Clinical Safety Evaluation of Cosmetics

DEPARTMENT OF PHARMACEUTICAL SCIENCE DR.
HARI SINGH
GOUR VISHVIDIYALA SAGAR M.P.
ASSIGNMENT ON
BY- SHRIKANT THAKUR
(M. PHARAMA III SEM.)
Under the Supervision of
PROF. N.K. JAIN
Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

S.NO. TITLE PAGE NO.
1 INTRODUCTION 1-2
2 SAFETY TERMINOLOGY 3-4
3 COSMETIC SAFETY REGULATION 5-9
(a) USA safety regulation

(b) European safety regulation
(c) Japan safety regulation
4 Exposure and risk assessment consideration for cosmetic product and 10-11
ingredient
5 Ingredient ,toxicology and product safety 11-12
6 PROTOCOL FOR CLINICAL SAFETY TESTING 12-46

TESTS FOR SKIN IRRITATION
A) SKIN CORROSION TESTING
B) IRRITANCY TESTS
1) 48-Hour Irritancy Tests
2) Cumulative Irritancy Tests
3) Facial-Stinging Test
C) ANIMAL MODELS
1) Draize Rabbit Models
2) Modified Draize Irritation Method
D) HUMAN MODELS
1) Single-Application Patch Testing
2) Cumulative Irritation Testing
3) The Chamber Scarification Test
4) The Soap Chamber Test
5) Immersion Tests
6) Wash Tests

5 OCULAR IRRITATION TESTING
A) THE DRAIZE EYE-IRRITATION TEST
B) MODIFICATIONS OF THE IN VIVO EYE-IRRITATION TEST
The Low-Volume Eye Test
C) REPLACING THE ANIMAL TEST WITH IN VITRO
METHODS
1) Isolated Whole Eyes
2) Isolated Cornea Models
3) Other Test Systems
5 SENSITIZATION TESTS
6 PHOTOSENSITIVITY
1) Sunlight
2) UV-Induced Erythema
3) Phototoxicity Reactions
4) Phototoxicity Assays
5) Photoallergic Reactions
6) Photoallergenicity Assays
Reference 47

CLINICAL SAFETY EVALUATION OF COSMETICS
(1) INTRODUCTION
The term ‘‘cosmetic’’ is defined in Section 201 (i) of the 1938 Food, Drug, and
Cosmetic Act (FD&C Act) as:
1) Articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or
otherwise applied to the human body or any part thereof for cleansing, beautifying,
promoting attractiveness, or altering the appearance, and
2) Articles intended for use as a component of any such articles; except that such term shall
not include soap . . .
One of the skin’s primary physiological functions is to act as the body’s first line of
defense against exogenous agents. However, the skin should not be viewed as a flawless
physicochemical barrier. Many low–molecular weight compounds are capable of penetrating
this barrier. When toxic agents (such as irritants or allergens in cosmetic products) permeate
it, the resulting adverse effects may cause considerable discomfort to the consumer. Even
minor disturbances of the skin surface can produce discomfort, especially in the facial area
which has an extensive network of sensory nerves. Moreover, because most cosmetics are
applied to the highly permeable facial skin, the majority of reported cosmetic reactions occur
in the face. Therefore, safety with regard to cosmetic products is a vital issue.
Animal testing for dermatotoxic effects has come under increasing scrutiny and
criticism from animal-rights activists for being inhumane and unnecessary. Legislation is
pending that would restrict the marketing of products containing ingredients that have been
tested on animals. The often conflicting needs to protect worker and consumer safety, comply
with regulatory statutes, and reduce animal testing procedures has led to a significant effort
within industry, government, and academia to develop alternative testing methods for
assessing the skin corrosion and irritation hazard of chemicals and product formulations
without reliance on animal test procedures .
United States and international regulations require that chemicals be properly classified,
labeled, packaged, and transported on the basis of their potential to damage or destroy tissue,
including the speed with which such tissue-destructive reactions occur . The most common
animal testing methods used over the years for the evaluation of chemical corrosion potential
are all based on the original method by Draize. We, as well as other laboratories, have been
active in the development of alternative procedures for skin-corrosion testing.

Recently, several test methods have been evaluated in an international validation program.
Certain of these methods should provide short-term and cost effective alternatives to the
Draize procedure, at the same time providing experimental systems for developing a better
mechanistic understanding of the process of skin corrosion.
Skin irritation, by definition, is a less severe response than corrosion, but can span a
range of responses from near corrosive at one extreme to weak cumulative or neurosensory
responses at the other. The development of alternatives for skin irritation testing has lagged
behind that of skin corrosion testing, likely because of the greater urgency of developing
alternatives for the more severe skin responses and because of the range of responses
encompassed within the ‘‘skin irritation’’ umbrella. Currently, the irritation hazard potential
of chemicals is often determined through use of the same Draize procedure used for corrosion
testing, the difference being mainly in the length of chemical exposure, with results used to
determine labeling requirements for chemicals and products according to European
Commission (EC) directives. For noncorrosive chemicals, there has been a recent effort to
develop and promote the use of clinical patch testing methods for a more relevant assessment
of chemical skin irritation potential than that provided by the rabbit test.
The manufacture, transport, and marketing of chemicals and finished products require
the prior toxicological evaluation and assessment of skin corrosivity and skin irritation that
might result from intended or accidental skin exposure. Traditionally, animal testing
procedures have provided the data needed to assess the more severe forms of skin toxicity, an
assessment requiring extrapolation of the data from the animal species to humans. Current
regulations may require animal test data before permission is granted for the manufacture,
transport, or marketing of chemicals, as well as for the formulations that contain them.
The most common animal testing methods used over the years for the evaluation of
chemical corrosion potential are all based on the original method by Draize. We, as well as
other laboratories, have been active in the development of alternative procedures for skin-
corrosion testing. Recently, several test methods have been evaluated in an international
validation program. Certain of these methods should provide short-term and costeffective
alternatives to the Draize procedure, at the same time providing experimental systems for
developing a better mechanistic understanding of the process of skin corrosion.
(2) SAFETY TERMINOLOGY

Contact dermatitis
This is a nonspecific term used to describe any inflammatory skin disease resulting from
contact with an irritant or allergenic substance. Whatever the causative agent, the clinical
features are similar: itching, redness, and skin lesions. It is also often used (inaccurately) as a
synonym for allergic contact dermatitis (ACD).
Irritant contact dermatitis (Irritation)
Irritant contact dermatitis (ICD) is a term given to a complex group of localized inflammatory
reactions that follow nonimmunological damage to the skin. The inflammation may be the
result of an acute toxic (usually chemical) insult to the skin, or of repeated and cumulative
damage from weaker irritants (chemical or physical). There is no definite laboratory test for
ICD—diagnosis is by clinical morphology, of course, and appropriate negative patch-test
results.
Allergic contact dermatitis
ACD occurs when a substance comes into contact with skin that has undergone an acquired
specific alteration in its reactivity as a result of prior exposure of the skin to the substance
eliciting the dermatitis. The skin response of ACD is delayed, immunologically mediated
(Type IV), and consists of varying degrees of erythema, edema, papules, and papulovesicles
Patch testing is the gold standard; it is imperative for proving ACD, determining the actual
allergen, predictive testing, i.e., determining ‘‘safe’’ materials for the consumer, and
exclusion of other diagnoses.
Photoirritant contact dermatitis (Photoirritation/Phototoxicity)
Photoirritant contact dermatitis (PICD) is a chemically induced nonimmunological skin
irritation requiring light. This reaction will occur in all individuals exposed to the chemical–
light combination. The clinical picture is that of erythema, edema, or vesiculation in sun-
exposed areas, resembling exaggerated sunburn. This may be followed by
hyperpigmentation, or if the exposure is repeated, scaling and lichenification may occur.
Bergapten, a component of bergamot oil, which used to be a popular ingredient in perfume, is
a potent photoirritant that causes berloque dermatitis.
Photoallergic contact dermatitis

Photoallergic contact dermatitis (PACD) is an immunological response to a substance that
requires the presence of light. The substance in the skin absorbs photons and is converted to a
stable or unstable photoproduct, which binds to skin proteins to form an antigen, which then

elicits a delayed hypersensitivity response. Examples of photoallergens present in cosmetics
are musk ambrette and 6-methylcoumarin, which are present in fragrances. Photopatch
testing is the diagnostic procedure for photoallergy.
Contact Urticaria Syndrome
Contact urticaria syndrome (CUS) represents a heterogeneous group of inflammatory
reactions that appear, usually within a few minutes to an hour, after contact with the eliciting
substance. Clinically, erythematous wheal-and-flare reactions are seen, and sensations of
burning, stinging, or itching are experienced. These are transient, usually disappearing within
a few hours. In its more severe forms, generalized urticaria or extracutaneous manifestations,
such as asthma, nausea, abdominal cramps, and even anaphylactic shock, may occur.
Diagnosis may be achieved by a variety of skin tests—the open test is the simplest of these
and is the ‘‘first-line’’ test. CUS may be divided into two categories on the basis of
pathophysiological mechanisms: nonimmunological and immunological. There are also
urticariogens that act by an uncertain mechanism.
Sensitive skin
This term is a neologism for consumers’ feelings about their intolerance to a variety
of topical agents, be it topical medicaments or cosmetics and toiletries. Individuals present
with very similar complaints, such as burning, stinging or itching sensations, on contact with
certain cosmetic products that most people do not seem to react to, sometimes accompanied
by slight erythema or edema. They frequently complain of a ‘‘tight feeling’’ in their skin,
secondary to associated dry skin.
Sensitive skin describes the phenotype noted by the consumer; mechanisms include
sensory irritation, suberythematous irritation, acute and cumulative irritation, contact
urticaria, allergic contact dermatitis, as well as photoallergic and phototoxic contact
dermatitis. Sensory irritation and suberythematous irritation are believed to be far more
common than the remaining mechanisms. Cosmetic Intolerance Syndrome The term cosmetic
intolerance syndrome (CIS) is applied to the multifactorial syndrome in which certain
susceptible individuals are intolerant of a wide range of cosmetic products.
CIS is thought to be caused by one or more underlying occult dermatological
conditions, such as subjective irritation, objective irritation, allergic contact dermatitis,
contact urticaria, or subtle manifestations of endogenous dermatological diseases, such as
atopic eczema, psoriasis, and rosacea. Status Cosmeticus Status cosmeticus is a condition in
which every cosmetic product applied to the face produces itching, burning or stinging,

rendering the sufferer incapable of using any cosmetic product. The patient’s history usually
includes ‘‘sensitivity’’ to a wide range of products. This diagnosis is only declared after a full
battery of tests have proved negative, and may be considered the extreme end of the spectrum
of sensitive skin.
(3) COSMETIC SAFETY REGULATION
(a) USA safety regulation
In the U.S., the Food, Drug and Cosmetic Act designate the Food and Drug Administration as
the agency responsible for cosmetic safety. For this purpose, the FDA has defined cosmetics
as "articles to be rubbed, poured, sprinkled, or sprayed on or introduced into, or otherwise
applied to the human body or any part thereof for cleansing, beautifying, promoting
attractiveness, or altering the appearance, and articles intended for use as a component of any
such articles; except that such term shall not include soap." The exemption for soap applies
only to a composition consisting of an alkali metal salt of a fatty acid that is intended only for
cleansing. Certain cosmetic products that are "intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease" are classified by FDA as drugs. This category
includes sunscreen products, anticavity toothpastes, antiperspirants (as opposed to
deodorants, which are "cosmetics"), and antidandruff preparations, "medicated" skin lotions
and liquids, skin protectants, and hair restorers. Under the regulations, the active ingredients
in cosmetic drug products must be safe and effective according to the appropriate monograph
covering the claimed indication.
The only category of cosmetic ingredient subject to FDA approval is colors used for
purposes other than dyeing the hair. There are only 36 "certified colors” and 23 "permitted"
colors available for general use in cosmetics, and a further 7 that are permitted only for
specific uses. In addition, a number of lakes of the soluble certified colors are available. For
all other cosmetic ingredients, the safety evaluation is the responsibility of the manufacturer.
In order to assist its members in this, the Cosmetic, Toiletry, and Fragrance Association
(CTFA) established in 1976 the Cosmetic Ingredient Review (CIR) to review all the available
data on an ingredient and todecide whether the ingredient is safe under the conditions of use.
The CIR expert panel comprises six independent scientists as voting members and a
nonvoting member representing each of the CTFA, the FDA, and the consumer. By 1996, the
CIR had reviewed over 600 ingredients, finding 64% to be safe as used, 27% to be safe for
use under defined conditions, 8% to have insufficient data, and five ingredients to be unsafe.

The CIR expert panel currently requests the following as a minimum data requirement for
reviewing the safety of the cosmetic ingredients that are on its priority list:
1. Current concentration of use data.
2. Chemistry data, including method of manufacture and impurities.
3. UV-absorption data; if absorption occurs in the UVA or UVB range, photosensitization
data are needed.
4. Skin-irritation and -sensitization data at concentrations of use in humans.
5. Dermal-absorption data; if significant dermal absorption occurs, 28-day dermal-toxicity
and development-toxicity data are needed.
6. Two genotoxicity studies, one using a mammalian system; if positive, a 2-year dermal
carcinogenicity assay performed using NTP methods is needed.
(b) European Union safety regulation

In the European Union, cosmetics are regulated under the Cosmetics Directive (76/768/EEC)
of 1976 and amendments thereto. For the purpose of regulation, cosmetics are more broadly
defined than in the United States, viz. "any substance or preparation intended to be placed in
contact with various external parts of the human body (epidermis, hair system, nails, lips and
external genitalia) and with the teeth or mucous membranes of the oral cavity with a view
exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or
correcting body odors and/ or protecting them or keeping them in good condition."
This definition covers many of the product categories that are considered to be over-the-
counter drugs in the U.S. In common with the United States, cosmetics do not require
premarket clearance in Europe. However, certain ingredients, notably colorants (other than
hair dyes), preservatives, and sunscreens, do require approval before they can be used in
cosmetic products. The list of permitted colorants (Annex IV) is broader than the U.S. list,
comprising 157 materials. The approval process involves the submission of data via
COLIPA, the European trade association, to the European Commission. The data is evaluated
by a group of independent experts who make up the Scientific Committee for Cosmetology
and Non-Food Products (SCCNFP) and who decide whether the ingredient can be accepted
for listing in the appropriate Annex. Data requirements are similar to those listed above for
the CIR.
The General Toxicological Requirements for Cosmetic Ingredients (1996 revision) state :

When requested, the manufacturer shall provide the Commission with the information set out
below:
1. Acute toxicity
2. Dermal absorption
3. Dermal irritation
4. Mucous membrane irritation
5. Skin sensitization
6. Sub-chronic toxicity
7. Mutagenicity
8. Phototoxicity and Photomutagenciny (in case of UVfight absorbing substances)
9. Human data (if available)
10. Toxicokinetics
11.Teratogenicity,Reproduction toxicity, Carcinogenicity, and additional Genotoxicity.
Additionally, there is now a legal requirement, forrealized in Article 7a of the sixth
amendment to the Cosmetics Directive, that cosmetic companies hold a technical dossier of
information on each of their products. This requirement applies to every cosmetic product,
both retail and professional, including imported products. This dossier must include
information or product composition, specifications, and method of manufacture, as well as an
assessment of product safety carfled out by an appropriately qualified expert. The assessment
must take account of the general toxicological profile of the ingredients, their chemical
structures, and the potential levels of exposure.
The SCC is also responsible for recommending the listing of substances prohibited for
use in cosmetics (Annex II) and substances that can be used with certain limitations with
regard to product category, concentration, and/or special labeling requirements (Annex III).In
addition to these requirements, the EU regulation for the Notification of New Substances
must be followed by companies introducing new chemicals. These regulations state that "a
material is considered to be a new substance within the EU if it is not listed in EINECS
(European Inventory of Existing Chemical Substances)." EINECS is a closed list (closed in
1981), that is, no new substances can now be added. Substances not in EINECS must be
notified according to Council Directive 92/32/EEC (seventh amendment to the EC Dangerous
Substances Directive) prior to marketing in the EU. Notified substances are issued in
ELINCS (European List of New Chemical Substances). ELINCS is an open list, that is, new
substances may be added once notified according to the Directive. The precise contents of a

notification depend on the quantity of the substance to be placed or already placed on the
market. The notification system is banded; the information required increasing as the quantity
of a substance placed on the market increases.
(C) JAPAN SAFETY REGULATION

The Japanese regulation of cosmetics is the most restrictive. While the definition of a
cosmetic is similar to that in the U.S., the number of product types considered to be "quasi-
drugs" (analogous to cosmetic drugs or O.T.C. products) is much greater. Thus, in addition to
the U.S. categories, products to combat bad breath, bath preparations, hair dyes, permanent
waves, talcum powder, depilatories, shaving lotions, and skin packs are considered "quasi-
drugs." In Japan, all cosmetic products are subject to premarket approval by the Ministry of
Health and Welfare and can contain only those ingredients included in the Comprehensive
Licensing Standards of Cosmetics by Category (CLS) and these must conform to certain
defined specifications. New ingredients can be added to the list on the basis of the submission
of appropriate toxicological data. For quasi-drugs, there are lists of permitted active
ingredients for use in hair dyes, permanent waves, and medicated toothpastes. Lists for other
categories are being developed. For countries other than those discussed here, ingredients
acceptable in the United States and/or Europe would usually be acceptable.
(4) EXPOSURE AND RISK ASSESSMENT CONSIDERATIONS FOR

COSMETICPRODUCTS AND INGREDIENTS
The toxicological evaluation of cosmetic ingredients serves usefully to identify potential
hazards, which then need to be put into appropriate perspective via a risk assessment that
incorporates an estimation of exposure and a calculation of the margin of safety by relating
the human exposure dose to the noobserved- adverse-effect level obtained from animaltests.
Key exposure parameters include the amount of product and the concentration of ingredient
applied, the route and site of application, and the frequency and duration (e.g., rinse off or
leave on) of application. Although, there are no good quantitative data that adequately reflect
the variations in consumer habits and practices, particularly for several of the newer cosmetic
types and forms, some conventions that are used for cosmetic exposure assessment are
given in Table. Because demographics and cultural differences would be expected to have a
significant influence, particularly on use frequency or on amounts, there may be considerable
variations in the actual exposures, which often may be remarkably overestimated using
conservative exposure assumptions.While the intended route of exposure for most cosmetic
products, by definition, is topical, inadvertent exposure may also occur via the mucous
membranes, orally, or via inhalation for aerosol or powdered cosmetic forms. Inhalation may
also be a potential exposure route for ingredients, including contaminants, that may be
volatilized under use conditions. In some cases, such as for hair cosmetics, skin (scalp)
exposure is an inadvertent but unavoidable consequence of product use. While it is important
to consider the relative contribution of various exposure routes to an ingredient on a case-by-
case basis, oral and inhalation exposures would generally be considered to potentially
engender higher risks than would topical application.
(5) INGREDIENT, TOXICOLOGY AND PRODUCT SAFETY

While the toxicologic potential of cosmetic ingredients, some of which have a broader use in
other consumer, industrial, and pharmaceutical products or in food applications, has
traditionally been characterized in conventional animal studies such as those listed earlier in
this chapter, the European Union sixth amendment to the Cosmetics Directive has brought
increased emphasis on the use of nonanimal study alternatives for both ingredients and
finished products.The use of selected individual in vitro tests or test batteries shows good
promise for cosmetic materials in certain areas, such as skin irritation, percutaneous
absorption, and photoirritation or photosensitization.
While considerable progress has been made in understanding their utility and
limitations, there is less of a consensus on alternative methods for testing eye irritation and,
particularly, skin sensitization. It is generally acknowledged that for the evaluation of
systemic effects, such as studies of chronic toxicity, reproductive toxicity, and immunotoxic
potential as currently required or requested by international cosmetic regulations and
guidelines, there appear to be no near-term alternatives to animal testing.

Fig.1 Complementary approaches for safety testing of cosmetics
In many circumstances, finished product testing may be accomplished without or with
minimal use of animals by a stepwise approach involving the thorough evaluation of the
available toxicological data on the ingredients and consideration of prior experience with
related ingredients and formulations ("paper" toxicology); the conservative application of
controlled clinical studies with appropriate ethical considerations and close medical
supervision; and the use of in vitro methods that have been validated for a specific purpose
with the appropriate known "benchmark" materials. This scheme, incorporating the option of
conducting limited animal tests under selected circumstances to resolve outstanding issues or,
alternate Cosmetic Toxicology (Development in show in figure.1)
An important element in such a process is the inclusion of feedback loops to address
issues that may develop during the various stages of product development, including the
exposure of a broader numbers of individuals under less controlled circumstances, such as
consumer-use tests or test marketing. Postmarket surveillance is an important element of
cosmetic product safety in that low-frequency events may only be ascertained after broad
consumer use. The viability of maintaining the option to include animal tests of finished
products, even on a selected basis, remains to be determined by future international
regulatory activities and by the policy and practices of individual cosmetic companies.

(6) PROTOCOL FOR CLINICL SAFETY TESTING
(a) TESTS FOR SKIN IRRITATION
The major problem of human testing for skin irritation or compatibility is the extended
duration and relatively high cost of this clinical testing. In vitro skin irritation test methods
could be used to rank chemicals or formulations for skin irritation potential, even at the low
end of the irritation spectrum. These methods (and others under development elsewhere)
might provide for short-term, cost-effective approaches for screening chemicals and product
formulations of interest, so that only those with satisfactory skin irritation profiles would
undergo longer and more costly clinical evaluations.
A) Skin corrosion testing
Assay Systems
Screening of chemicals for skin corrosion properties in vitro has followed three general
formats. These include
1) Changes in electrical conductance across intact skin (rat or human),
2) Breaching of noncellular biobarriers, and
3) cellular cytotoxicity in skin or epidermal equivalent cell culture systems.
Each of these systems has been subject to intra- and interlaboratory development, evaluation,
and validation.
Skin corrosivity has been distinguished from skin irritation in two important ways.
 First, corrosive skin reactions generally occur soon after chemical exposure and are
irreversible.
 Second, it is thought that the major processes leading to chemical corrosivity are more
commonly physicochemical in nature rather than the result of inflammatory biological
events, although inflammation is a common consequence of skin corrosion.
Initial efforts to develop a screening test for skin corrosivity examined the effects of chemical
exposure on barrier function of skin through assessment of changes in the resistance of the
exposed skin to transmission of electric current. This test method, called transcutaneous
electrical resistance (TER), was based on early studies of the electrical resistance properties
of skin and has been developed as a corrosivity assay over the past 15 years using either rat or
human skin.

 In the TER assay, full thickness skin is stretched over a hollow tube opening with
the stratum corneum side exposed to the lumen. Test materials are applied to the
skin surface for varying periods of time while the skin is immersed in buffer.
 After chemical exposure, the electrical resistance of the skin is measured. TER
values empirically established as corrosion thresholds have been set at 4 K ohms
for rat skin and 11 K ohms for human skin.
 The current validation status of this assay is described in the following section.
The biobarrier destruction assay approach for corrosivity testing is exemplified by
the commercial Corrositex assay system manufactured by In Vitro International
(Irvine, CA).
 Like the TER assay, the premise here is physicochemical destruction of a barrier
by direct chemical action of a test material. Instead of intact stratum corneum, the
Corrositex assay relies on a macromolecular protein matrix as the barrier.
 Chemicals that breach this barrier come into contact with an underlying chemical
detection system (CDS).
 A color change indicates penetration of the test material into the CDS. The speed
with which the color change occurs after application of the chemical to the
biobarrier is proportional to the severity of corrosive action.
These have included simple submerged cell cultures, submerged cell co cultures
incorporating more than a single cell type, and, more recently, the development of full
thickness skin and epidermal equivalent systems. The latter are characterized by stratified
epidermal cell layers and a multilayered stratum corneum. The full-thickness culture systems
also have different types of cellular and macromolecular matrices serving as a dermal
element.
B) Irritancy tests
Ethically, any human test procedure must be approached with the safety of the subjects as a
priority. To this end, procedures have been developed that minimize the risk of serious
cutaneous reactions. These procedures use a stepwise approach of increasing times o f
exposure and bioengineering methods to minimize the danger and discomfort to the subjects
involved and to minimize the potential risk of irreversible skin damage. In most situations,
however, the product being tested is not novel and is related to previously studied
formulations. In this situation, the irritant potential can be determined by simple patch tests,

such as the 48-h patch test or the 2- to 3-week cumulative irritancy test.
1. 48-Hour Irritancy Tests
The 48-h irritancy test consists of two sequential 24-h applications under occlusion to the
same site. The break in the application is to premit assessment and possible termination of the
application, should unexpected severe reactions occur. Assessments are made at the end of
the 48-h period, and again at 72 h. The latter assessment time serves to check that any
reactions are subsiding satisfactorily and to catch any late reactions that may develop only
several hours after the removal of the patch. It may be appropriate to include both negative
and positive controls in this type of test, particularly if experience of human testing with the
class of product is minimal.
Negative controls usually consist of the empty application chamber or the adhesive base
without active agent. Positive controls may be known irritants, such as 0.5% sodium lauryl
sulfate. It may also be desirable to compare results with existing or marketed products at this
stage, so that if some reactions occur the relation to what is currently used is clear. An
important variable, which is not possible to exclude from this type of test, is intraindividual
variability in terms of cutaneous reactivity. This may be due to an individual’s skin type, age,
or hormonal status. To compensate for this, a reasonable number of individuals, usually 25–
30, must be used in this type of study.
The 48-h irritancy test is sometimes referred to as a primary irritancy test. This is based
on the definition that a primary irritant produces a reaction on first contact, and a secondary
irritant produces a reaction only after repeated exposure. This is confusing because,
depending on the concentration, a product can be both a primary and secondary irritant.
Irritant potential is always dependent on conditions of exposure and is never absolute.
Prolonged application of something apparently as innocuous as water can produce a
dermatitis, given the right circumstances.
The interpretation of the results of the 48-h irritancy test is important. A negative result
does not necessarily mean that the product is safe to use. Many irritants reveal themselves
only after repeated application. As many products are, by virtue of their function, intended to
be repeatedly applied to the skin, this short-term type of test is patently inadequate as a safety
test. The 48-h test does however serve as a simple screening device and this is its primary
function. It will indicate those products with a high potential for causing cutaneous irritancy.
Several products can be tested at the same time, and information can be obtained quickly
during product development.

2. Cumulative Irritancy Tests
Cumulative irritancy assays permit a more-sophisticated ranking of products and enable the
comparison and classification of the weaker types of irritants, into which category most
topical formulations and transdermal systems fall. There are several variations on the
cumulative irritancy assay.
The main variations are the type of application patch, the number of subjects, and the
duration of the study. The central feature of these assays is that the product is repeatedly
applied to the same site such that the exposure is exaggerated. In addition, the site is
occluded, such that the barrier function of the skin is compromised, allowing greater
penetration of potential irritants. By virtue of this exaggerated exposure, it is hoped that the
information gained from a relatively small panel of individuals (usually 25–30 subjects) will
predict the likely behavior under conditions of everyday consumer use.
The cumulative irritancy assay normally consists of either a 14- or 21-day application
period. There is still some debate over whether the shorter time period is sufficient to detect
very weak irritants, and the decision between the two time periods is usually made according
to the product being tested and its proposed usage. Thus, for products that are in prolonged
daily contact with the skin (e.g., dermal and transdermal therapeutic systems), the longer test
procedure may be desirable to give the necessary reassurance of product safety.
Another variable in the cumulative assay is the number of applications over the test
period. Some protocols call for daily applications and assessments (Monday to Friday, but
not weekends), others for application and assessment every 2 to 3 days (Monday,
Wednesday, and Friday). In both protocols the subjects continue to wear the patches over the
weekend. There is very little to choose between the two schedules because both give
continuous contact over the test period. The advantage of the daily regimen is that if severe
reactions develop quickly, then the application can be stopped and, therefore the subject is
exposed to minimal risk.
However, this is not likely with the types of products generally tested, especially if
they have been subjected to a 48-h test previously. The more frequent patch application and
removal schedule has the distinct disadvantage that trauma to the skin surface by repeatedly
applying and removing adhesive dressings may lead to severe ‘‘plaster’’ reactions that mean
that the subject is no longer able to continue with the study. This is obviously more likely to
happen in the 21-day protocol.
The nature of the application patch is also a variable in this assay. Many of the original assays

used gauze pads (Webril pads) covered with impermeable tape such as Blenderm. We prefer
an alternative system, 12-mm aluminum Finn chambers on Scanpore tape. The advantages
and disadvantages of this system are as follows:
 Scanpore tape is not occlusive and minimizes the ‘‘plaster’’ reactions seen
with occlusive tapes, such as Blenderm; the chambers have a fixed volume and
when used with filter papers can be used for liquids; an impression of the
raised rim is clearly seen on removal of the patch, giving the reassurance that
occlusion was continuous and that the subject had complied with the
conditions of the study. The chambers are available commercially as strips of
chambers at fixed intervals that are perforated to allow subdivision.
 A possible disadvantage of the Finn chamber system is that the area of
2
application (12-mm diameter = 113 mm ) is less than the gauze pad. However,
although the area of application is important in terms of the intensity of the
2
response, above a certain level (approximately 100 mm ) this effect is
minimal. With transdermal systems the patch itself is occlusive, and by its
nature will produce cutaneous reactions such as folliculitis.
 An additional problem when testing transdermal systems is the
pharmacological load on the subject. Topical formulations for treating skin
diseases are designed to deliver relatively small amounts of drug locally. In
contrast, transdermal systems are designed to deliver large doses
systematically through a relatively small area of skin.
 The amount absorbed from a topical agent in a 12-mm–Finn chamber is
usually negligible as far as the wellbeing of the subject is concerned. In
contrast, a transdermal system will deliver a large dose over a short time
period, and this must be taken into account when designing the study.
Thus, a transdermal estrogen product is ideally tested for irritancy in postmenopausal
women. In this situation the study almost becomes a phase I clinical study and pre and
poststudy medical screening is advisable. An alternative approach is to reduce the size of the
transdermal patch, such that the pharmacological dose carries negligible risk, but the area is
such that the irritant properties can be assessed. Evaluation and interpretation of any irritant
response also varies according to the test procedure. Grading scales for erythema vary from
the simplest 0–4 scale (none, mild, moderate, severe), to more complex scales with half-point
divisions.

The scale we used has evolved over several years experience in assessing these types of
reaction. The irritancy potential of a product is generally assessed by the number and severity
of the reactions occurring during the study, in particular the number of grade 2 or higher
reactions. A cumulative index of irritancy can be derived, and this can be related to previous
assays and assays on marketed or competitor products. An alternative approach is to
determine the time at which half of the subjects have reacted to a product in a procedure
analogous to the LD50determination. A range of concentrations needs to be tested and the
proportion of subjects reacting to each concentration is plotted against the time of reaction
and, by a curve fitting procedure, the time when half the subjects have shown a reaction is
recorded. A concentration at which half of the subjects react in this type of test can be
determined and absolute comparisons made.
Human Patch Test-Grading Scale
0 No reaction
0.5 Slight, patchy erythema
1 Slight uniform erythema
2 Moderate, uniform erythema
3 Strong erythema
4 Strong erythema, spreading outside patch
5 Strong erythema, spreading outside patch with either swelling or vesiculation
6 Severe reaction with erosion
3. Facial-Stinging Test
Products may pass tests such as the cumulative irritancy test, but still cause problems for
the consumer. Disagreeable sensations may occur, particularly when products are applied
to sensitive areas such as the face. A not infrequent complaint is that of stinging or burning
or itching after application. Signs of irritation, such as erythema or scaliness, may not
necessarily occur in this situation. Part of the reason for such sensations is that facial skin
is very permeable and has a rich nerve supply. It is also exposed to weathering and a
constant bombardment of cosmetics and cleansers.These may all contribute to increased
sensitivity of this area. A method to assess the stinging capacity of topical materials has
been described. Subjects who are sensitive to the stinging sensation of lactic acid are
selected as panelists for testing new products. Their subjective responses are noted, and a
cumulative score is derived that is considered indicative of the potential for stinging in the

general population.
C) ANIMAL MODELS
1) Draize Rabbit Models
The Draize model and its modifications are commonly used to assay skin irritation using
albino rabbits. Various governmental agencies have adopted these methods as standard test
procedure. The procedure adopted in the U.S. Federal Hazardous Substance Act (FHSA) is
described and compares this method some other modifications of the Draize model.
Draize-FHSA Models
Number of animal’s 6 albino rabbits (clipped)
Test sites 2 _ 1 inch2 sites on dorsum
One site intact, the other abraded, e.g.,
with hypodermic needle
Test materials Applied undiluted to both test site
Liquids: 0.5 mL
Solids/semisolids: 0.5g
Occlusion 1 inch2 surgical gauze over each test site
Rubberized cloth over entire trunk
Occlusion period 24 hours
Assessment 24 and 72 hours
Visual scoring system

Draize used this scoring system to calculate the primary irritation index (PII). This is
calculated by averaging the erythema scores and the edema scores of all sites (abraded and
nonabraded). These two averages are then added together to give the PII value. A value of
less than 2 was considered nonirritating, 2 to 5 mildly irritating, and greater than 5 severely
irritating. A value of 5 defines an irritant by Consumer Product Safety Commission (CPSC)
standards. Subsequent laboratory and clinical experience has shown the value judgments (i.e.,
non-, mild, and severely irritating) proposed in 1944 requires clinical judgment and
perspective, and should not be viewed in an absolute sense. Many materials irritating to the
rabbit may be well tolerated by human skin.
Draize-FHSA Scoring System
Erythema and eschar formation Score
1) No erythema 0
2) Very slight erythema (barely perceptible) 1
3) Well-defined erythema 2
4) Moderate to severe erythema 3
5) Severe erythema (beet redness) to slight eschar for-
mation (injuries in depth) 4
Edema formation
1) No edema 0
2) Very slight edema (barely perceptible) 1
3) Slight edema (edges of area well defined by definite 2
raising)
4) Moderate edema (raised _1 mm) 3
5) Severe edema (raised _1 mm and extending beyond 4
the area of exposure)
Examples of Modified Draize Irritation Method

Requirment/ Draize FHSA DOT FIFRA OECD
Condition
Number of animals 3 6 6 6 6
Abrasion/intact Both Both Intact 2 of each Intact

Dose liquids 0.5 mL 0.5mL 0.5mL 0.5 mL 0.5mL
undiluted undiluted undiluted
Dose solids in 0.5 g 0.5g moistened 0.5g 0.5 g 0.5 g
solvent moistened
Exposure period (h) 24 24 4 4 4
Examination (h) 24, 72 24, 72 4, 48 0.5,1, 24, 0.5, 1, 24,

48, 72 48, 72
Removal of test Not Not Specified Skin washed Skin wiped Skin washed
materials Specified
Excluded from — — — Toxicmaterials Toxicmaterials
Testing pH<2 >11.5 pH <2 or 11.5
Abbreviations: FHSA, Federal Hazardous Substance Act; DOT, Department of

Transportation; FIFRA, Federal Insecticide, Fungicide and Rodenticide Act; OECD,
Organization for Economic Cooperation and Development.
Although the Draize scoring system does not include vesiculation, ulceration, and severe
eschar formation, all of the Draize-type tests are used to evaluate corrosion as well as
irritation. When severe and potentially irreversible reactions occur, the test sites are further
observed on days 7 and 14, or later if necessary.
2) Modified Draize Irritation Method
Modifications to the Draize assay have attempted to improve its prediction of human
experience. The model is criticized for inadequately differentiating between mild and
moderate irritants. However, it serves well in hazard identification, often overpredicting the
severity of human skin reactions. Therefore, Draize assays continue to be recommended by
regulatory bodies for drugs and industrial chemicals.
D) HUMAN MODELS
Human models for skin irritation testing are species relevant, thereby eliminating the
precarious extrapolation of animal and in vitro data to the human setting. As the required test
area is small, several products or concentrations can be tested simultaneously and compared.
Inclusion of a reference irritant substance facilitates interpretation of the irritant potential of
the test substances. Prior animal or in vitro studies, depending on model relevance and

regulatory issue, can be used to exclude particularly toxic substances or concentrations before
human exposure.
1) Single-Application Patch Testing
The National Academy of Sciences (NAS) outlined a single-application patch test procedure
determining skin irritation in humans. Occlusive patches may be applied to the intrascapular
region of the back or the volar surface of the forearms, using a relatively nonocclusive tape
for new or volatile materials. More occlusive tapes or chambers generally
increase the severity of the responses. A reference material is included in each battery of
patches.
The exposure time may vary to suit the study. NAS suggests a 4-hour exposure period,
although it may be desirable to test new or volatile materials for 30 minutes to 1 hour. Studies
longer than 24 hours have been performed. Skin responses are evaluated 30 minutes to 1 hour
after removal of the patch, using the animal Draize scale (Table 2) or similar.
Kligman and Wooding described statistical analysis on test data to calculate the IT50 (time
to produce imitation in 50% of the subjects) and the ID50 (dose required to produce irritation
in 50% of the subjects after a 24-hour exposure).
Robinson et al. suggested a 4-hour patch test as an alternative to animal testing. Assessing
erythema by visual scoring, they tested a variety of irritants on Caucasians and Asians. A
relative ranking of irritancy was obtained using 20% SLS as a benchmark.
McFadden et al. investigated the threshold of skin irritation in the six different skin types.
Again using SLS as a benchmark, they defined the skin irritant threshold as the lowest
concentration of SLS that would produce skin irritation under the 4-hour occluded patch
conditions. They found no significant difference in irritation between the skin types.
2) Cumulative Irritation Testing

Lanman et al. and Phillips et al. described a cumulative irritation assay, which has become
known as the ‘‘21-day’’ cumulative irritation assay. The purpose of the test was to screen
new formulas before marketing. A 1 inch square of Webril was saturated with liquid of 0.5 g
of viscous substances and applied to the surface of the pad to be applied to the skin.The patch
was applied to the upper back and sealed with occlusive tape. The patch is removed after 24
hours, and then reapplied after examination of the test site. This is repeated for 21 days and
the IT50 can then be calculated. Note that the interpretation of the data is best done by
comparing the data to an internal standard for which human clinical experience exists.

Modifications have been made to this method. The chamber scarification test (see the
following) was developed to predict the effect of repeated applications of a potential irritant
to damaged skin, rather than healthy skin. The cumulative patch test described above had
failed to predict adverse reactions to skin damaged by acne or shaving, or sensitive areas such
as the face. Wigger-Alberti et al. compared two cumulative models by testing skin reaction to
metalworking fluids (MWF). Irritation was assessed by visual scoring, transepidermal water
loss, and chromametry. In the first method, MWF were applied with Finn Chambers on the
volunteers’ midback, removed after 1 day of exposure, and reapplied for a further 2 days. In
the second method, cumulative irritant contact dermatitis was induced using a repetitive
irritation test for 2 weeks (omitting weekends) for 6 hours per day. The 3-day model was
preferred because of its shorter duration and better discrimination of irritancy.
For low-irritancy materials in which discrimination is not defined with visual and palpatory
scores, bioengineering methods (i.e., transepidermal water loss) may be helpful.
3) The Chamber Scarification Test
This test was developed to test the irritant potential of products on damaged skin. Six to eight
1 mm sites on the volar forearm were scratched eight times with a 30-gauge needle without
causing bleeding. Four scratches were parallel and the other four are perpendicular to these.
Duhring chambers, containing 0.1 g of test material (ointments, creams, or powders), were
then placed over the test sites. For liquids, a fitted pad saturated (0.1 mL) may be used.
Chambers containing fresh materials are reapplied daily for 3 days. The sites are evaluated by
visual scoring 30 minutes after removal of the final set of chambers. A scarification index
may be calculated if both normal and scarified skin are tested to reflect the relative degree of
irritation between compromised and intact skin; this is the score of scarified sites divided by
the score of intact sites. However, the relationship of this assay to routine use of substances
on damaged skin remains to be established. Another compromised skin model, the arm
immersion model of compromised skin, is described in the following immersion tests section.
4) The Soap Chamber Test
Frosch & Kligman proposed a model to compare the potential of bar soaps to cause
‘‘chapping.’’ Standard patch testing was able to predict erythema, but unable to predict the
dryness, flaking, and fissuring seen clinically.
 In this method, Duhring chambers fitted with Webril pads were used to apply
0.1 mL of an 8% soap solution to the human forearm.

 The chambers were secured with porous tape, and applied for 24 hours on day
1. On days 2 to 5, fresh patches were applied for 6 hours.
 The skin is examined daily before patch application and on day 8, the final
study day. No patches are applied after day 5.
 Applications were discontinued if severe erythema was noted at any point.
Reactions were scored on a visual scale of erythema, scaling, and fissures.
 This test correlated well with skinwashing procedures, but tended to
overpredict the irritancy of some substances.
5) Immersion Tests
These tests of soaps and detergents were developed in order to improve irritancy prediction
by mimicking consumer use.
Kooyman & Snyder describe a method in which soap solutions of up to 3% are prepared in
troughs. The temperature was maintained at 105°F while subjects immersed one hand and
forearm in each trough, comparing different products (or concentrations). The exposure
period ranged from 10 to 15 minutes, three times each day for 5 days, or until irritation was
observed in both arms.
The antecubital fossa was the first site to show irritation, followed by the hands. Therefore,
antecubital wash tests (see the following) and hand immersion assays were developed.
 Clarys et al. used a 30-minute/4-day immersion protocol to investigate the
effects of temperature as well as anionic character on the degree of irritation
caused by detergents. The irritation was quantified by assessment of the
stratum corneum barrier function (transepidermal water loss), skin redness (a*
color parameter), and skin dryness (capacitance method). Although both
detergents tested significantly affected the integrity of the skin, higher anionic
content and temperature, respectively, increased the irritant response.
 Allenby et al. describe the arm immersion model of compromised skin, which
is designed to test the irritant or allergic potential of substances on damaged
skin. Such skin may show an increased response, which may be negligible or
undetectable in normal skin. The test subject immersed one forearm in a
solution of 0.5% sodium dodecyl sulfate for 10 minutes, twice daily until the
degree of erythema reached 1 to 1_ on visual scale. This degree of damage
corresponded to a morning’s wet domestic work. Patch tests of various

irritants were applied to the dorsal and volar aspects of both the pretreated and
untreated forearms, and also to the back. Each irritant produced a greater
degree of reaction on the compromised skin.
6) Wash Tests
Hannuksela and Hannuksela compared the irritant effects of a detergent in use testing and
patch testing. In this study of atopic and nonatopic medical students, each subject washed the
outer aspect of the one forearm with liquid detergent for 1 minute, twice daily for 1 week.
Concurrently, a 48-hour chamber patch test of five concentrations of the same detergent was
performed on the upper back. The irritant response was quantified by bioengineering
techniques: transepidermal water loss, electrical capacitance, and skin blood flow. In the
wash test, atopics and nonatopics developed irritant contact dermatitis equally, whereas
atopics reacted more readily to the detergent in chamber tests. The disadvantage of the
chamber test is that, under occlusion, the detergent can cause stronger irritation than it would
in normal use. Although the wash test simulates normal use of the product being tested, its
drawback is a lack of standard guidelines for performing the test.
Charbonnier et al. included squamometry in their analysis of a hand-washing model of
subclinical irritant dermatitis with SLS solutions. Squamometry showed a significant
difference between 0.1 and 0.75% SLS solutions whereas visual, subjective, capacitance,
transepidermal water loss, and chromametry methods were unable to make the distinction.
Charbonnier suggests squamometry as an adjunct to the other bioengineering methods.
Frosch describes an antecubital washing test to evaluate toilet soaps, using two washing
procedures per day. Simple visual scoring of the reaction (erythema and edema) allows
products to be compared. This comparison can be in terms of average score, or number of
washes required to produce an effect.
(b) OCULAR IRRITATION TESTING
A) THE DRAIZE EYE-IRRITATION TEST
The standard Draize eye-irritation test uses either three or six albino rabbits. Statistical
studies conducted to determine the effect of reducing the number of animals used in a single
study from six to three showed that a three-animal test provides eye-irritation classification
similar to that obtained by using six rabbits.
 Standard Draize eye-irritation test protocols normally require that 100 μL of a
test material is placed in the lower culde-sac of one eye, and the eyelids are
held shut for a brief period of time.

 The untreated contralateral eye is used as the control. The eyes are sometimes
rinsed after treatment to determine the effect of irrigation on the extent of
irritation or to remove test substances trapped within the cul-de-sac.
 Generally the eyes are examined using a pen light and graded by a technician
forirritation on days 1, 2, 3, 4, and 7 after dosing and weekly thereafter.
 However, times at which the eyes are examined for irritation after dosing may
vary because of differences in government regulations and preferences of
different toxicologists.
 In some cases, the eyes are examined at time points earlier than day 1 (e.g., 1h,
3h). Similarly the maximum period allowed to determine recovery may vary
(e.g., 3–5 weeks). Eyes are generally not examined once they have returned to
normal.
 Examinations are sometimes augmented by fluorescein staining and slit-lamp
examinations to better assess corneal changes. A grading scale has been
proposed based on examinations with a slit lamp.
The Draize test uses a systematic numeric grading system to quantify the eye irritation
response (Table 6). Changes associated with the cornea, conjunctiva, and iris are assessed by
using a pen light.
 Scores are assigned for the various changes. The scores for the
cornea,conjunctiva, and iris are weighted such that changes associated with the
cornea are given the most weight, with the maximum score for the cornea
being 80 out of a total possible score of 110.
 A test substance’s potential to cause ocular irritation is then determined by
assessing the individual animal scores, the maximum average score (highest
mean group score during the study), and days to recovery.
 In general, innocuous or slightly irritating materials tend to affect only the
conjunctiva, and the eye recovers in 1 to 2 days; mildly to moderately
irritating materials affect the conjunctiva and cornea, and the eye recovers in
days to weeks; and moderately to severely irritating materials affect the
cornea, iris, and conjunctiva, and the eye recovers in weeks or not at all.

These results are often further classified according to various regulatory classification
schemes in use around the world. The interested reader should consult Chan and Hayes for a
summary of regulatory considerations. Although the Draize eye-irritation test and slight
variations of it have remained the standard procedure for determining ocular-irritation
responses, the use of this test has not continued without significant criticism.
The sensitivity and relevance of the Draize test have been questioned because the
dose given is greater than the volume of the conjunctival cul-de-sac of the rabbit eye (30 μl),
thereby considerably exceeding the dose received in human accidental eye exposure.
Additionally, the in vivo tests have been criticized for their subjectivity, lack of repeatability,
overprediction of human responses, and by animal welfare advocates because they require the
use of animals. Therefore, efforts have been made to develop and validate significantly
modified in vivo test protocols as well as develop in vitro tests to reduce and perhaps
ultimately eliminate the use of animals in ocular-irritation testing.
B) MODIFICATIONS OF THE IN VIVO EYE-IRRITATION TEST
The Low-Volume Eye Test
In the early 1980s, modifications made in the amount of test material dosed and site of
application resulted in a refined version of the classical Draize test, called the low-volume
eye test (LVET). The LVET has been reported to be less stressful to rabbits and more
predictive of human ocular irritancy potential than the standard Draize procedure. This
method of application and the dose applied much more closely simulates accidental human
exposures. Normally either three or six rabbits are used per test substance. Statistical studies
similar to those conducted for the Draize test indicate that results from three rabbits provide
eye-irritation classification similar to that obtained from studies using six rabbits, so that
animal use in this test can be minimized.
The LVET differs from the standard Draize eye-irritation test in three ways:
(1)The volume of test substance applied is 10 μL instead of 100 μL
(2) The test substance is placed directly on the corneal surface instead of into the lower
conjunctival cul-de-sac; and
(3) The eyes are not held shut after the test substance is applied.
Scale of Weighted Scores for Grading the Severity of Ocular Lesions Ocular effects
Grade

Cornea
(A) Opacity-degree of density (area that is most dense is taken for reading)
Scattered or diffuse area—details of iris clearly visible 1
Easily discernible translucent areas, details of iris clearly visible 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris invisible 4
(B) Area of cornea involved
One quarter (or less) but not zero 1
Greater than one quarter—less than one half 2
Greater than one half—less than three quarters 3
Greater than three quarters—up to whole area 4
Score = AXB X 5 (range, 0 to 80)
Iris
(A) Values
Fold above normal, congestion, swelling, circumcorneal injection (any one or all of these or
combination of any thereof), iris still reacting to light (sluggish reaction is positive)
1
No reaction to light, hemorrhage; gross destruction (any one or all of these) 2
Score = A X 5 (range, 0 to 10)
Conjunctivae
(A) Redness (refers to palpebral conjunctivae only)
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red 3
(B) Chemosis
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial eversion of the lids 2
Swelling with lids about half closed 3
Swelling with lids about half closed to completely closed 4
(C) Discharge
Any amount different from normal (does not include small amounts observed in inner
canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adjacent to the lids 2
Discharge with moistening of the lids and considerable area around the eye3
Score = (A + B+ C) X 2 (range, 0 to 20)
C) REPLACING THE ANIMAL TEST WITH IN VITRO METHODS

1) Isolated Whole Eyes
At the first stage of reduction, in vitro tests use isolated whole eyes usually obtained from an
abattoir. Examples of such tests include the Isolated Rabbit Eye Test (IRE) and the Chicken
Enucleated Eye Test (CEET). In these model systems, test substances are applied directly to
the cornea of an isolated eye for short time periods (usually around 10 sec). Subsequently,
several measurements are made to estimate the severity of the resulting injury. These
measurements are generally similar to those that can be made in the whole animal, including
corneal opacity, corneal swelling, and fluorescein retention.
Histopathological examination of the injured tissue can also be conducted. Both isolated
eye models have generally performed quite well in identifying severely irritating materials; in
fact, the IRE is accepted by regulatory agencies in the United Kingdom for the classification
of severely irritating materials, as is the CEET in the Netherlands. Both test methods are
compatible with solid and liquid test articles.
2) Isolated Cornea Models
The substrate used at the next level of reduction is isolated corneas. The most common source
of corneas for these studies is bovine eyes obtained from the abattoir. These corneas are used
in an assay called the bovine cornea opacity and permeability (BCOP) test. In this assay, test
maerials are applied directly to the anterior surface of corneas mounted in the center of a
dual-sided organ culture chamber. After the designated exposure time, the test substance is
washed away and the resulting corneal opacity and changes in epithelial barrier function,
evaluated by increased permeability to fluorescein, are measured.
An advantage of this model is that the corneal opacity can be measured quantitatively
with a photometer because the organ chamber has transparent glass covers on each end. As
with the isolated whole eye, it has been shown that assessment of histopathological changes
provides additional useful information.
3) Other Test Systems
There are several in vitro tests that have been evaluated extensively as alternatives for eye-
irritation testing that do not fit entirely within the reductionist scheme just described. The

most significant tests in this category are the chorioallantoic membrane (CAM) assays.
surrogate for conjunctival tissue.
To this end, they developed a model called the hen’s egg test–CAM (HET–CAM). In this
procedure, test substances are placed directly on the CAM exposed directly underneath the air
cell. The resulting hemorrhage, coagulation, and lysis appearing on the CAM are measured at
defined timepoints after the test article is applied. Results from this test are accepted by
regulatory agencies in Germany as adequate for identifying severe irritants. A complementary
test called the CAM vascular assay (CAMVA) has also been developed. The CAMVA differs
from the HET–CAM in several ways, including the site of the egg shell that is opened (side
of the egg instead of the air cell), the endpoint measured (changes in characteristics of the
CAM vasculature), and the dosing scheme (serially diluted test substances instead of a single
test concentration). Both the HET–CAM and the CAMVA assay are reviewed in detail in the
U.S. IRAG evaluations. Results from evaluation of this test in several international validation
studies have been reported.
(c) SENSITIZATION TESTS

Allergic reactions to topically applied materials are much less common than irritant reactions.
Many sensitizers will produce allergy only in a small percentage of the population, possibly
less than 1%. As such, it is difficult to devise a test that will accurately reflect the incidence of
sensitization when used by the population at large. The need for predictive tests was
recognized more than 30 yeas ago, and the essential features of such tests were established.
More recent examples of sensitization protocols indicate only small variations on the original
procedures. Thus, there is an induction period, during which repeated exposure to the
product occurs. This is followed 1–2 weeks later by a challenge to a previously unexposed
site to determine sensitization. The test site for the induction phase is usually the back,
although the upper arm is favored in some laboratories.
The challenge should take place on previously unexposed skin, either at a site on the
back, well removed from the original test site, or preferably on the upper arm. A control
application of either vehicle without the active ingredient or a transdermal patch without drug
is essential in interpreting any reactions that occur.
 Induction Phase
The induction phase varies according to the protocol, but essentially consists of multiple

applications, under occlusion, that are either continuous, as in the cumulative irritancy
protocol, or intermittent (24-h on–24-h off). Typical examples are as follows:
1. An induction period of 42 days consisting of 21 X 48-h applications, each to a fresh
site, as used for a transdermal nicotine patch.
2. An induction period of 21 days consisting of 9 X 24-h applications, 24-h on–24-h off,
applications to the same site.
3. An induction period of 15 days consisting of 6 X 48–72-h applicationsto the same site
(i.e., continuous application with inspection and reapplication).
We favor the latter variation because it gives continuous exposure to the same site and is
probably equivalent, in terms of the immunological challenge, to an intermittent exposure
over a longer period or to the longer exposure to fresh sites. Continuous exposure to the same
site under occlusion maximizes the percutaneous penetration of any potential allergen.
Intermittent exposure allows for barrier recovery and, therefore, may not be as severe a
provocation. A major variation to this induction procedure is the use of chemical agents to
damage the skin before patch testing. The most common departure is the use of sodium lauryl
sulfate in the maximization procedure. Briefly, an area of skin is treated with a 5% sodium
lauryl sulfate solution for 24 h to induce a moderate inflammatory response. This is followed
by a 48-h application of the test material to the same site.
The sequence of 24-h irritant and 48-h test material application is repeated five times
to the same site. This procedure is designed to maximize penetration of potential allergens.
However, severe cutaneous reactions can develop with this approach, and this may be
unacceptable to volunteer subjects. In addition the possibility of interaction between any
sodium lauryl sulfate that has penetrated the skin and the test material cannot be ruled out.
Such an interaction may alter the immunogenic potential of the test material.
 Challenge Phase
In the challenge phase there is less variation in protocols. The main differences are the use of
a single 48-h challenge or two consecutive 48-h challenges (back-to-back). The latter is
claimed to be more sensitive in terms of detecting weak allergens. What is perhaps more
critical is the use of appropriate controls and the time of assessment. Controls consisting of
vehicle or transdermal system without active drug must be tested alongside the product of
interest to interpret any reactions. At least two observations should be made, one assessment
within 15–30 min after patch removal, and a second assessment 48 h later to determine late
reactions. Sensitization challenges are usually carried out under occlusion and must be at sites

remote from the initial induction site (e.g., the upper arm if the back has been used in the
induction phase). The only other variation is the maximization procedure, which again uses
pretreatment of the challenge site with sodium lauryl sulfate solutions.
 Population Size
The number of subjects used in sensitization procedures varies from 25, in the
maximization test, to 200 in the Draize test. The sample size must be large enough to give
confidence, in terms of predicting what will happen in general use, but not so large that
adoption of the procedure becomes expensive and time-consuming. The mathematics of
extrapolating from a small test population to many consumers has been discussed. Briefly, if
there are no reactions in a panel of 200 subjects, then as many as 15 out of every 1000 of the
general population could react (95% confidence limits). For 100 and 50 subjects the figures
are 30 and 58 out of every 1000 of the population.
The possibility that the sensitization test may not predict a potential sensitizer may
result from a variety of factors, such as skin site, variation in the frequency of application
during the induction phase, poor release from the vehicle, and the dose applied. Clearly, to
reduce the level of potential reactors in the general population to fewer than 1:1000 would
require an enormous number of volunteers.
The compromise is to use 100 or 200 subjects, which gives a reasonable level of
confidence in the test. The choice of 100 or 200 subjects rests, to a certain extent, on the type
of product. A cosmetic product that may be a reformulation or that does not contain any new
chemical entities may require only 100 subjects. It may be more prudent to test a new
pharmaceutical agent in a panel of 200 subjects. Any possible regulatory requirement of
countries where the product is to be sold must also be taken into account.
 Dosage
Undoubtedly the induction and elicitation of an contact sensitization reaction is dose
dependent. The higher the concentration, the more likely it is that individuals who are
predisposed will become sensitized. With final formulations the test concentration is
predetermined. When a range of therapeutic doses is being considered, the highest dose likely
to be used clinically should be tested. With raw ingredients, the highest nonirritating
concentration may be tested, depending on the likely exposure. As with irritancy, the area
over which an allergen is applied is important.
With a potent sensitizer, such as 2,4-dinitrochlorobenzene (DNCB), higher dose–

response curves were obtained for a constant dose per unit area when the area of application
2
was increased from 8 to 80 mm . However, in a separate study with DNCB, at a similar dose
2
per unit area, no difference was found between areas of 180 and 710 mm , neither in terms of
the number of subjects sensitized nor in the challenge dose response curves. Thus, with the
potent sensitizer DNCB, the critical surface threshold area appears to be between 80 and 180
2
mm .
(d) PHOTOSENSITIVITY
It is possible that topically applied or systemically administered chemicals or drugs that have
little or no potential to promote an irritant or allergic reaction in the skin, may do so in the
presence of sunlight. Although photosensitive reactions may be rare, relative to irritant or
sensitization reactions, the development of such reactions can lead to withdrawal from the
market (e.g., the antirheumatic drug, benoxaprofen).
1. Sunlight
Electromagnetic radiation is classified according to wavelength ranges. Electromag
netic radiation emitted by the sun ranges from very short wavelength cosmic rays to very
long wavelength radio waves. Short-wavelength, high-energy forms of radiation such as
gamma and x-rays, are termed ionizing radiation, whereas longer lower energy radiation
(more than 800 nm) is nonionizing and increases molecular motion, giving a thermal effect.
Wavelengths between 200 and 800 nm are capable of causing chemical changes if absorbed
by molecules in the skin. Ultraviolet radiation falls into this category and is between 100 and
400 nm. Fortunately for us, no wavelengths shorter than about 288 nm reach the earth’s
surface, because these are filtered out by the earth’s atmospheric ozone layer. Damage to this
important protective layer by man-made chemicals such as chlorofluorocarbons (CFCs) is
currently under review and the subject of much discussion.
UVR is subdivided into UVC (100–280 nm), UVB (280–320 nm), and UVA (320–
400 nm). UVA can be further subdivided into UVA II (320–340 nm) and UVA I (340–400
nm) .
Nonionizing Radiation
UVC 100–280 nm
UVB 280–320 nm

UVA 320–400 nm
UVA II 320–340 nm
UVA I 340–400 nm
Visible 400–800 nm
Although sunlight that reaches the earth is essential to most living organisms and has many
beneficial effects for humans, it is UVB and UVA that are responsible for most of the
cutaneous photobiological events. These include DNA and RNA damage, inhibition of
protein synthesis, damage to liposomal and cellular membranes, mutagenic and carcinogenic
effects and erythematous responses. Chronic UV exposure is also responsible for dermal
connective tissue injury (elastosis) leading to the changes known as photoaging.
2. UV-Induced Erythema
It is important to remember that sunlight is a continuous spectrum and that it is in its entirety
that it causes erythema or sunburn in exposed skin. The paler the skin the greater the
erythematous response for any given dose of UVR. Thus, skin type-I burns far more readily
than skin type-VI (Table 8). UVB is primarily responsible for most of the erythema seen in
human skin following excessive sun exposure. This response is apparent approximately 6 h
following exposure and is maximal at 20–24 h. In contrast to UVB, UVA can induce an
immediate erythema that usually diminishes within 2 h of exposure. It can also give a delayed
erythematous response that reaches a peak at 6 h. UVA contributes to about 10–20% of
sunburn and passes through window glass, in contrast to UVB, which is essentially blocked
by window glass. UVA can also induce an immediate pigment darkening response (IPD).
This reaction appears within seconds of exposure and fades within a few minutes. The
pigment darkening is due to a photochemical reaction involving the oxidation of a low
molecular weight form of melanin in melanosomes. The response is clearest in skin type-III,
IV, or V individuals who have higher levels of melanin. Prolonged exposure to UVA can lead
to persistent pigment darkening (PPD) or tanning of the skin. The effects described so far are
the results of UVR itself; however, photosensitivity reactions can occur when an exogenous
or endogenous chemical (chromophore) absorbs UVR or visible light. These reactions may
be either phototoxic (photoirritant) or photosensitive (photoallergic).
3. Phototoxicity Reactions
Phototoxicity refers to skin irritation that is produced through the interaction of
chemical substances and radiant energy in the ultraviolet and visible ranges. Phototoxic or

photoirritant effects are immediate and nonimmunological. When testing for the phototoxic
potential of topically applied chemicals, the output from the required radiation source is UVA
only. This is best accomplished by using a suitable filtered solar simulator.
Photo–Skin Types
Skin type I Always burns and never tans
Skin type II Always burns and tans with difficulty
Skin type III Often burns and tans moderately
Skin type IV Burns minimally and tans easily
Skin type V Rarely burns and tans profusely
Skin type VI Insensitive, never burns, deeply pigmented
4. Phototoxicity Assays
The development of procedures for assessing both the phototoxic (and photoallergic)
potential of chemicals has, in general, been carried out in laboratory animals. A wide range of
animal species have been used, including rabbits, mice, guinea pigs, squirrel monkeys,
opossums, and swine. All methods require the skin to be irradiated with UVA following
application of the test substance. However the measurable end result is not always similar to
that obtained from human experiments. Increases in guinea pig or mouse ear thickness has
been used to quantify phototoxic responses.
Dermatitis and the increase in weight of the mouse tail has also been used. However,
hairless mice and albino guinea pigs have been used where simple erythema was the
toxicological endpoint for assessing a phototoxic response.The results obtained from such
experiments may be false-negative or false-positive when extrapolated to humans.
Kaidbey and Kligman suggested a method for identifying potential topical phototoxic
agents in humans, although the title of this publication misguidedly refers to photosensitizing,
rather than phototoxic agents. Human testing is ethical as only small areas of skin are
irradiated and clinical experience of phototoxic reactions indicate that when the stimulus is
removed the dermatitis subsides. As with all clinical trials the informed consent of the subject
and ethical committee approval must be obtained. The method we recommended is based on
that of Kaidbey and Kligman and is suitable for assessing the phototoxic potential of topically
applied drugs, chemicals, transdermal, and skin care products.
In the protocol for phototoxicity testing, a test panel consisting of a minimum of 12
healthy white adults with untanned back skin is required. As the dermatitis of a phototoxic

agent can be produced in almost every subject, given sufficient exposure, it is not necessary
to employ a large test panel. Two sets of test products and appropriate controls (usually
vehicle or base alone) are applied occlusively to the midback area (one set on the left and one
set on the right). In our experience 12mm aluminum Finn chambers on Scanpore tape are
suitable for product application.They are occlusive and ensure optimal product contact with
the skin. An empty chamber should also be fixed to each side of the back.
Six hours after administration of the materials and chambers, these should be removed
and one set of applications only irradiated immediately with UVA. The chemical-to-skin
contact time is relatively short compared with, for example, the 24 h needed in a
vasoconstriction assay. If longer times are used (e.g., 24 h), light exposure will often produce
2
negative results. The test sites should be irradiated with 20 J/cm of UVA. The radiation
source should ideally be a xenon arc lamp solar simulator.
Such a system could typically consist of a 1000-W ozone-free xenon arc lamp, the
output from which is filtered with a Schott WG 345 filter of 2-mm thickness. This filter will
block all erythemogenic UVC and UVB wavelengths below 320 nm. In addition unwanted
longer wavelength visible and infrared radiation can be removed using a combination of a
suitably coated dichroic mirror, water filter, and UG11 filter. If this is not accomplished then
the subjects may feel heat and or pain from the irradiations. It is often convenient to deliver
the UVA radiation to the skin surface using an 8-mm–diameter liquid light guide.
A broad-spectrum thermopile should be used to measure the output of energy from
2
the solar simulator, typically expressed in milliwatt per square centimeter (mW/cm ). The
thermopile should be calibrated against a known standard from, for example, The National
Physics Laboratory in the United Kingdom. To calculate the time of irradiation necessary to
2
administer a dose of 20 J/cm to the skin, the formula in Table 9 should be used.
Skin assessments should be made immediately following irradiation and at 24 and 48
h after photoexposure. The grading system in Table 10 is suitable for recording any
cutaneous reactions. The irradiated sites should be compared in each subject with the
nonirradiated sites. If the response in any one subject at the irradiated site is greater than that
seen at the nonirradiated site, then that product or chemical is deemed phototoxic.
Phototoxicity is relatively easy to detect and therefore prevent; however, phototoxic
reactions may sometimes mask or contribute to photoallergic reactions
Calculation of Irradiation Times
t(s) =2mJ/cm2 / mW/cm2
Therefore, if a reading of 200 mW/cm2is obtainedfrom the thermopile and a dose of 20
J/cm2(20,000 mJ/cm2) is required then: t(s) =20,000/200 and t(s) = 100 s
Photoallergic Reactions
Photoallergy (or photosensitization) refers to a dermatitis that is produced through the
interaction of chemical substances and radiant energy, in the UVR and visible wavelengths,
to produce an allergen. The chemical substance may be orally ingested or topically applied
(photocontact allergy). Unlike phototoxic reactions, photoallergic reactions are often delayed,
immunological, and less dose-dependent. Photoallergic and particularly photocontact
photoallergic reactions are relatively uncommon when compared with phototoxic reactions.
Clinically, photocontact allergic reactions produce a dermatitis that resembles allergic contact
dermatitis, appearing as an acute dermatitis affecting primarily, but not exclusively, light-
exposed skin.
A characteristic histological feature of photocontact allergy is a dense perivascular
round cell infiltrate in the dermis, which helps distinguish this dermatitis from a phototoxic
reaction. A second and rare type of photoallergic reaction is solar urticaria. This occurs after
only brief exposure to light and is characterized by an immediate urticarial wheal-and-flare
reaction within minutes of exposure. The reaction usually subsides within 1–2 h, is associated
with degranulation of mast cells at the site of exposure, and the release of neutrophil
chemotactic factors and histamine into venous blood near the reaction sites.Photoallergic
reactions, when they occur, may apparently be triggered by irradiation alone (in the absence
of known sensitizers) or may be due to exogenous chemicals and UVR.
Phototoxicity Grading Scale
Grade Cutaneous rection
0 No reaction
1 Mild erythema, possibly with scaling
2 Moderate or strong erythema
3 Moderate or strong erythema with a papular response
4 As grade 3, but with definite edema
5 Vesicular or bullous eruption.
Photoallergy, whether photocontact allergic dermatitis (delayed hypersensitivity: type
IV reaction) or solar urticaria (immediate hypersensitivity: type I reaction), is an acquired
reactivity dependent on cell-mediated hypersensitivity or antigen–antibody interaction.Test
procedures designed to identify potential photosensitizing chemicals were developed in

response to the outbreak of reactions caused by the use of antibacterial halogenated
salicylanilides in the early 1960s. A minority of affected individuals developed a persistent
photodermatitis that lasted several years despite the avoidance of contact with
photosensitizing phenolic compounds. It therefore became clear that there was a requirement
for a laboratory test to detect potential photosensitizing agents and avoid such situations.
Several photocontact sensitizers have been identified, including coumarins and
coumarin derivatives, musk ambrette, fentichlor, bromochlorosalicylanilide, chloro2-
phenylphenol, and benzocaine. Certain sunscreens have also been reported to produce
photocontact allergic dermatitis, most notably para-aminobenzoic acid and derivatives,
benzophenone 3, mexenone, and cinnamates. Several essential oils have also produced
photoallergic reactions (e.g., sandalwood oil). Also, 8-methoxypsoralen, which is used in
PUVA (psoralen UVA), can also be photoallergic as well as phototoxic.
6. Photoallergenicity Assays
Test procedures to identify potential photosensitizers have received less attention
thanmethods designed to detect either ordinary contact sensitizers or phototoxic chemicals.
Landsteiner and Chase demonstrated that low molecular weight haptens can produce contact
dermatitis in guinea pigs. They also observed that allergic contact dermatitis could be
conferred on immunologically naive guinea pigs by the passive transfer of mononuclear cells
from nonsensitized animals. Furthermore guinea pigs develop edema and erythema after
contact with topically applied sensitizers and, to some extent, develop a response similar to
the clinical response in humans.
These observations became the cornerstone of photocontact allergic dermatitis
research over many years and led to the guinea pig becoming the most commonly used
animal in photoallergy studies.
On the other hand, mouse ear swelling is claimed to be a more sensitive model, but is
even further removed from the human response than that of the guinea pig. To induce contact
photosensitivity in any animal it has to be repeatedly exposed to the test molecule in the
presence of UVR. For this induction phase a broad-spectrum source is necessary, which
should include UVB as well as UVA.
The period of induction should be similar to that for testing contact sensitizers,
followed by a rest period and then a challenge on a previously untested site with the test
chemical and UVA alone. The photomaximization test for the prediction of photosensitizers

is conducted on humans and is similar in design to a sensitization test, but with the addition
of exaggerated UVR exposure of both the chemical and the skin to which it is applied. In an
ideal world, this type of test would be carried out on a large number of subjects (>100) to
more accurately predict the incidence of photosensitization reactions in the population at
large.
From a practical point of view this is not possible because of the demanding nature of
the protocol; therefore, a test panel of 26 is normally recommended. The method described
here is based on that of Kaidbey and Kligman and is suitable for identifying topical
photocontact sensitizers.
The photomaximization test is a 6-week study and is divided into an induction phase
and an elicitation phase. A solar simulator, as described previously, is an ideal source of
UVR.
During the induction phase a 1-mm–WG320 filter and a 2-mm–UG11 filter should be
used, allowing both UVA and UVB (290–400 nm) to reach the subject’s skin. For the
elicitation phase, a 2-mm–WG345 and a 1-mm–UG11 filter should be used to allow only
UVA (320–400 nm) to reach the skin. The test chemical together with the vehicle control is
applied occlusively to the midback of each subject using 12-mm aluminum Finn chambers on
Scanpore tape. Twenty-four hours later, the patches are removed and the test sites wiped
clean and allowed to air-dry for approximately 30 min. Each site is then exposed to twice the
subjects minimal erythema dose (MED) of solar-simulated UVR. The sites are then left
uncovered and exposed to the air for approximately 48 h.
This procedure of application and irradiation is repeated such that each subject has six
applications and irradiations over a 6-wk period. The sites are evaluated 24 h after each
irradiation and, if the reaction becomes severe such that further application and irradiation is
undesirable, then application of the material and subsequent irradiation is carried out at an
adjacent site.
Following completion of the induction phase, there is a 2-wk rest period, with no
applications or irradiations. Approximately 14 days after the end of the induction phase, two
sets of test materials are applied to previously untreated sites on the midback, again using 12-
mm–Finn chambers. Twenty-four hours later the patches are removed, the skin wiped dry
2
with a gauze swab, and one set of applications irradiated with only 4 J/cm of UVA. The sites
are then evaluated at 24, 48, and 72 h after the elicitation irradiation. The grading system for

all irradiations (both induction and elicitation) can be seen in Table 11.
If one or more subjects develops a reaction at an irradiated site during the elicitation
phase that is greater than the corresponding unirradiated site, then that chemical is considered
to be a photosensitizer. In practical terms there are usually many or no reactors in a test panel
making the decision as to whether a product is a photoallergen relatively easy.
Grading System for Photoallergy Test: Induction and Elicitation Phase

0 No reaction
1 Reaction readily visible, but mild unless letter grade appended. Mild reactions include
weak, but definite erythema, and weak superficial skin responses such as glazing,
cracking, or peeling.
2 Definite papular response.
3 Definite edema.
4 Definite edema and papules.
5 Vesicular–bullous eruption.
E Strong erythema at patch site.
F Strong effects on superficial layers of the skin including fissures, a film of dried
serous exudate, small petechial erosions, or scabs.
S Reaction spreading beyond test site.
I Itching.
B/S. Burning or stinging.
(Applications must be either terminated or moved to an adjacent nonirradiated site if a
reaction score of 2 or higher occurs). Descriptive letter designations may be added to the
numerical score if experienced at the test site. (Any other signs or symptoms (e.g., wheal-
and-flare responses) may be described separately).

REFERENCE
1. Peter J. D., Anthony D. P., Safety Considerations for Dermal and Transdermal
Formulations.
2. Paye M., Maibach H. I., Barel A. O., Handbook of Cosmetic Science and Technology
Marcel Dekker, New York.
3. Wilhelmus K. R., Mindel J.,The Draize Eye Test, Survey ophthalmology (2000)
45, 6.
4. John F., Corbett., Raj K. Sharma., William E. Dressler., cosmetic Toxicology, chapter
6,899-915.
5. FDA. (1994). "Cosmetic Injury Reports from Consumers as Reported to Office of
Cosmetics & Colors Division of Programs and Enforcement Policy Cosmetic
Programs and Regulations Branch, Food and Drug Administration, Washington, D.C.
20204, July 1987 through 1995." U.S. Food and Drug Administration, Washington,
D.C.
6. FDA. (1996) "Regression Line and Other Pertinent Statistical Data Based on
Cosmetic Product Experience Information ReportedVoluntarily by Some Cosmetic
Firms under 21 CFR 730 for the Years 1989-1992" U.S. Food and Drug
Administration, Washington,D.C.
7. www.google.com
8. www.scribd.com

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Clinical Safety Evaluation of Cosmetics

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DEPARTMENT OF PHARMACEUTICAL SCIENCE DR.

BY- SHRIKANT THAKUR

(M. PHARAMA III SEM.)

Under the Supervision of

PROF. N.K. JAIN

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(a) USA safety regulation

5 Ingredient ,toxicology and product safety 11-12

6 PROTOCOL FOR CLINICAL SAFETY TESTING 12-46

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(2) SAFETY TERMINOLOGY

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Photoallergic contact dermatitis

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(a) USA safety regulation

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(b) European Union safety regulation

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(C) JAPAN SAFETY REGULATION

(4) EXPOSURE AND RISK ASSESSMENT CONSIDERATIONS FOR

(5) INGREDIENT, TOXICOLOGY AND PRODUCT SAFETY

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(a) TESTS FOR SKIN IRRITATION

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Examples of Modified Draize Irritation Method

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Examination (h) 24, 72 24, 72 4, 48 0.5,1, 24, 0.5, 1, 24,

Abbreviations: FHSA, Federal Hazardous Substance Act; DOT, Department of

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

2) Cumulative Irritation Testing

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

C) REPLACING THE ANIMAL TEST WITH IN VITRO METHODS

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

(c) SENSITIZATION TESTS

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Grading System for Photoallergy Test: Induction and Elicitation Phase

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

Department of Pharmaceutical Sciences, Dr H.S. Gour Central University, Sagar (M.P.)-470003

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