489
53 In Vivo Models of Skin Irritation
F. Anthony Simion
53.2
Contents
53.1
53.2
53.3
Introduction . . . 489
Theoretical Models of Irritation . . . 489
The Initial Effects of Surfactants on the Skin . . . 490
53.3.1
Sensory Irritation . . . 490
53.3.2
Squamometry . . . 491
53.3.3
Superhydration of the Stratum Corneum . . . 492
53.4
The Role of Skin Condition on the Irritant Response . . . 492
53.5
Models for Assessing Skin Irritation . . . 493
53.5.1
Closed Patch Testing for Assessing Hazard . . . 493
53.5.2
Exaggerated Usage Tests . . . 494
53.5.2.1 Exaggerated Wash Tests . . . 494
53.5.2.2 Use Testing . . . 495
53.6
Models for Measuring the Moisturizing Potential of Cleansers . . . 496
53.6.1
Testing on Dry Skin . . . 496
53.6.2
Measuring the Clinical Effects of Moisturizing Cleansers on the Skin . . . 496
53.7
Bioengineering Measurements
of Skin Condition . . . 498
References . . . 498
53.1
Theoretical Models
of Irritation
Theoretical, in vitro and in vivo models have been
developed to assess many discreet forms of irritation,
and some will be discussed below. As they all affect
the same substrate, the skin, they frequently have
characteristics in common.
There are two theoretical models that help us better understand how the skin reacts to a variety of irritants. The trauma model of irritation proposed by
Malten suggests that visible irritation occurs when
the intensity of the insult exceeds a threshold [1]. A
single large insult (a), or a series of small ones (b),
that cumulatively exceed the threshold, can cause this
(Fig. 1). As the skin repairs itself, the intensity is reduced, eventually falling below the threshold, and the
clinical signs disappear.
Introduction
Irritation is the response of skin to noxious chemicals, trauma, or other insults from the environment.
The response can take many forms, both visible and
sensory, but by definition is unpleasant to the person
experiencing it. The mechanisms by which different
forms of irritation are produced can vary greatly. To
develop optimal strategies to prevent or ameliorate
the different forms of irritation being experienced,
understanding what is happening and the factors
that enhance or reduce the irritation is needed. This
is best achieved by examining each form of irritation
separately in a model system, where that type of irritation is the primary type induced.
Fig. 1. Theory of traumiterative irritation
One key implication from this model is that the
skin can respond before clinical signs are apparent.
This is the basis of the “invisible dermatoses” proposed
by Kligman [2]. He demonstrated that the skin might
be damaged at the histological level, even though
nothing is visible at the surface. For instance, in photobiology, half the minimal dose of UV required to
produce erythema (1/2 MED) will cause cell death in
the epidermis, i.e., produce “sunburn” cells. Another
490
F. Anthony Simion
example is patching the skin with 0.5% sodium lauryl sulfate (SLS) for 24 h. In many subjects, erythema
was not observed, nor were there any histological
changes apparent in hematoxylin-eosin stained sections. However, thin sections showed a great deal of
epidermal damage, with swollen keratinocytes and
edematous intercellular spaces.
This also explains why damaged or compromised
skin is more responsive. It already has a significant
but subclinical level of damage, thus requiring a
smaller insult to produce a visible sign that impact skin in good condition. Freeman and Maibach
showed a greatly elevated TEWL response to a second
SLS patch, applied to the same site as a first patch one
week before, even though the TEWL rate had apparently returned to baseline in the intervening time [3].
Another implication of this model is that different
forms of irritation have different thresholds. Therefore, a mild insult may produced only a few, mild
forms of irritation, whereas with a greater insult, the
threshold for more forms of irritation is surpassed, so
they too are expressed.
The second model relates skin strata to the type
and degree of irritation (Fig. 2). Each strata produces
irritation that is characteristic of that level: for instance, the stratum corneum and the upper epidermis
can reduce sensory irritation and dryness. Erythema,
which involves increased blood flow, requires dermal
involvement.
If the stratum corneum is damaged, then the irritants can penetrate to lower strata and produce a
more intense irritation than is expected. This is the
basis of the enhanced response of compromised skin.
53.3
The Initial Effects
of Surfactants on the Skin
Surfactants and other irritants initially interact with
the stratum corneum. Thus, in normal skin it is the
stratum corneum and the structures within the upper
epidermis that initially respond to chemical irritants.
These responses can take several different forms, including:
•
•
•
Sensory irritation
Damage to the surface corneocytes
Superhydration of the stratum corneum
However, as the exposure to the irritant becomes
more exaggerated, such as increased intensity, is prolonged, or the stratum corneum barrier is damaged,
the lower skin structures will become involved, and
other signs of irritation will appear.
53.3.1
Sensory Irritation
Exposure to many chemicals or products can produce unwanted sensations such as the feelings of dryness, stinging, itching, or skin burning, even in the
absence of visible signs of irritation. Epidemiological
studies have indicated that half of the adverse reactions caused by personal care products fall into this
category [4]. There are many different mechanisms by
which such sensations are produced. Some personal
care products such as sunscreens and lactic acid-containing lotions were shown to cause a facial stinging
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53 In Vivo Models of Skin Irritation
in a responsive subpopulation of consumers. Placing a 10% lactic acid solution on the face can identify these individuals while they are sweating [5]. The
responsiveness of panelists can be increased by facial
washing with soap and decreased by repeated applications of a good moisturizer. This suggests that the
skin of lactic acid stingers is somewhat damaged, although the mechanism by which stinging is produced
is not well understood.
Exposure to capsicin, the hot component in Chili
peppers, can reduce a burning sensation. Green and
his colleagues have been able to measure this phenomenon using a labeled magnitude scale and have
shown that although there is a large person-to-person
variation in response, there is good reproducibility of
the measurements within individuals [6]. The relative
sensitivities to other chemical irritants such as lactic
acid (stinging) and ethanol may be different in different individuals.
The mechanisms by which these sensory irritations
are produced are unclear. There appear to be several
sensory mediators such as histamine and substance
P. Indeed, intradermal injections of histamine can induce itching in many subjects [7].
As the unmyelinated C fibers appear to play an
important role in the detection of chemical irritancy
via the sensations of itching and stinging, it is likely
that histamine stimulates them. These fibers also can
detect heat and cold. It has been hypothesized that
stimulation of a few fibers results in the perception of
itching. As more of the fibers are stimulated, the signal is interpreted as stinging. The response of the C
fiber can be blocked. Maibach and his colleagues have
shown that a variety of anti-irritants such as menthol
and anesthetics can modify the ability of the C fibers
to detect heat and cold, itching and stinging [8].
Sensory irritation can frequently be detected before
clinical signs can be observed. Simion et al. showed
that in an exaggerated forearm wash test panelists
could detect differences between soap and a milder
synthetic detergent bar before a trained observer
could differentiate the products [9]. This is consistent
with the results of the epidemiological study by DeGroot et al., which reported that many people experience sensory irritation in the absence of visible signs,
and discontinue use of that product before visible irritation appears.
53.3.2
things happen as a result. Firstly the corneocyte sheet
begins to break into smaller sheets and individual
cells. These cells will take up hydrophilic stains more
readily than undamaged corneocytes. Both processes
can be assessed. Whether sheets of corneocytes are
present can be determined by visual inspection under
a light microscope. Dye uptake is readily quantified
by colorimetric assessment. This is the basis of both
squamometry and corneosurfometry. The latter is
the in vitro approach where the corneocytes are harvested first using a sticky tape and then exposed to
surfactants. Squamometry involves treating the skin
first and then harvesting the corneocytes with sticky
tape and dyeing them.
Periard, Paye, and their colleagues have used squamometry to assess the effects of cleansing products
on the conditions that resemble normal usage. Paye
and Cartiaux showed that the daily usage of the of
dishwashing liquids combined with a 5-min soak for
4 consecutive days at normal usage concentrations
(0.25%) gave the same line-up for skin damage as the
highly exaggerated Frosch and Kligman soap chamber test (occlusive patching for 24 h with a 2.25% solution of the dishwashing liquid, followed by 6-h occlusive patches on the next 4 consecutive days [10].
Periard et al. have extended this methodology
beyond surfactant-induced irritation. They showed
squamometry was extremely sensitive in its ability
to detect the effects of a fabric softener in reducing
the degree of skin surface damage caused by repeated
rubbing with wet towels. In this it was more discriminating than an observer, TEWL, or capacitance [11].
Squamometry can also be used to assess moisturizer efficacy. The effective moisturizers can stimulate
the desquamation of damaged surface corneocytes.
This results in the reduced dye uptake as measured
by a lower C * value (Fig. 3). Polyol-based moisturizers are more effective than those without polyols at
reducing C* and enhancing skin conductance. This
suggests that the polyol-containing moisturizers are
more effective at removing damaged aggregates of
corneocytes otherwise known as dry skin scales or
flakes.
Squamometry
Any short-term exposure of the skin surface to surfactants can damage the surface corneocytes. Two
Fig. 3.
After Lotion Treatment
No Product Treatment
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F. Anthony Simion
53.3.3
Superhydration
of the Stratum Corneum
Short-term (minutes) exposure of the stratum corneum to aqueous solutions of anionic surfactants
causes it to swell. Wilhelm et al. showed that this
swelling is related to the primary irritation potential of the surfactant [12]. When examined in an ex
vivo model, Rhein et al. speculated swelling occured
when the hydrophobic tails of the surfactants bound
to the stratum corneum [13]. The negatively charged
head groups would then repel each other. This would
have two effects – firstly it will enable the small hydrophilic molecules such as the natural moisturizing
factors (nmfs) to leach out, resulting in the skin’s reduced ability to hold moisture. Secondly the bound
surfactants are not readily desorbed. As they remain
in the upper stratum corneum, they damage the skin
for example donator. Imokawa and his colleagues
have proposed that surfactant binding to the skin is
a major cause of skin roughness and perceived tightness [14, 15].
It should be noted that nonionic and cationic surfactants do not cause the stratum corneum to swell;
yet cationic surfactants can be just as irritating as
their anionic analogs.
53.4
The Role of Skin Condition
on the Irritant Response
The condition of the skin is a crucial factor on the
type and intensity of the response to a set insult.
Firstly, it must be realized that skin on different parts
of the body will react with different intensities to the
same stimulus. Cua et al. showed that the thigh was
the most responsive anatomical site to SLS exposure,
whereas the palms were least responsive. TEWL was
found to be a more sensitive measure of irritation
than visual scoring [16].
The basis for these differences in responsiveness
is unclear. They may be related to the ease at which
molecules can diffuse through the stratum corneum.
Rougier et al. showed that there was a correlation between the skin’s permeability to water exiting and the
absorption of hydrophobic molecules such as benzoic acid, acetyl salicylic acid, and caffeine [17]. The
correlation coefficient (R) ranged from 0.92 down to
0.72. This may be a function of corneocyte size. The
idea that the skin’s permeability to irritants is related
to its responsiveness is not only intuitively reasonable
but is supported by experimental data, especially for
ionic irritants.
1. Predamaging the stratum corneum by immersing the skin in dilute surfactant solutions increases
the erythema induced by subsequent patching with
SLS [18].
2. Predamaging the skin by physically abrading
the stratum corneum with a needle significantly decreases the threshold concentration of Triton X-100,
formalin, or nickel ions required to elicit irritation.
This scarification procedure has much less effect on
the skin’s responsiveness to hydrophobic irritants
such as lauric or benzoic acids [19].
3. Panelists who had a stronger than average vasodilation response due to the percutaneous penetration
of methyl nicotinate also had a stronger irritant response to SLS [20].
4. Skin responds more strongly in the winter to
patching with SLS than in the summer [21]. The
basal level of transepidermal water loss is higher in
the winter, indicating the stratum corneum barrier is
more permeable, i.e., damaged.
5. Agner showed that patients with atopic dermatitis,
where the stratum corneum barrier is compromised
(basal TEWL rate is higher), show a stronger TEWL
response to SLS-induced irritation than nonatopic
controls [22].
6. Pinnagoda et al. extended Agner’s observation to
a nonatopic population. They showed that the basal
(pretreatment) TEWL rate correlates with the elevated rate observed after SLS exposure [23]. I have
observed that TEWL rate after 24 h of surfactant exposure is a strong predictor of the TEWL rates after
a 2nd day of occlusive patching using the modified
soap chamber test [24]. This suggests that the “leakiness” of the stratum corneum to water loss will make
it more vulnerable to surfactant-induced irritation.
In the skin strata model, shown in Fig. 2, damage to
the stratum corneum implies that the irritants can
penetrate more deeply into the skin and produce
53 In Vivo Models of Skin Irritation
more intense forms of irritation than if the stratum
corneum were intact.
53.5
Models for Assessing
Skin Irritation
53.5.1
Closed Patch Testing
for Assessing Hazard
Closed patch testing is used to assess the overall dermal primary irritation potential (toxicological hazard) of chemicals, including surfactants, and products.
Frequently the Draize test in rabbits has been used as
the standard, especially for regulatory assessments.
However, there is experimental data that questions
how predictive rabbit skin is of human skin’s response.
Phillips et al. assessed the primary irritation potential
(hazard) of a large variety of chemicals in human volunteers by occlusive patching for up to 21 consecutive days, i.e., the cumulative irritation test [25]. They
found that while the Draize test could differentiate
strong irritants from chemicals that were not irritating to humans, it was not effective at comparing materials of mild and moderate irritation potential. For
more refined comparison, human models appear to
be required. For cosmetic ingredients and products,
Burger and Bowman reduced the original 21-day
cumulative irritation test to only 14 days, by demonstrating that the relative magnitude of irritation does
not change between 14 and 21 days. Reducing study
duration greatly reduces the risk of tape reactions.
Inclusion of positive and negative controls (0.1% SLS
and a blank, respectively) can be used to standardize the results between studies. Another, similar approach that can be used in product safety assessments
is to assess irritation potential of a material from the
induction phase of the Maibach-Marzulli Human Repeated Insult Patch Test (HRIPT). In this procedure,
100 panelists or more are occlusively patched almost
continually for 3 weeks. The erythema produced is
recorded, when patches are replaced, three times a
week.
Recently a 4-h human exposure test has been developed as an assessment of hazard [26]. Volunteers
are patched occlusively with test material and a standard (positive control, 20% SLS aqueous solution) for
up to 4 h. At specified times, the test site is checked to
determine if erythema has been induced. Once this
occurs, that particular chemical is removed from the
test site. The response is then statistically compared
with the positive control. Initially, 20% SLS was used
for this purpose, as in European Regulations this solution is defined as an irritant (R38). Those materials
that are not statistically different from 20% SLS in this
procedure are also regarded as irritants.
Frequently companies are interested in the primary
irritation potential of cosmetics and cleansers, both
as a measure of consumer acceptability in the marketplace and as the basis of commercial claims support.
Since the intrinsic hazard of these products is actually
very similar, they are difficult to differentiate using
tests designed to assess intrinsic hazard. Instead, the
sensitivity of the test must be increased, and the type
of response expected must be a focus.
One example of a high-resolution test is the soap
chamber method developed by Frosch and Kligman
[27]. This requires a panel of sensitive skin individuals – defined as people who will give a strong erythema reaction (≥1.5 on a 0–4 scale) when patched
overnight with either 1% sodium lauryl sulfate (SLS)
or 5% soap. Panelists are then occlusively patched for
24-h occlusive patch with 5%–8% soap solution (inuse concentration). This is followed by a series of four
6-h patches on subsequent days. The skin is evaluated for erythema and dryness (scaling and fissures)
3 days after the application of the last patch. This
method differentiated soap bars from a synthetic detergent based on sodium cocoyl isethionate (Dove1),
the latter inducing less primary irritation (erythema)
and dryness. Dove and soap are frequently used as
the mild and irritating controls, respectively, to ensure adequate test sensitivity. This methodology has
also been applied to differentiate the irritation potential of dishwashing liquids.
A modified soap chamber test was developed to
decrease testing time without reducing the ability to
differentiate between soap and synthetic detergent
bars based on irritation potential only [28]. This
methodology involves a single 24-h exposure only –
Day 1 of the Frosch-Kligman soap chamber test. Erythema is assessed by a trained observer and by use of
a colorimeter, and stratum corneum barrier damage
is measured as the increase in transepidermal water
loss (TEWL) rate using an evaporimeter. To differentiate between products that are milder than Dove,
exposure time may be increased to two consecutive
days of patching. This closed patch test only produces
1 Zest and Ivory are registered trademarks of the Procter and
Gamble Co. and Dove is a registered trademark of Lever Bros.
Co.
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F. Anthony Simion
dryness if there is sufficient irritation and then only
several days after patching is completed. This suggests that dryness produced by this method is a result of the primary irritation – perhaps part of the
repair response. In subjects with darker skin tones,
especially FitzPatrick types IV, V, and VI, hyperpigmentation is also a major response to primary dermal
irritation [29].
Skin responses in the closed patch test are very
dependent on climate and season. Agner and Serup
showed that during the summer, the erythema and
TEWL responses to SLS are greatly diminished. This
emphasizes the importance of running mild and irritating controls, in order to ensure that the test has
sufficient resolving power. If the soap and syndet bar
cannot be distinguished, other null results should be
strongly questioned.
Patch testing is an assessment of hazard, the maximum potential primary irritation that could be produced. It takes no account of the way the product is
used, which may modify the amount of irritation induced. To develop a better understanding of the type
and severity of irritation produced in normal usage,
alternative approaches such as open application and
exaggerated usage tests were developed.
53.5.2
Exaggerated Usage Tests
Intuitively we understand that the closer a clinical
test mimics the way it is used by consumers, the more
predictive of in-use problems, such as irritation, it
will be. This has led to development of exaggerated
wash tests for personal cleansers and immersion testing for dishwashing liquids.
For personal cleansers, the physical nature of a
product, such as lubricity or the presence of abrasive
beads, and the method or tool used for product application will greatly influence the level of irritation
experienced by consumers. For dishwashing liquids,
chemical composition, dosage, and water temperature are key determinants of irritation potential.
53.5.2.1 Exaggerated Wash Tests
Initially, Frosch used an exaggerated half-face wash
method to distinguish soap and synthetic detergent
based cleansing bars. After 2-min washes twice a day
for 4–5 days, Dove was demonstrated to be milder
than Zest or Ivory based on lower observable erythema and panelist self-assessed tightness and stinging.
Since then, two types of exaggerated arm wash
studies have been developed. Strube, Sharko, and
their colleagues at Unilever have developed methods that focus on irritation (erythema and increased
TEWL rates) as the primary endpoints, parameters
to differentiate between products. These methods are
characterized by longer periods (minutes) of washing the skin. In contrast, the methods developed by
Lukacovik, Ertel, and their colleagues at Procter and
Gamble (P&G) focus on skin dryness as the primary
endpoint. These methods are characterized by short
washes (seconds) after which the lather remains on
the skin for more than 1 min, before it is rinsed away.
Arm Wash Methods
Using Irritation as the Primary Endpoint
The antecubital flex test developed by Strube et al.
uses repeated washes with an abrasive applicator to
damage the stratum corneum and produce erythema
in the fold of the elbow [30]. The erythema is evaluated by a trained observer and can be measured instrumentally using a colorimeter, e.g., a chromameter.
Measuring increases in TEWL rates using an evaporimeter assesses stratum corneum barrier damage.
The flex test is relatively sensitive to product differences, since it can distinguish between soap and
bars that have about 10% of the soap replaced with a
milder synthetic detergent such as sodium cocoyl isethionate. The soap chamber test was not able to differentiate between these bars. The irritation response
to the products in the flex test does not vary greatly
with season. This is an advantage over closed patch
testing (see above) and the arm wash method of Lukacovic et al. [31], where the response is reduced by
higher humidity in the summer.
As the test is aggressive, the effects of damage to
the outer stratum corneum are readily overwhelmed
and dryness is not observed. The flex test has been
criticized for being overly traumatic and very dependent on the roughness of the accessory, e.g., sponge,
used to apply the product to the skin [32].
Sharko et al. developed a method able to detect
differences in both dryness and primary irritation
(erythema and TEWL rates) induced by a soap and
Dove, a synthetic detergent bar, after 4.5 days of
twice-daily treatment [33]. For smaller product differences, Sharko, Nicoll, and their colleagues showed
that this method could distinguish between a soap
bar and a bar soap and a low level of sodium cocoyl
isethionate based on erythema and TEWL rates but
not on observed dryness scores [34]. The reason
for this greater discriminatory power for primary
irritation rather than dryness is uncertain. Lather is
applied to the volar forearms by rubbing with gloved
hands for 1 min or more, several times a day. The
53 In Vivo Models of Skin Irritation
increased rubbing may slightly damage the stratum
corneum, enabling the surfactants to penetrate more
readily. Thus irritation rather than drying potential is
the main basis for differentiating between products.
Furthermore the rubbing may mechanically remove
the scaling of upper stratum corneum, so flaking is
less apparent.
(e.g. hairdressers, bar tenders and kitchen workers)
have a significantly higher incidence of hand irritation than the general population [37, 38],. Therefore
it is important to be able to model these effects in
vivo. Below three approaches are described: immersion testing, repeated hand washing, and open application tests.
Washing Studies Using Dryness
as the Primary Endpoint
In the method developed by Lukacovic et al., lather is
applied to the forearms with a towel or muslin cloth
for 10 s and remains there for an additional 90 s. The
surfactant remains on the surface of the skin, and
primarily damages the outer stratum corneum. This
leads to visible dryness and skin roughness. Without
the additional abrasion, little surfactant penetrates
into the viable epidermis and primary irritation is not
induced. Thus soap and mild syndet bars are differentiated based on observable dryness, tactile softness,
and when the differences between products are large,
on erythema as well. This methodology produces
lower responses than that used by Sharko et al. and
appears to differentiate products more on their ability to induce dryness, rather than on irritation potential (method II with products C and D in Nicoll et
al. 1995). It is, however, very sensitive to prevailing
weather conditions, especially humidity. Increasing
the number of wash cycles each day may overcome
this limitation.
In the past, the number of samples that could be
tested simultaneously has been limited for both approaches. Original published reports had focused on
running a single product on each arm. In contrast,
the soap chamber test could evaluate eight samples
simultaneously on the same panelist. Two approaches
have been described to overcome this limitation, especially for less exaggerated methods where dryness,
not primary irritation (observed in closed patch
tests), is the key endpoint. First, Ertel et al. described
modifications to the original method that allowed up
to eight products to be tested simultaneously, four on
each leg [35]. Another approach is to combine different studies using meta-analysis [36].
Immersion Testing
To fully assess the in-use effects of the dishwashing
liquids a realistic exposure, immersion testing should
be used. Repeated short-term (15- to 30-min) immersions of the hands and/or forearms are used to
assess primary irritation and dryness [39, 40]. Paye
et al. showed that two products that could be differentiated in a Frosch and Kligman soap chamber test
could also be differentiated in a hand immersion test,
if the dominant and nondominant hands are assessed
separately. The products could also be differentiated
using bioengineering methods such as skin conductance, and squamometry [41]. Interestingly, the dominant hand was observed to have a lower conductance,
at baseline, than the nondominant hand. Similarly
Grammer-West et al. showed that the Closed patch
(soap chamber) test differentiates the primary irritation potential of anionic- and nonionic surfactantbased dishwashing liquids [42].
This enables a formulator to screen up to eight samples at one time, making formula optimization based
on irritation potential more efficient. The method of
usage or the applicator does not usually play a significant role in the amount of irritation produced in an
in-use situation. However, the intensity of skin effects
is dependent on the products’ composition, concentration, and temperature [43], as well as the reactivity
of the subjects’ skin.
53.5.2.2 Use Testing
A major cause of irritation in both the home and the
work place is repeated exposure to dilute detergent
solutions used for dishwashing and housekeeping, i.e.,
wet work. Epidemiological studies indicate that occupations that involve a great deal of hand washing,
such as nursing, or repeated exposures to surfactants
Repeated Hand Washing
Repeated hand washing with soap has been used generate skin dryness [44]. Initially, dryness and surface
corneocyte damage is produced. This can be assessed
by a trained observer, by conductance measurements,
and by squamometry. With more washings, erythema
and stratum corneum barrier damage, measured by
TEWL, are produced [45]. However, this method is
more frequently used to assess the efficacy of moisturizers to prevent dryness than to compare the ability of different surfactants to elicit it.
Open Application Tests
Repeated exposure of a small test site to surfactantbased cleansers or other cosmetics can produce irritation even when the skin is left open to the environment, i.e., not occluded. This has been used as a
495
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F. Anthony Simion
diagnostic tool in identifying products or ingredients
that have caused adverse reactions (repeated open application test – ROAT) [46]. It has also been used predictively and as a test model. Wigger-Albert, Elsner,
and their colleagues have used the repeated irritation
test to elicit dryness and stratum corneum barrier
damage and assess the ability of protective (barrier)
creams to inhibit the irritation [47].
In two related papers Wilhelm et al. compared the
response of different surfactants to induce irritation
and dryness in open and closed patch testing [48, 49].
They showed that closed patch testing produced more
erythema rather than the dryness observed in open
patch exposure. In open patching, the response was
observed at higher surfactant concentration: 7.5%
compared with 0.5% in occlusive patches. Furthermore, in occlusive patches the anionic surfactant SLS
gave stronger stratum corneum barrier disruption
as measured by TEWL and dryness as measured by
conductance. However, the erythema response was
similar with observer and colorimeter measurements.
the flex wash or the volar forearm wash test have the
potential of removing skin flakes, resulting in a loss
of sensitivity.
53.6.1
In order to demonstrate that the cleanser delivers a
benefit to the skin, the skin must start out in poor
condition. As with moisturizer efficacy studies, the
skin should be dry at baseline (dryness score of 2 or
more on a 0–4 scale). It is best to run the test on a
body site that readily showed skin dryness, such as
the lower legs or the dorsal aspect of the forearms.
The former has sufficient area to enable multiple
products (and a no-product control) to be tested simultaneously. Using a within-subject design enables
potentially large person-to-person variations to be
eliminated. There are three main ways of producing
dry skin:
•
53.6
Models for Measuring
the Moisturizing Potential
of Cleansers
•
•
Previously, most evaluations of cleanser effects on the
skin have been to assess primary irritation or drying
potential. Such studies start with the skin in good
condition and the extent by which parameters such
as erythema and dryness worsen is evaluated. However, moisturization potential has the implication that
the skin condition is improved. Therefore, a different
experimental design is required. Such studies incorporate various aspects of moisturizer efficacy testing,
especially with regard to:
•
•
Starting with dry skin, to enable improvement to
be observed
Using moisturizer end-points, such as assessments
of skin dryness and skin hydration
Testing on Dry Skin
Rely on cold weather frequently occurring during
the winter to produce dryness.
Select people that have a predisposition to dry
skin. As people age, they exhibit more dry skin,
especially at the extremities.
Prewash their test area with a drying cleanser.
Combining the first and second methods is probably
the best approach. Relying on the weather alone can
be risky, as a few warm, humid days will significantly
reduce the level of dryness observed.
Giving the panelists a drying soap bar for regular
cleansing has two great disadvantages. Firstly, the
soap bar may interfere with the effects of the moisturizing cleanser, and consumers do not use two cleansing products on the same body sites. Secondly, Ertel et
al. suggested that artificially drying out the skin with
soap reduces the response compared with naturally
dry skin. The basis of this is unclear, but it contrasts
with the enhanced irritation response observed when
subclinically or mildly irritated skin is exposed to an
irritant.
Together with:
•
Application methods that reflect how the cleansing products are used.
Ideally the application method should not greatly
affect dry skin, especially removing it. Thus the
method initially described by Lukakovic et al. in
1988 is probably most appropriate method. Methods
that involve rubbing for a longer time, for example,
53.6.2
Measuring the Clinical Effects of
Moisturizing Cleansers on the Skin
Based on the approaches used to assess moisturizer
efficacy, the two main parameters to assess the moisturizing potential of cleansing products are:
•
Skin dryness
53 In Vivo Models of Skin Irritation
Table 1. Overview of bioengineering instruments used to assess skin irritation
Skin characteristic
Interpretation
Issues
Instrumentation
Transepidermal water loss
Measure of stratum corneum
barrier integrity/damage
Measures water regardless of source, e.g., sweat
Must use in temperature and humidity controlled environment
Evaporimeter
DermaLab TEWL probe
Tewameter
Skin color/redness
Erythema
Chromameter
Erythema meter
Dermaspectrometer
Blood flow
Irritation increases blood blow Due to laser, cannot
in the superficial dermis
be used near eyes
Laser Doppler velocimeter
Desquamation index
Skin dryness (scaling/flaking)
Reproducible sampling of the skin
D-Squame tape
Image analysis
Skin conductance/capacitance Skin hydration
Materials that change dielectrics of skin will effect reading
Skicon 200
DermaLab
Nova meter
Stained D-Squame
(squamometry)
Reproducible sampling of the skin
High levels of damage can disintegrate cells
and cause loss of dye
D-Squame tape
Polymultichrome stain
Chromameter
•
Skin surface integrity
Skin hydration
It is always advisable to use multiple methods for assessing efficacy, as each individual method has potential shortcomings. The use of a panoply of methods
will yield a fuller assessment of skin condition.
Skin Dryness. Traditionally, a trained observer, using
an ordinal scale, has evaluated skin dryness. However,
this approach has two major drawbacks. Firstly, it is
very dependent on the evaluator, and great care must
be taken to ensure reproducibility between evaluators,
studies, and between different testing laboratories. In
this, a standardized photographic scale is very helpful.
Secondly, there are many factors that can reduce the
appearance of dryness without any benefit to the skin.
These include short-term humidity and occlusive
lotions that matte the dry skin flakes down without
removing them. These problems can be overcome by
using a sticky tape to sample the skin's surface, such
as DeSquame tape (CuDerm Inc. Dallas TX). The
tape is pressed on to the skin's surface and then removed. The greater the scaling, the more skin flakes
are removed by the tape. These can be quantified by
using an analog scale or by image analysis [50]. The
tape will remove the flakes even if they are matted
down or obscured by hydration.
The use of DeSquame tape has been expanded to
assess the damage to surface corneocytes, i.e., squamometry. This is discussed in greater detail above, but
has been shown to detect damage to the skin's surface
before any visible dryness is apparent [51].
Conductance and Capacitance. Conductance and/
or capacitance are frequently used to measure skin
hydration. This approach has been supported empirically by Morrison and Scala [52], who showed a
strong correlation between dryness and reduction in
skin conductance (measured by a Skicon 200) and capacitance (measured by a Nova dermal phase meter).
There are two explanations of how skin conductance
measures dryness. Firstly, as the skin becomes drier,
the concentration of water in the stratum corneum is
reduced. As water is a good conductor compared with
the more hydrophobic stratum corneum, a reduction
in water activity will reduce conductance. Another
possible mechanism by which dryness reduces conductance is that as scales develop, air pockets are
formed in the damaged stratum corneum. As air is
a poor conductor, this scaling also results in reduced
conductance. Clearly these two mechanisms are not
mutually exclusive and may occur simultaneously.
It should be stressed that residues left on the skin's
surface may modify conductance in the absence of
497
498
F. Anthony Simion
dryness. For instance, petrolatum, silicones, and mineral oil are good insulators and can reduce conductance even as they moisturize the skin. Conductance
data should be evaluated based on the product's composition and an understanding of which ingredients
may remain on the skin after rinsing.
53.7
Bioengineering Measurements
of Skin Condition
The last 20 years have seen a great expansion in the
number and sophistication of bioengineering instruments to assess skin condition. These instruments
provide a quantitative assessment of a single characteristic of the skin. Based on our knowledge of skin
physiology and irritation processes, this is used as a
measure of irritation. Like all metrics, bioengineering methods can be misleading, when used inappropriately. They do give objective responses that can
be reduced to a single number or series of numbers.
However, each measurement can be effected by parameters that have nothing to do with skin irritation. For instance an evaporimeter measures water
loss from the skin's surface and cannot differentiate
water loss due to sweating from water loss caused by
disruption of the stratum corneum barrier. Thus the
environmental temperature must be kept below that
causing most panelists to sweat (70°F).
For most bioengineering methods, the environmental and other experimental conditions must be
tightly controlled and the data carefully interpreted.
Guidelines published for many instrumental methods
such as transepidermal water loss should be followed.
Table 1 shows the bioengineering methods most
frequently used to measure irritation.
References
1. Malten KE. Thoughts on irritant contact dermatitis. Contact Dermatitis 1981; 7:238–247
2. Kligman AM. The invisible dermatoses. Arch Dermatol
1991; 127:1375–1382
3. Freeman S, Maibach HI. Study of irritant contact dermatitis produced by repeated patch testing with sodium lauryl
sulfate and assessed by visual methods, transepidermal water loss and laser Doppler velocimtery. J Am Acad Dermatol 1988; 19:496
4. De Groot AC, Nater JP, van der Lende R, Ricken B. Adverse
effects of cosmetics and toiletries – a retrospective study in
a general population. Int J Cosmet Sci 1987; 9:255
5. Frosch PJ, Kligman AM. A method for appraising the stinging capacity of topically applied substances. J Soc Cosmet
Chem 1977; 28:197
6. Green BG, Bluth J. Measuring the chemosensory irritability of human skin. J Toxicol Cut Ocular Toxicol 1995;
14:23–48
7. Yosipovitch G, Maibach HI. Effect of topical pramoxine on
experimentally induced pruritus in humans. J Am Acad.
Dermatol 1997; 37:278–280
8. Leopold CS, Maibach HI. Percutaneous penetration of local anesthetic bases: Pharmacodynamic measurements. J
Invest Dermatol 1999; 113:304–307
9. Simion FA, Rhein LD, Morrison BM Jr, Scala DD, Salko
DM, Kligman AM, Grove GL. Self-perceived sensory responses to soap and synthetic detergent bars correlate
with clinical signs of irritation. J Am Acad Dermatol 1995;
32:205–211
10. Paye M, Cartiaux Y. Squamometry: a tool to move from exaggerated to more realistic application conditions for comparing the skin compatibility of surfactant based products.
Int J Cosmet. Sci 1999; 21:59–68
11. Pierard GE, Arrese JE, Rodriguez C, Daskaleros PA. Effects
of softened and unsoftened fabrics on sensitive skin. Contact Dermatitis 1994; 30:286–291
12. Wilhelm K-P, Cua AB, Wolff HH, Maibach HI. Surfactant
induced stratum corneum hydration in vivo: prediction of
the irritation potential of anionic surfactants. J Invest Dermatol 1993; 101:310–315
13. Rhein LD, Robbins CR, Fernee K, Cantore R. Surfactant
structure effects on swelling of isolated human stratum
corneum. J Soc Cosmet Chem 1986; 37:125–139
14. Imokawa G, Mishima Y. Cumulative effect of surfactants
on cutaneous horny layers: adsorption onto human keratin
layers in vivo. Contact Dermatitis 1979; 5:357–366
15. Kawai M, Imokawa G. The induction of skin tightness by
surfactants. J Soc Cosmet Chem 1984; 147–156
16. Cua A, Wilhelm KP, Maibach HI. Cutaneous sodium lauryl sulfate irritation potential: age and regional variability.
Br J Dermatol 1990; 123:607–613
17. Rougier A, Lotte C, Maibach HI. In vivo relationship between absorption and transepidermal water loss. In: Bronaugh RL, Maibch HI (eds) Topical absorption of dermatological products. Marcel Dekker, New York, p 115–128
18. Allenby CF, Basketter DA, Dickens A et al. An arm immersion model of compromised skin. (I) Influence on irritation reactions. Contact Dermatitis 1993; 28:84–88
19. Frosch PJ, Kligman AM. The chamber scarification test for
irritancy. Contact Dermatitis 1976; 2:314–324
20. Berardesca E, Cespa M, Farinelli N, Rabbiosi G, Maibach
H. In vivo transcutaneous penetration of nicotinates and
sensitive skin. Contact Dermatitis 1991; 25:35–38
21. Agner T, Serup J. Seasonal variation of skin resistance to
irritants. Br J Dermatol 1989; 121:323–328
22. Agner T. Susceptibility of atopic dermatitis patients to irritant dermatitis caused by sodium lauryl sulfate. Atcta
Derm Vernerol (Stockh) 1990; 71:296–300
53 In Vivo Models of Skin Irritation
23. Pinnagoda J, Tupker RA, Ceonraads PJ, Nater JP. Prediction of susceptibility to an irritant response by transepidermal water loss. Contact Dermatitis 1989; 20:341–346
24. Simion FA, Rhein LD, Grove GL, Wojtowski J, Cagan RH,
Scala DD, Sequential order of skin responses to surfactants
in a soap chamber test. Contact Dermatitis 1991; 25:242–
249
25. Phillips L, Steinberg M, Maibach HI, Akers WA. A comparison of rabbit and human skin response to certain irritants. Toxicol Appl Pharmacol 1972; 21:369–382
26. Robinson MK, McFadden JP, Basketter DA. Validity and
ethics of the human 4-h patch test as an alternative method
to assess acute skin irritation potential. Contact Dermatitis
2001; 45:1–12
27. Frosch PJ, Kligman AM. The soap chamber test: a new
method for assessing irritancy of soaps. J Am Acad Dermatol 1979; 1:35–41
28. Babulak SW, Rhein LD, Scala DD, Simion FA, Grove GL.
Quantitation of erythema in a soap chamber test using a
Minolta Chroma (Reflectance) meter: comparison of instrumental results with visual assessments. J Soc Cosmet
Chem 1986; 37:475
29. Morrison BM, Babulak SW, Scala DD, Simion FA, WooMing G, Gyening I, Kenney JA, Kligman AM. Evaluation
of the response of African-American skin to facial cleansing products using a soap chamber test. Scientific exhibit
at the 51st American Academy of Dermatology Annual
Meeting San Francisco CA, 1992
30. Strube DD, Koontz SW, Murahata RI, Theiler RF. The flex
wash test: a method for evaluating the clinical mildness of
cleansing products. J Soc Cosm Chem 1988; 39:355
31. Lukacovic MF, Dunlap FE, Michaels SE, Visscher MO,
Watson DD. Forearm wash test to evaluate the clinical
mildness of cleansing products. J Soc Cosm. Chem 1988;
39:355
32. Keswick BH, Ertel KD, Visscher MO. Comparison of exaggerated and normal use techniques for assessing the
mildness of personal cleansers. J Soc Cosmet Chem 1992;
43:187
33. Sharko PT, Murahata RI, Leyden JJ, Grove GL. Arm wash
with instrumental evaluation – a sensitive technique for
differentiating irritation potential of personal washing
products. J Dermal Clin Eval Soc 1991; 2:19
34. Nicoll GA, Murahata RI, Grove GL Barrows J, Sharko PT.
The relative sensitivity of two arm-wash methods for evaluating the mildness of personal washing products. J Soc
Cosmet Chem 1995; 46:129–140
35. Ertel KD, Neumann PB, Hartwig PM, Rains GY, Keswick
BH. Leg wash protocol to assess the skin moisturization
potential of personal cleansing products. Int J Cosmet Sci
1999; 21:383–397
36. Neumann PB, Ertel KD, Keswick BH, Rains GY. Meta
analysis is a cost effective tool for estimating mildness differences. J Soc Cosmet Chem 1997; 48:283–288
37. Halkier-Sorensen L. Notified occupational skin diseases in
Denmark. Contact Dermatitis 1996; 35 [Suppl 1]:1
38. Wall LM, Gebauer KA. Occupation skin disease in Western Australia. Contact Dermatitis 1991; 24:101–109
39. Kooyman DJ, Snyder FH. Tests for mildness of soap. Arch
Dermatol 1942; 46:846
40. Justice JD, Travers JJ, Vinson LJ. The correlation between
animal tests and human tests in assessing product mildness. Toilet Goods Assoc Proced of the Scientific Section
1961; 35:12
41. Paye M, Gomes G, Zerwick CR, Pierard GE, Grove GL. A
hand immersion test under laboratory controlled usage
conditions: the need for sensitive and controlled assessment methods. Contact Dermatitis 1999; 40:133–138
42. Grammer-West NY, Fitzpatrick JE, Jackson RL, Horton
H, Daminano MA. Comparison of the irritancy of hand
dishwashing liquids with modified patch testing methods.
J Am Acad Dermatol 1996; 35:258–260
43. Clarys P, Manou I, Barel AO. Influence of temperature on
irritation in the hand/forearm immersion test. Contact
Dermatitis 1997; 36:240–245
44. Highley DR, Savoyka VO O’Neill JJ, Ward JB. A stereomicroscopic method for determination of moisturizing efficacy in humans. J Soc Cosmet Chem 1976; 27:351–363
45. Simion FA, Babulak SW, Morrison BM Jr, Rhein LD, Scala
DD. Experimental method for soap-induced dryness in
absence of erythema. Scientific Exhibit at the American
Academy of Dermatology 50th Annual Meeting. December 1991
46. Hannuksela M, Salo H. The repeated open application test.
Contact Dermatitis 1986; 14:221–227
47. Wigger-Alberti W, Krebs A, Elsner P. Experimental irritant
contact dermatitis due to cumulative exposure to sodium
lauryl sulphate and toluene: single and concurrent application. Br J Dermatol 2000; 143:551–556
48. Wilhelm KP, Freitag G, Wolff HH. Surfactant induced skin
irritation and skin repair: evaluation of an acute human
irritation model by non-invasive techniques. J Am Acad
Dermatol 1994; 30:944–949
49. Wilhelm KP, Freitag G, Wolff HH. Surfactant induced skin
irritation and skin repair: evaluation of a cumulative human irritation model by non-invasive techniques. J Am
Acad Dermatol 1994; 31:981–987
50. Schatz. H, Kligman AM, Manning S, Stoudemayer T.
Quantification of dry (xerotic) skin by image analysis of
scales removed by adhesive discs (D-Squames). J Soc Cosmet Chem 1993; 44:53–63
51. Simion FA, Witt PS, Rau AH, Thueneman PJ, Peters HE.
Stratum corneum damage produced by surfactants during dry skin induction. Scientific exhibit at the American
Academy of Dermatology Meeting, 1995
52. Morrison BM Jr, Scala DD. Comparison of instrumental
measurements of skin hydration J Toxicol Cut Ocular Toxicol 1996; 14:305–414
499