This research protocol outlines a study to evaluate the cumulative irritation and sensitization potential of topical products. The study will involve: 1) Applying test products to the backs of 50 volunteers over 14 applications to assess irritation and sensitization. 2) Evaluating reactions using an international scale after each application and a rest period. 3) Conducting a single blinded application to a new skin site to confirm sensitization. The goal is to evaluate the safety of products before market launch by determining their potential to cause cumulative skin irritation or induce an allergic sensitization reaction.

1. RESEARCH PROTOCOL 1 of 8
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Allergisa pesquisa dermato-cosmética ltda.
ASSESSMENT OF THE CUMULATIVE IRRITATION
AND SENSITIZATION POTENTIALS
(Repeated Insult Patch Test)
RESEARCH PROTOCOL
CONTENTS
1. Introduction 2 of 8
2. Objective 3 of 8
3. Methods 4 of 8
3.1. Population and Sample 4 of 8
3.2. Ethical Aspects 4 of 8
3.3. Product 4 of 8
3.4. Required Material and Equipments 4 of 8
3.5. Study Length 5 of 8
3.6. Methodology 5 of 8
3.7. Study Design 6 of 8
3.8. Results 6 of 8
4. References 7 of 8
History of Revision 8 of 8
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1. INTRODUCTION
Over the last few years, the cosmetic and pharmaceutical industry has grown
considerably, same as its concern in developing effective and safe products. The
creation of the Consumer Defense Code, the requirements of the National Agency of
Sanitary Surveillance of the Ministry of Health and the competition itself have led the
companies to adopt a more careful attitude as to the action and benefits of their products
by trying to associate their claims to scientific works.
Industry awareness and consumer requirements caused cosmetic manufacturers to
adopt a new procedure: before marketing a product, nowadays companies are
concerned to conduct clinical tests on safety and efficacy, which are coordinated by
dermatologists. This procedure provides cosmetic companies with greater safety,
credibility and reliability from their consumers.
A growing concern of the cosmetic industry is the prevention of possible adverse
reactions in users of its products. After all, consumers are much more critical about skin
irritation caused by a cosmetic product than about irritation caused by topical medication.
An adverse reaction is considered to be any signs or symptoms triggered by a
topical product used correctly (FISHER, 1995). Among cutaneous adverse reactions
caused by these products, the major ones are contact eczematous dermatitis, urticaria,
acne and stains (SAMPAIO, & RIVITTI, 2000).
The irritation potential of a product depends on a series of variables: components
used, ingredient concentration, absorption, amount applied, skin condition, application
mode and frequency, and cumulative effect (DOOMS-GOOSSENS, 1993).
As human variability is very large, no food, medication or cosmetic products are fully
innocuous. The term hypoallergenic is used for products with very little capacity to
induce an allergic reaction. Therefore, as a principle, all cosmetic products should be
hypoallergenic.
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Tests conducted with humans are governed by very strict laws in order to protect and
safeguard people. These laws vary from country to country. In Brazil, these researches are
allowed, provided that their protocols are approved by a Medical Ethics Committee, and
comply with the precepts of the Helsinki Declaration (CONSELHO NACIONAL DE SAÚDE
[NATIONAL HEALTH COUNCIL], 1996).
Patch tests are the main tool used to diagnose reactions caused by cosmetics and
in allergenicity researches. As the key potential risks of using a new product are
irritation, sensitization allergy, phototoxicity and photoallergy, the following clinical tests
are involved in allergenicity researches: primary and accumulated dermal irritation,
cutaneous sensitization, phototoxicity and photoallergy. These consist of repeated
applications of the product to the skin. The purpose is to detect any possible irritations or
induction to sensitization (KLIGMAN & WOODING, 1967, FISHER, 1995).
If the product passes the patch tests, it is important to carry out usage tests with the
finished product before it is introduced in the market (BARAN & MAIBACH, 1994).
Besides allergenicity, this test can also assess sensorial characteristics of the product,
and detect additional complaints and comments as to its performance.
The company finds out about any possible considerations and complaints that will
arise when the product is marketed, so it can develop strategies such as specific training
for its Consumer Service prior to product launch (BARAN & MAIBACH, 1994).
2. OBJECTIVE
The objective of this study is to evaluate by repetitive epidermal contact (Repeated
Insult Patch Test - RIPT) the allergenic (sensitization) potential and/or the cumulative
cutaneous irritation potential of topical products and products like non-woven fabrics and
fibers.
ALLERGISA pesquisa dermato-cosmética ltda. Cumulative Irritation and Sensitization
Potentials - RIPT

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3. METHODS
3.1. POPULATION AND SAMPLE
At least fifty voluntary subjects of both sexes, phototypes I to IV (PATHAK, 1983), in
the 18-80 age range will be selected, should they have already been approved in a
medical screening. Exclusion criteria will be: localized or generalized dermatological
diseases, presence of lesions or moles on the back of the subjects, use of any topical
and/or oral steroids and/or antihistamines for at least seven days prior to the start of the
study, personal history of adverse reactions to the type of product going to be tested, as
well as pregnancy or nursing.
The subjects will be submitted to an interview and to a dermatological examination.
3.2. ETHICAL ASPECTS
The subjects will be given explanations and guidance on the objectives and methods
of the research. They will be given proper instructions and sign a participation consent term
elaborated according to Helsinki Declaration and approved by the Research Ethics
Committee (CONSELHO NACIONAL DE SAÚDE [NATIONAL HEALTH COUNCIL], 1996).
They will be followed-up by specialist physicians in case they present any adverse
reactions to the test product.
3.3. PRODUCT
A sample of the test product will be catalogued and kept in our files for one year.
The informations given by the contractor company on the test product will be attached
to the final report.
3.4. REQUIRED MATERIAL AND EQUIPMENTS
• Hypoallergenic adhesive cards for the patch test with 1 sq.-cm filter paper discs
duly identified.
• Semi-occlusive hypoallergenic tape.
• Saline solution.
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3.5. STUDY LENGTH
This study will be accomplished in eight weeks.
3.6. METHODOLOGY
3.6.1. APPLICATION TECHNIQUE
Cosmetics and other topical products:
The product will be spread on a filter paper disk (0.05g/cm2), and the saline solution
(control) on another disk. The disks will be attached to the right or left side of the back and
covered with semi-permeable hypoallergenic tape. Some products, such as hair coloring or
hair straightening products, shampoos and toilet soaps, must be prepared and diluted
previous to being applied on the disks.
Non-woven fabrics and fibers:
Prior to the initiation of the study the test product will be cut into squares of
approximately 2.0 x 2.0 cm.
The squares will be moistened with saline solution, attached to the right or left sides of
the backs of the subjects and covered with a semi-occlusive hypoallergenic tape.
3.6.2. EVALUATION
The rating scale set by the International Contact Dermatitis Research Group (ICDRG)
will be used (WILKINSON, 1970; FISHER, 1995).
REACTION RESULT
0- none Negative (-)
1- slight erythema Doubtful (?)
2- clear erythema Positive (+)
3- erythema + edema + papules Positive (++)
4- erythema + edema + papules + vesicles Positive (+++)
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3.6.3. CUMULATIVE IRRITATION AND SENSITIZATION RESEARCH - RIPT (KLIGMAN
& WOODING, 1967; MARZULLI & MAIBACH, 1975)
The patches will be applied always to the same duly protected areas (either right or left
side of the backs of the subjects). The product will be applied every day, and the first
application will remain in contact with the skin for 48 hours. On the weekend, the patch will
remain on the site for 72 hours. The research will last three weeks, totaling fourteen
applications.
The product will be re-applied always to the same sites, and the reactions will be
recorded. After fourteen consecutive applications, a rest period of at least 14 days will be
followed, during which no patches will be applied. After this rest period, a single patch with
the sample will be applied either on the right or left side of the back of the subjects where
no patches have been applied before (virgin area). This patch will be removed after 48
hours of contact with the skin. The reactions will be recorded 30 minutes and 24 hours after
removal.
3.7. STUDY DESIGN
This will be a clinical single-blind randomized controlled study.
3.8. RESULTS
Any adverse reaction happening throughout the study period will be described on the
final report.
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4. REFERENCES
BARAN, R. & MAIBACH, H.I., (ed.). Cosmetic Dermatology, Baltimore, Willians &
Wilkins, 1994.
CONSELHO NACIONAL DE SAÚDE [NATIONAL HEALTH COUNCIL]. Resolução
196/96 do Ministério da Saúde. Diário Oficial, 16/10/1996.
DOOMS-GOOSSENS, A. Cosmetics as causes of allergic contact dermatitis. Cutis.
52: 316-320, 1993.
FISHER, A.A. Contact Dermatitis, 2a edition, Philadelphia, Lea & Febiger, 1995.
KLIGMAN, A.M. & WOODING, W.M. A method for the measurement and evaluation of
irritants of human skin. J.Invest.Derm. 49:78-94, 1967.
MARZULLI, F.N. & MAIBACH, H. I. The rabbit as a model for evaluating skin irritants: a
comparison of results obtained on animals and man using repeated skin exposures.
Fd. Cosmet. Toxicol. 13: 533-540, 1975.
PATHAK, M.A. & COL. Photobiology of melanin pigmentation: dose/response of
skin to sunlight and its contents. J.Am.Acad.Dermatol. 9: 724-733, 1983.
SAMPAIO, S.A.P. & RIVITTI, E.A. Dermatologia Básica, 2a edição, São Paulo, Artes
Médicas, 2000.
WILKINSON, D.S. & COL. Terminology of contact dermatitis. Acta Dermatovener
(Stockholm) 50: 287-292, 1970.
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Potentials - RIPT

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HISTORY OF REVISION
Revision Revision Date Event Description
04 11/27/2006 Inclusion of one more reading in the item 3.6.3.
Elaborated by: Approved by:
ALLERGISA pesquisa dermato-cosmética ltda. Cumulative Irritation and Sensitization
Potentials - RIPT