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CHD-FA (pH 5.5):

ACUTE DERMAL IRRITATION IN THE RABBIT

PROJECT NUMBER: 41004593

AUTHOR: J Bradshaw

STUDY SPONSOR: TEST FACILITY:

Fulhold Limited Harlan Laboratories Ltd

Legh Coach House Shardlow Business Park
Wilmslow Road Shardlow
Mottram-St-Andrew Derbyshire
Cheshire DE72 2GD
SK10 4QH UK
UNITED KINGDOM
Telephone: +44 (0) 1332 792896

Facsimile: +44 (0) 1332 799018

41004593.doc/JS
 PROJECT NUMBER: 41004593 PAGE 2

QUALITY ASSURANCE REPORT

This study type is classed as short-term. The General Study Plan for this study type was
reviewed for compliance once only on initial production. Inspection of the routine and
repetitive procedures that constitute the study is carried out as a continuous process
designed to encompass the major phases at or about the time this study was in
progress. In addition, inspection of general facilities not specifically related to this study
are done monthly or annually in accordance with QA Standard Procedure.

This report has been audited by the Quality Assurance Unit, and is considered to be an
accurate account of the data generated and of the procedures followed.

In each case, the outcome of QA evaluation is reported to the Study Director and
Management on the day of evaluation. Audits of study documentation, and process
inspections appropriate to the type and schedule of this study were as follows:
19 October 2010 General Study Plan Compliance Audit
07 December 2010 Test Item Preparation
07 December 2010 Animal Preparation
07 December 2010 Dosing
07 December 2010 Assessment of Response
§ 25 January 2011 Draft Report Audit
§ Date of QA Signature Final Report Audit

§ Evaluation specific to this study

............................................................................... DATE: ................................................

For the Quality Assurance Unit*

*Authorised QA Signatures:
Senior Audit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA,
G Wren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQA
 PROJECT NUMBER: 41004593 PAGE 3

GLP COMPLIANCE STATEMENT

The work described was performed in compliance with UK GLP standards (Schedule 1,
Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by
SI 2004/0994)). These Regulations are in accordance with GLP standards published as
OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17);
and are in accordance with, and implement, the requirements of Directives 2004/9/EC
and 2004/10/EC.

This report fully and accurately reflects the procedures used and data generated.

............................................................................. DATE: ...................................................

J Bradshaw BSc (Hons)
Study Director

This report may be presented in final form as a digital (pdf) document. Such documents are prepared by scanning the paper original,
and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the
hand-signed paper original, held in secure archives, is the definitive document.
 PROJECT NUMBER: 41004593 PAGE 4

CONTENTS
QUALITY ASSURANCE REPORT 2
GLP COMPLIANCE STATEMENT 3
CONTENTS 4
SUMMARY 5
1. INTRODUCTION 6
2. TEST ITEM 6
2.1 Description, Identification and Storage Conditions 6
2.2 Preparation of Test Item 7
2.3 Measurement of pH 7
3. METHODS 7
3.1 Animals and Animal Husbandry 7
3.2 Procedure 8
3.3 Interpretation of Results 9
4. ARCHIVES 10
5. RESULTS 11
5.1 Skin Reactions 11
5.2 Bodyweight 11
6. CONCLUSION 11
Table 1 Individual Skin Reactions 12
Table 2 Individual Bodyweights and Bodyweight Changes 13
Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 14
 PROJECT NUMBER: 41004593 PAGE 5

CHD-FA (pH 5.5):

ACUTE DERMAL IRRITATION IN THE RABBIT

SUMMARY

Introduction. The study was performed to assess the irritancy potential of the test item
to the skin of the New Zealand White rabbit. The method was designed to be compatible
with the following:

OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal
Irritation/Corrosion” (adopted 24 April 2002)

Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC)

No. 440/2008

Results. A single 4-hour, semi-occluded application of the test item to the intact skin of
three rabbits produced no evidence of skin irritation.

Conclusion. The test item produced a primary irritation index of 0.0 and was classified
as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive
effects were noted.
 PROJECT NUMBER: 41004593 PAGE 6

CHD-FA (pH 5.5):

ACUTE DERMAL IRRITATION IN THE RABBIT

1. INTRODUCTION

The study was performed to assess the irritancy potential of the test item following a
single, 4-hour, semi-occluded application to the intact rabbit skin. The method was
designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal
Irritation/Corrosion” (adopted 24 April 2002)

Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC)

No. 440/2008

The albino rabbit has been shown to be a suitable model for this type of study and is
recommended in the test method. The results of the study are believed to be of value in
predicting the likely skin irritancy potential of the test item to man.

The study was performed between 07 December 2010 and 08 January 2011.

2. TEST ITEM

2.1 Description, Identification and Storage Conditions

Sponsor's identification : CHD-FA (pH 5.5)
Description : brown liquid
Batch number : 5059/10
Purity : not supplied
Date received : 15 November 2010
Expiry date : 15 November 2012
Storage conditions : approximately 4°C in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item is
the responsibility of the Sponsor.
 PROJECT NUMBER: 41004593 PAGE 7

2.2 Preparation of Test Item

For the purpose of the study the test item was used as supplied.

The absorption of the test item was not determined.

2.3 Measurement of pH

The pH of the test item was determined prior to commencement of the study and found
to be as follows:

pH Measurement
Preparation
immediately after 10 minutes

Undiluted as Supplied 5.1 not applicable

90% v/v aqueous preparation of the test item 5.2 5.2

3. METHODS

3.1 Animals and Animal Husbandry

Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan
Laboratories U.K. Ltd., Loughborough, UK. At the start of the study the animals weighed
2.53 to 3.05 kg and were twelve to twenty weeks old. After an acclimatisation period of
at least five days each animal was given a number unique within the study which was
written with a black indelible marker-pen on the inner surface of the ear and on the cage
label.

The animals were individually housed in suspended cages. Free access to mains
drinking water and food (2930 Teklad Global Rabbit diet supplied by Harlan Laboratories
U.K. Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water
were considered not to contain any contaminant of a level that might have affected the
purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to
70% respectively. Any occasional deviations from these targets were considered not to
have affected the purpose or integrity of the study. The rate of air exchange was at least
fifteen changes per hour and the lighting was controlled by a time switch to give twelve
hours continuous light (06:00 to 18:00) and twelve hours darkness.
 PROJECT NUMBER: 41004593 PAGE 8

The animals were provided with environmental enrichment items which were considered
not to contain any contaminant of a level that might have affected the purpose or integrity
of the study.

3.2 Procedure

On the day before the test each of a group of three rabbits was clipped free of fur from
the dorsal/flank area using veterinary clippers. Only animals with a healthy intact
epidermis by gross observation were selected for the study.

One rabbit was initially treated. A quantity of 0.5 ml of the test item was applied directly
to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in
position with a strip of surgical adhesive tape. To prevent the animal interfering with the
patch, the trunk of the rabbit was wrapped in an elasticated corset and the animal was
returned to its cage for the duration of the 4-Hour exposure period.

Four hours after application the corset and patch were removed from the animal and any
residual test item removed by gentle swabbing with cotton wool soaked in distilled water.

After consideration of the skin reactions produced in the first animal, two additional
animals were similarly treated.

Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours
later, the test sites were examined for evidence of primary irritation and scored according
to the following scale:

EVALUATION OF SKIN REACTIONS

Erythema and Eschar Formation Value

No erythema .................................................................................................... 0
Very slight erythema (barely perceptible)......................................................... 1
Well-defined erythema ..................................................................................... 2
Moderate to severe erythema .......................................................................... 3
Severe erythema (beef redness) to eschar formation preventing grading of
erythema.......................................................................................................... 4
 PROJECT NUMBER: 41004593 PAGE 9

Oedema Formation Value

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 millimetre) .................................... 3
Severe oedema (raised more than 1 millimetre and extending beyond the area
of exposure)..................................................................................................... 4

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the
observation period.

3.3 Interpretation of Results

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the
Draize Scheme
The scores for erythema and oedema at the 24 and 72-hour readings were totalled for
the three test rabbits (12 values) and this total was divided by six to give the primary
irritation index of the test item. The test item was classified according to the following
scheme devised by Draize J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of
the United States, Austin, Texas, p.46-59:

Primary Irritation Index Classification of Irritancy

0 Non-irritant
> 0 to 2 Mild irritant
> 2 to 5 Moderate irritant
> 5 to 8 Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study
Director, which include ulceration and clear necrosis or signs of scar tissue, the test item
is classified as corrosive to rabbit skin. Classification according to Draize may, therefore,
not be applicable.
 PROJECT NUMBER: 41004593 PAGE 10

4. ARCHIVES

Unless instructed otherwise by the Sponsor, all original data and the final report will be
retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which
instructions will be sought as to further retention or disposal.
 PROJECT NUMBER: 41004593 PAGE 11

5. RESULTS

5.1 Skin Reactions

The individual scores for erythema/eschar and oedema are given in Table 1.

No evidence of skin irritation was noted during the study.

5.2 Bodyweight

Individual bodyweights and bodyweight changes are given in Table 2.

All animals showed expected gain in bodyweight during the study.

6. CONCLUSION

The test item produced a primary irritation index of 0.0 and was classified as
NON-IRRITANT to rabbit skin according to the Draize classification scheme. No
corrosive effects were noted.
 PROJECT NUMBER: 41004593 PAGE 12

CHD-FA (pH 5.5) : ACUTE DERMAL IRRITATION IN THE RABBIT

Table 1 Individual Skin Reactions

Observation Time Individual Scores – Rabbit Number and Sex

Skin Reaction (following patch Total
removal) 69815 Male 69834 Male 69862 Male

Immediately 0 0 0 (0)

1 Hour 0 0 0 (0)
Erythema/Eschar
24 Hours 0 0 0 0
Formation
48 Hours 0 0 0 (0)

72 Hours 0 0 0 0

Immediately 0 0 0 (0)

1 Hour 0 0 0 (0)
Oedema
24 Hours 0 0 0 0
Formation
48 Hours 0 0 0 (0)

72 Hours 0 0 0 0

Sum of 24 and 72-hour Readings (S) : 0

Primary Irritation Index (S/6) : 0/6 = 0.0

Classification : NON-IRRITANT

( ) = Total values not used for calculation of primary irritation index

 PROJECT NUMBER: 41004593 PAGE 13

CHD-FA (pH 5.5) : ACUTE DERMAL IRRITATION IN THE RABBIT

Table 2 Individual Bodyweights and Bodyweight Changes

Rabbit Number Individual Bodyweight (kg)

Bodyweight Change (kg)
and Sex Day 0 Day 3

69815 Male 2.82 2.89 0.07

69834 Male 3.05 3.08 0.03

69862 Male 2.53 2.61 0.08

 PROJECT NUMBER: 41004593 PAGE 14

Appendix 1 Statement of GLP Compliance in Accordance with Directive

2004/9/EC