LAN DISTRJBOTION 

The fill a l study plnn (o ri g inal copies) wi ll be distributed as (o llows: 

Sponsor: S oft copy of the signed S tud y pla n in PDF fo nllal and one 

sig ned study plan in o ri g inal (Copy l'hL 1/1 ). 

St udy D irector: One signed study p lan i ll o ri g inal (Copy No. III). 

S P

C OP)' No. 1/ 1 

Page 1411 () 

Copy No. 212 

Page 37/46 
- 

APPENDIX 2 contd. Study plan 

15. REPORT DISTIUBUTlON 

The final report (orig inal copies) will be d istributed !IS follows: 

Test Fac ility Archives: One sign ed tinal report in o ri ginal (COpy 

No.1 /2). 

Sponsor: One signed fina l report in original (Cop y 'l"'os.2/2) and One 

Soft copy of the rc p nrt in PDF forma t. 

16. HEFERENCES 

I. LJraizc, .1.11., W oodward, G and Cnlvcry, H.O (1944) . 

.I .Pharm aeol. Exp.The r.83: 384. 

2. Dra ize . .l..I 1 (1959) : Dermal Toxicity In : Appraisal o [tl", snrcty 

o f chemicals in Foods~ Drugs and COSll1 c ticS. Association o f 

Food a nd Drug Officia ls of the United S tates, Austin , Texns. 

p.47. 

3. OECD (2000) Guidance Document on the R ecognition. 

Assessrnent and Usc of Clinical Signs as I-lunlanc Endpoints for 

Experi me ntal A nimals U~c d in Safely Evalua tion Enviro nmell ta l 

Health and Safety ~I]onog 'a ph S eries on Testin g and Assessment 

No 19 . (ENVI.IM /MONO(2000)7. 

S I'- S KIN 

C('" y No. Til 

P lI J!. 4.' 1511 6 

Copy No. 2/2 

I'age 38/46 
- 

APPENDIX 2 contd. Study plan 

17. AGREEMENT 

This Study I'lal1 fo r Study No.: : 

A cute dermal irritatioll / corrosion study in Rahbits" has been 

mutuall y agn.!(!d and signed. 

S ponsor Sludy plan AIlp/'ovIII: 

The finnl vel'sion or the study plan was approved by email fi'om the 

study monito!' on 17 February 201 1 for Study Director's Signature. 

for TEST FACILITY 

STllD Y DIRECT OJ{ 

Date: III ~" r eb ?o II 

Q UALITY ASSUJ{i\ NCF. UNIT 

Oa tc : I r, t " (-eb ru..""1 2 01 1 

S P

Cop)' No. I / J 

Page ' M i ll 

·SKIN 

Copy No. 212 

Page 39/46 
- 

Copy No. 212 

15. ANNEXURES 

Page 40/46 
- 

ANNEXURE 1. Analysis Report - Animal Diet Sample 

ADVINUS THERAPEUTICS LIMITED 

21 & 22, PEENY A INDUSTRIAL AREA, II PHASE 

BANGALORE • 560 058 

ANALYSIS REPORT· ANIMAL DIET SAMPLE 

From: Analytical R &D Department 

Advinus, Bangalore·560 058 

To: Department of Safety Assessment 

Advinus, Bangalore-560 058 

Our Ref. No.: WCrrFS/216 1 Date: 27.12.2010 

Sample Details: Name : Rabbit Feed Sampling Date: 03.12.2010 

Batch No. : 60602 

Supplier : Pranav Agro Industries Ltd., Bangalore 

Manufacturer : Pranav Agro Industries Ltd., Bangalore 

SI. No. Parameter 

I. Moisture 

2. Crude protein (N x 6.25) 

3. Crude fat (Ether extract) 

4. Crude fibre 

6. Acid insoluble ash 

7. N itrogen free extract 

8. Ca lcium (Ca) 

9. Phosphorus (P) 

Copy No. 2/2 

ANALYSIS RESULTS 

(Analysis On "as is basis") 

(%) 

12.59 

22.51 

3.88 

1.97 

3.62 

48.93 

0.56 

0.10 

Analytical R&D Department 

Page 41/46 
- 

ANNEXURE 2. Analysis Report - Wate r Sample 

ADVINUS THERAPEUTICS LIMITED 

21 & 22, PEE NY A INDUSTRIAL AREA, II PHASE 

BANGALORE 560 058 

ANALYSIS REPORT - WATER SAMPLE 

Fr0m: Annlyti(,f'll R& n l)pr~ rtm f" nt 

Advinus, Bangalore-560 058 

To' np;pMlmf':nl of S~\fp.,y A~Sf' "Smf'.nl 

Advinus, Bangalore-560 058 

Our Ref. No: wcrrWS/256 Date: 24/01120 I I 

Sample Details: SOllIce of Collection: Outlet of the Aquaguard (At use point) 

Date of Collection: 10101120 II 

ANALYSIS RESULTS 

SI. Parameter 

No. 

I. Colour 

2. Odour 

3. Turbidity 

4. PH 

5. Electrical Conductivity, 

dSm" 

6. Tota l solids, (ppm) 

7. Suspended solids, (ppm) 

8. Dissolved solids, (ppm) 

9. Dissolved oxygen (ppm) 

10. Biochemical Oxygen 

Demand 5 days at 20°C, 

- (ppm) 

II. Chemical Oxygen 

Demand (ppm) 

SKJN 

Copy No. 212 

Content 

Colourl ess 

Odourless 

Clear 

6.61 

1.373 

797 

18 

779 

4.1 

2.4 

12.1 

SI. Parameter Content 

No. (ppm) 

12. Total hardness as 495 

CaCO, 

13. Calcium as CaH 105 

14. Magnesium as Mgl+ 57 

15. Chlorides as cr 334 

16. Sulphates as S04.L- 58 

17. Carbonates as C03-- -

18. Bicarbonates as HeO]- 370 

19. Sodium as Na 79.86 

20. Potassium as K 6.39 

6cpattmcnt 

Page 42/46 
- 

ANNEXURE 3. GLP Certificate - Germany 

d I: 

/. 

Ii 

; 

I 

I 

S KI N 

Copy No. 2/2 

Bundesinstitul fLlr Risikobewertung I 

Federal Institute for Hisk Assessment F'Jt BfR fti .. ",." •• lC"''''' _ G .......... ' .. ,I ."h !il«o 

GUTE LABORPRAXIS / 

GOOD LABORATORY PRACTICE 

(gernaB I according to § 19b Abs.2 Nr.3 Chemikaliengesclz J German Chemicals Act) 

Eine 3LP-!nspeklion wurde uurcl .gefOhrt in I A GlP inspectio,l was carried out;;at 

Prlifclnric htung I Test Facifity 

A.d vinus ThetdpP'JtiCs Limitc<t 

(fo rmer na me until October 2010: A dvinus Therapeutics Private Limited) 

2 1 & 22 Phase II, Pccn ya Industrial A rea 

Bangaloro - 560 058, INDIA 

Prl.lfkatogoricn I Area of Expertise 

Profungen zur Bestirnrnung der physikalisch-chemischen Eigenschaften und 

Gehaftsbestirmnungcn , Physical-chemical testing 

PrOfungen zur Bestimmung der toxikotogischen Eigenschaften I Toxicity studies 

PrOfungen zur Bestimmung erbgutver~ndernden Eigenschaften (ill vitro, ill vivo) I 

Mvtagenlcity s tudies 

Okc:oxikOlogisct,e PrOfungen ?ur Bestimmung der Auswirkl mgen auf aqualische 

und terrestrische Organismen I En vironmental toxicity studies on aquatic and 

terrestrialorganisniG 

Prilfungen zum Verholtcn im Bodon. im Waccnr und in dcr Luft: Bionkkwnulntion I 

Studic:;; I)rl behaviour in soil, water aod air: bioaccumulation 

PrOfungen zur Bestirnmung von ROckstanden I Residue studies 

Analytische PrOfungen an biologisChen Malerialien I Analylicat and dinical 

chemistry testing 

Sonslige PrOfungen - Bionanalylik Pharmakokinetlk, Sicherheilspharmakologie I 

other sludies - bioanalytics , pharmacokinetics and safety pharmacology 

Datum der Inspektion I Date of Inspection 

July 07 - 10,2009 

Auf dcr Grund10ge des Inspektionsberichtes und dcr Besprechung Obcr zu crfolgendc 

Mal1nahmen wird hiermil bcst~tigt. doss in dieser PrOfeinrichtung die oben gensnnlen 

PrOfungel' unter Einhaltung der GLP-Grundsl:itze durchgefOhrt werden kOnner'!. { 

Based on the inspection report tlnd the d iscussion of follow up activities it can be 

confirmed, thai the test facility is able to conduct Ihe aforementioned studies in 

compliance with the Prir,ciples of GLP. 

Eine OberprOfung dieser GLP·Bestl.tligung isl sp~ te tens drei Jahre nach der o. g. 

Inspektion zu beantragen, Ohne d iesen Antrag wird nach Ablauf der Frist die 

PrOfeinrichlung 8US dam deutschen GLP·Oberwachur,gsprogramm genommen und diese 

GLP-Bestatigung verliert ihre GOltigkeit. I 

Verification of this GLP Certificate has to be applied three years afler the above 

mentioned inspection at the lates\. Elapsing this term. Ihe test facility will be taken oul of 

the German GLP Monitoring Programme and this GLP Certificate becomes invalid. 

November 26, 2010 

/'! ;-".... ';-: . ) 

.: ..J. 

/

> auUdft$;n$t,tvt fur ~-o'lvng t,:6clftta11r6i1i\u1e for Risk A!;Ii6Sllrnenl 

. , . Th,elaUee 86·92 C' " 1410S 0onin .. GERMANY 

'1 

I! 

'I I 

I 

I ,I 

! 

I 

I 

I 

I 

II 

, 

I 

.1 

, 

i 

il 

il 

!I 

!I 

-, 

I 

Pa ge 43/46 
- 

ANNEXURE 4. GLP Certificate - The Netherlands 

vOedfIJl en w aren autoriteit 

ENDORSEMENT OF COMPLIANCE 

WITH THE OECD PRINCIPLES OF 

GOOD LABORATORY PRACTICE 

Pursuant to the Netherlands GLP Compliance Monitoring Programme and 

according to Directive 2004191EC the conformity with the OEeD Principles of GLP 

was assessed on 4·- 7 May 2009 at 

Advinus Therapeutics Private Limited 

21 & 22 Phase II, Peenya Industrial Area 

Bangalore - 560 058 INDIA 

It is herewith confirmed ttlat the afore-mentioned test facility is currently operating 

in compliance with the OECD Principles of Good I.aboratory Practice in the 

following areas of expertise: physical-chemical testing, toxicity studies, mutagenicity 

studies, environmental studies on aquatic and terrestrial animals, and analytical Dnd 

clinical chemistry. 

C opy No. 212 

Food .)nd Consumer Product Safely Authority 0/'NA) 

Prtnses Beafrixlaan 2. 2595 AL Den Haag 

Poslbus 19500,2500 eM Den Hnag, The Netherlands 

Page 44146 
- 

ANNEXURE 4 contd. GLP Certificate - The Netherlands 

Food and Consumer Product Safety 

Authority 

h1inisIIY ot Agricullllre, Nawre and 

Food Qualiry 

,. PO Box 19506 ~soo C.N [)en llall4} The Netnerl,lIw:ls 

Advinus Thempeutics Limited 

for the attention of Dr. S. Natesan 

Vice President 

21 & 22, Phase II, Peenya Industrial Area 

Bangalore - 560 058 

INDIA 

Date 18 November 2010 

Re. Change of name 

Dear Dr. Natesan, 

I have received your letter of 26 October 2010 in good order. In yOllr Ictter you 

explained that Advinus Therapeutics Private Limited has changed its name into 

Advinus Therapeutics Limited. Further, you stated that the change of name has no 

consequences for Hie ownership and t hat the test facility wifl continue to function 

as earlier. 

The GLP Compliance Monitoring Authority of The Netherlands has periodically 

inspected Advinus Therapeutics Private limited. The most recent inspection was 

Ctlrried out on 4 - 7 May 2009. Subsequently, the inspectorate issued an 

Endorsement of GLP Compliance on 27 August 2009. This endorsement reflects 

the name of the test facility at the time of the inspection and will not be renewed 

until a new inspection has been conducted. 

Yours sincerely, 

Manager GLP Compliance Monitodng Program 

C opy No. 2/2 

n~cs Bcalr;xlllMt ] 

2!.i9!lIlL Den ia~9 

PO 001( 19506 

25-:: 0 e M Dcn Haag 

I he Nlltherlanos 

wW .V""".1I1 

Contact 

Dr. R.M.II. J,lspe'$ 

T ... 31 (0)70 '1484]69 

M ")10 

f ·, ) 1 (0)](14'1847'17 

ob lilSPtlrs~ vwa nl 

Our ref. 

Tfl.CVVYN20 I 01':933 J 

Page 45/46 
- 

ANNEXURE 5. GLP Certificate - India 

NATIOl'tAL GLP COMPLIANCE MONITORING AUTHORITy 

GOOD LABORATORY PRACTICE 

GLP CERTIFICATE 

GLP Inspection was carried out at Advinus Therapeutics Private Limited, Plot Nos. 21 & 

22, Phase-il, Post Box No. 5813, Peenya Industrial Area, Bangalore-560 058, India in the 

following areas of expertise: . 

• physical-cbentical testing 

• toxicity studies 

• mutagenicity studies . 

• environmental toxicity studies on aquatic & terrestrial organisms 

• studies on behaviour in water, Boil and air, bioaccumulation 

• residue studies 

• UJJlllYUQlI ~u CUD.lCUI cnemJstry lesnng 

Based on the InspectWn &e,>ort <Uld the fullow-up actWns taken by the test facilil)', it is 

confumed that the test facility is capable of conducting the above-mentioned tests/studies in 

compliance with OEm Principles of Good Laboratory Practice (GLP) and Norms, as 

adopted by the National GLP Compliance Monitoring Authority. 

Ths GLP Certificate is valid for a period of three yeprs from November 7, 2008 subject to the 

condition thaI the test facility complies with the Term. & Conditions of the National GLP 

Compliance Monitoring Authority'. Document Number GLP-I0l. 

Certificate No.: GLP/C-019 

J.ssue Date: 

SKlN 

Copy No. 2/2 

24-12-2008 

Helld 

National GLP Compliance MonJtoring Authority 

Depltrtmcnt of S,cience & Technology 

Technology Bbav811 New Delhi-110016 

Page 46/46