ArticlePDF Available

In Vitro Assessment of Skin Irritation Potential of Surfactant-based Formulations by Using a 3-D Skin Reconstructed Tissue Model and Cytokine Response


Abstract and Figures

The personal care industry is focused on developing safe, more efficacious, and increasingly milder products, that are routinely undergoing preclinical and clinical testing before becoming available for consumer use on skin. In vitro systems based on skin reconstructed equivalents are now established for the preclinical assessment of product irritation potential and as alternative testing methods to the classic Draize rabbit skin irritation test. We have used the 3-D EpiDerm™ model system to evaluate tissue viability and primary cytokine interleukin-1α release as a way to evaluate the potential dermal irritation of 224 nonionic, amphoteric and/or anionic surfactant-containing formulations, or individual raw materials. As part of our testing programme, two representative benchmark materials with known clinical skin irritation potential were qualified through repeated testing, for use as references for the skin irritation evaluation of formulations containing new surfactant ingredients. We have established a correlation between the in vitro screening approach and clinical testing, and are continually expanding our database to enhance this correlation. This testing programme integrates the efforts of global manufacturers of personal care products that focus on the development of increasingly milder formulations to be applied to the skin, without the use of animal testing.
Content may be subject to copyright.
The current efforts of the consumer products
industry are focused on finding in vitro methods
that can reliably assess the enhanced or improved
mildness of final formulations designed for topical
application to the skin. Traditionally, a combined
strategy based on the Draize rabbit primary der-
mal irritation test (1) and human clinical testing
was used to demonstrate enhanced mildness.
However, ethical concerns over unnecessary ani-
mal use, in addition to the need for efficient, cost-
effective preclinical screening assays for prototype
exploration, have promoted the development of
alternative methods for determining skin irrita-
tion (2–10).
As a fully immunocompetent organ, the skin
responds with an inflammatory reaction to the var-
ious chemicals or formulated products it comes in
contact with, either accidentally or intentionally.
The local inflammatory reaction following expo-
sure to an irritant is believed to start with the
release of primary cytokines (such as interleukin
[IL]-1α) that stimulate the synthesis and release of
multifunctional secondary cytokines, which then
escalate and drive the inflammatory cascade or
serve to repress the reaction through negative
feedback. The release of pro-inflammatory medi-
ators from skin equivalents in response to an irri-
tant has been shown to be useful for the
prediction of skin irritation potential, when in
vitro results were compared with the human clini-
cal response (2, 3). To that end, skin equivalents
like EpiDerm™ (MatTek Corporation, Ashland,
MA, USA) have become the prime choice for alter-
native testing methods.
Personal care cleansers typically contain multi-
ple different surfactant species in solution. The dif-
ferent surfactants self-assemble into mixed
micelles, typically spherical structures containing
the different surfactants. These mixed micelle sur-
factant systems, which often behave differently to
the individual surfactants, can change the thermo-
dynamics of the surfactant system and the extent
to which surfactants penetrate and disrupt the
skin (11–14). Therefore, the resultant properties of
the cleansers are determined by the way the sur-
factant system works. Baby cleansers are typically
In Vitro Assessment of Skin Irritation Potential of
Surfactant-based Formulations by Using a 3-D Skin
Reconstructed Tissue Model and Cytokine Response
Russel M. Walters,1Lisa Gandolfi,2M. Catherine Mack,1Michael Fevola,1Katharine Martin,1
Mathew T. Hamilton,3Allison Hilberer,4Nicole Barnes,4Nathan Wilt,4Jennifer R. Nash,4Hans
A. Raabe4and Gertrude-Emilia Costin4
1Johnson & Johnson Consumer Inc., Skillman, NJ, USA; 2Clariant Corporation, Charlotte, NC, USA;
3Wellspring Worldwide Inc., Chicago, IL, USA; 4Institute for In Vitro Sciences Inc., Gaithersburg, MD, USA
Summary — The personal care industry is focused on developing safe, more efficacious, and increasingly
milder products, that are routinely undergoing preclinical and clinical testing before becoming available for
consumer use on skin. In vitro systems based on skin reconstructed equivalents are now established for the
preclinical assessment of product irritation potential and as alternative testing methods to the classic Draize
rabbit skin irritation test. We have used the 3-D EpiDerm™ model system to evaluate tissue viability and
primary cytokine interleukin-1αrelease as a way to evaluate the potential dermal irritation of 224 non-
ionic, amphoteric and/or anionic surfactant-containing formulations, or individual raw materials. As part of
our testing programme, two representative benchmark materials with known clinical skin irritation poten-
tial were qualified through repeated testing, for use as references for the skin irritation evaluation of for-
mulations containing new surfactant ingredients. We have established a correlation between the in vitro
screening approach and clinical testing, and are continually expanding our database to enhance this cor-
relation. This testing programme integrates the efforts of global manufacturers of personal care products
that focus on the development of increasingly milder formulations to be applied to the skin, without the
use of animal testing.
Key words: 3-D skin equivalents, IL-1
, in vitro, irritation, surfactants.
Address for correspondence: Russel M. Walters, Johnson & Johnson Consumer Inc., 199 Grandview Rd,
Skillman, NJ, USA.
ATLA 44, 523–532, 2016 523
mild to the skin, and they often contain blends of
anionic, amphoteric and non-ionic surfactants.
Adult face cleansers are typically also mild, and
may contain blends of anionic, amphoteric and
non-ionic surfactants, whereas adult shampoos are
usually more aggressive and contain primarily
anionic surfactants. The penetration of surfactants
into the stratum corneum of the skin, and the ways
in which surfactants interact with the endogenous
skin lipids, can affect the barrier function of the
skin (11, 13–16) and result in increases in
transepidermal water loss (TEWL; 17–19), which
can be assessed clinically.
A critical component in the development of reli-
able in vitro screening methods for skin irritation is
the correlation of in vitro test results with clinical
skin irritation performance. The more prevalent in
vitro tests used to assess surfactant aggressiveness,
such as the Zein solubilisation test (5, 6) or transep-
ithelial permeation test (also known as fluorescein
dye leakage test; 4, 8, 9), have not been successfully
correlated to in vivo skin irritation models. Thus,
significant effort was put into generating a reliable
correlation between data provided by in vitro assays
based on reconstructed tissue models and clinical
studies for the assessment of skin irritation poten-
tial of surfactant-based formulations.
By employing 3-D reconstructed skin tissues
with tissue viability and IL-1αexpression analysis
as endpoints, we have developed an in vitro test
method to evaluate the potential dermal irritation
of non-ionic, amphoteric and/or anionic surfactant-
containing cleansing formulations or individual
surfactants. An exaggerated patch test with the
TEWL endpoint was used as the clinical assess-
ment for correlation with our in vitro test method.
Due to the successful correlation between in vitro
and clinical results, the EpiDerm-based dermal
irritation test can be used to compare and analyse
the potential dermal irritation of new formulations
as a guideline for formulation development for new
mild(er) skin-cleansing products.
Materials and Methods
Test articles
Sixteen individual surfactants (considered to be
‘raw materials’), 46 commercial surfactant-based
cleansers, and 162 other prototype surfactant-
based cleansers (non-commercialised formula-
tions), were used as test articles, making a total of
224 different test articles.
The test articles were tested as 10% dilutions
(weight/volume percentage [w/v]) in sterile,
deionised water (Quality Biological, Gaithersburg,
MD, USA), unless otherwise specified. As typical
mild surfactant-based cleansers contain about 10%
(w/v) surfactant, the 16 individual surfactants were
diluted to 10% (w/v) in sterile, deionised water, then
tested at 10% (w/v) dilutions, for a final concentra-
tion of 1% (w/v).
The 10% (w/v) dilution mimics the end-user expo-
sure to wash-off products such as shampoos, condi-
tioners, etc. Two test articles were included in nearly
every study and were qualified as benchmark mate-
rials (Mild Cleanser 1 and Mild Cleanser 2). They
were tested 32 and 35 unique times, respectively;
each individual test consisted of three independent
tissue samples. These two benchmark materials
were mild cleansers containing a mixture of anionic,
amphoteric and non-ionic surfactants. Triton®
X-100 (Fisher Scientific, Pittsburgh, PA, USA) was
used as the assay positive control to assess the qual-
ity of the tissue lots used in the experiments. Sterile,
deionised water (Quality Biological) was used as the
assay negative control.
Reconstructed tissues
The 3-D human reconstructed epidermal model
EpiDerm Skin Model (EPI-200) provided by
MatTek Corporation (Ashland, MA, USA) was
used in our experiments. The EpiDerm tissues are
based on normal, human-derived epidermal ker-
atinocytes cultured to form a multilayered, highly
differentiated model of the human epidermis (20).
The EpiDerm tissues were cultured in a Dulbecco’s
Modified Eagle Medium-based culture medium
provided by the tissue supplier. Since the tissues
have a functional stratum corneum, the test arti-
cles were applied directly to the culture surface, at
the air interface.
Treatment of the EpiDerm tissues
The EpiDerm tissues were stored at 2–8°C until
used. The day before treatment, the EpiDerm tis-
sues were cultured in six-well plates containing a
hydrocortisone free-assay medium (HCF-AM), and
equilibrated at 37 ± 1°C in a humidified atmo-
sphere of 5 ± 1% CO2in air (standard culture con-
ditions) overnight.
Each EpiDerm tissue was considered an inde-
pendent sample. At least 16 hours after initiating
the tissue cultures, the medium was removed from
under the tissues and a 0.9ml aliquot of fresh, pre-
warmed HCF-AM was added to each well. Each
test article (100μl) was applied onto three tissues,
and the negative control (100μl sterile, deionised
H2O) was added to the other three tissues in the
six-well plate. At the end of the 1-hour exposure
period, each tissue was rinsed five times with
approximately 0.5ml per rinse of calcium-free and
magnesium-free Dulbecco’s phosphate-buffered
saline (CMF-DPBS; Quality Biological). After rins-
524 R.M. Walters et al.
ing, each tissue was placed in the designated well
of a new six-well plate containing 0.9ml of fresh
HCF-AM, and incubated under standard culture
conditions for the post-exposure incubation period
(24 hours).
Viability assay
Tissue viability was determined by using a method
based on the reduction of the yellow tetrazolium
salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) to the purple formazan dye
by mitochondrial succinate dehydrogenase in
viable cells (21). A 1mg/ml solution of MTT in
warm MTT addition medium was prepared no
more than 2 hours before use.
Upon completion of the 24-hour post-exposure
incubation, the tissues were removed from their
incubation medium, rinsed with CMF-DPBS,
blotted dry and transferred into pre-labelled 24-
well plates containing 300μl MTT solution per
well. The medium from under each tissue was
quick-frozen (≤ –60°C) for subsequent cytokine
After 3 ± 0.1 hours of incubation in MTT, the
EpiDerm tissues were blotted on absorbent paper
and transferred into 24-well plates containing 2ml
of isopropanol per well and shaken at room tem-
perature. After 2 hours, the absorbance of a 200μl
aliquot of tissue extract was measured at 550nm
(Vmax® Kinetic ELISA microplate reader;
Molecular Devices, Sunnyvale, CA, USA). The via-
bility of the tissues exposed to the test articles was
calculated and expressed as a percentage relative
to the viability of the negative control (i.e.
deionised water-treated tissues). The tissue viabil-
ity value was taken as the mean value from the
three independent wells tested in each experi-
The IL-1αconcentration was determined by using
a kit from R&D Systems (Minneapolis, MN, USA),
according to the manufacturer’s instructions.
Thawed media samples, collected as described pre-
viously, were tested neat and as 1:10 dilutions, to
keep the readings within the linear range of the
assay. The IL-1αvalue reported for each test was
the mean value from the three independent tissues
used per test article in each experiment and plated
in duplicate.
Clinical study: Exaggerated patch test
Adult subjects (aged 18–65 years), who had been
diagnosed with atopic dermatitis, and therefore
had skin with impaired barrier function, were
recruited into the study. Each subject gave written
informed consent to participate, and the protocol
for the study was approved by an institutional
review board. Each subject was exposed to the test
articles (diluted to 50% with distilled water) on the
volar forearm under an occlusive patch for
24 hours (n= 25 subjects/panel). Typical in-use
exposure to personal care cleansers occurs over a
varying range of cleanser dilution during the
course of the washing period. A 50% dilution is
likely a relatively high cleanser concentration, and
was used in our clinical study in order to differen-
tiate between relatively mild surfactant systems.
After 24 hours, the patch and cleansing solution
were removed, and a new patch with the same
treatment was reapplied; this operation was
repeated for a total of four days. The skin barrier
function was measured before and after four days
of patching. The TEWL value was measured by
using a VapoMeter (Delfin Technol ogies, Kuopio,
Finland), as described previously (22). Clinical
TEWL results represented the change from the
baseline (i.e. before patching of the test article)
TEWL at Day 4. Each of the personal care cleans -
ers was tested on a panel of subjects; 27 subjects
were recruited into each testing panel, and at
least 25 subjects in each panel completed the
study. The data set of 28 cleansers combines the
results from three different panels that occurred
at different times of the year.
Test system reproducibility: Tissue viability
and IL-1αendpoints
To assess the test system reproducibility and to
determine the actual shapes of the distributions,
the tissue viability (MTT endpoint) and cytokine
release (IL-1αendpoint) data were analysed for
two mild cleanser formulas, two positive controls
and one negative control that were repeatedly
tested. Figure 1a shows the experimentally deter-
mined distribution of tissue viability after treat-
ment with the two benchmark materials. In
statistical analysis, commonly normal distribu-
tions are assumed, while biological systems often
produce distributions that are not normal. Due to
the repeated measurement, the distribution can be
experimentally generated for the four different
exposures. For each exposure, the set of responses
were binned (bin width of 5%), and the fraction of
all responses are shown for each bin. The mean per-
centages (± SD) of viable tissues treated with Mild
Cleanser 1 and Mild Cleanser 2 were 100.1 ± 7.1%
and 96.9 ± 6.4%, respectively. Also shown in
Figure 1a are the results of the positive control,
Skin irritation potential of cleansers 525
Figure 1: Tissue viability and IL-1αendpoints
20 30 40 50 60 70 80 90 100 110
tissue viability (%)
fraction of tests
31 39 49 61 77 96 120 150 187 234 293
Log IL-1α(pg/ml)
fraction of tests
50 75 100 125 150 175 200 225 250 275
fraction of tests
= Positive control (8 hours); = positive control (4 hours); = Mild Cleanser 2; = Mild Cleanser 1;
= negative control in c).
Graph a) shows the shape of the distribution and the reproducibility of tissue viability (%). The results of the positive
control, Triton X-100 (4-hour and 8-hour exposures), are also shown. Graph b) shows the IL-1
concentration (pg/ml)
for tissues treated with Mild Cleanser 1 and Mild Cleanser 2. IL-1
results are shown on a linear scale (graph b) and
log scale (graph c).
526 R.M. Walters et al.
Triton X-100-treated tissue, which, when exposed
for 4 and 8 hours, demonstrated a reduction in via-
bility of 18.9 ± 10.6% and 82.5 ± 10%, respectively.
IL-1αrelease from the same tissues is shown in
Figure 1b, on both linear and log scales. Also shown
in Figure 1b are the results of the negative control
(i.e. deionised water-treated tissues), after the 1-
hour exposure time. Although the viability assay
showed no difference between the two benchmark
materials (Mild Cleansers 1 and 2), there was a
clear difference in IL-1αproduction between them,
indicating that the sensitivity of the IL-1αendpoint
was greater than that of the MTT viability end-
point, and also suggesting that Mild Cleanser 2 had
slightly greater irritation potential than Mild
Cleanser 1. Mean values for IL-1αrelease after
exposure to Cleanser 1 (n= 32) and Cleanser 2 (n=
35) were 69 ± 25pg/ml and 149 ± 53pg/ml, respec-
tively, and the difference between the cleansers in
IL-1αrelease was significant (P < 0.001). Mean val-
ues for IL-1αrelease after exposure to the assay
positive control (Triton X-100) for 4 and 8 hours
were 100 ± 44pg/ml and 320 ± 124pg/ml, respec-
tively, and 20 ± 10pg/ml after exposure to deionised
water, the assay negative control. Therefore, the IL-
1αresponse was used to assess potential dermal
irritation of raw materials and final formulations in
this testing programme.
Skin irritation continuum for surfactant-
based test articles with rinse-off product
The sensitivity of the test system was also investi-
gated for a wide range of surfactant-based cleans-
ing formulations. Figure 2a shows the distribution
of tissue viability values obtained for 224 different
surfactant-based formulations. Most of these test
articles do not impact tissue viability, and the
results are distributed as expected, at around
100%. There is a small set of results with viability
values from 0 to 15%, and a smaller fraction (4% of
the total) with viability from 20% to 70%. The
observed distribution from these surfactant-based
cleansing formulations of tissue viability appears
to be largely binary, either viable or not viable.
Tissue viability does not appear to be a continuous
variable and, as such, traditional statistical
descriptions are not valid. Figure 2b shows the dis-
tribution in the IL-1αresponse across the range of
surfactant-based cleansers. The distribution is
clearly non-linear; there is a high fraction of
results that exhibit low IL-1αresponses and a long
tail of rare results that exhibit very high IL-1α
responses. The insert shows a power law distribu-
tion, which appears to be a more appropriate fit to
the data.
Figure 2c shows the correlation between tissue
viability and IL-1αobtained for the same set of test
articles. In the skin irritation continuum assessed
by the two endpoints, for over 90% of the test arti-
cles, the exhibited tissue viability was around
100%. Within this majority of test articles, a wide
range of IL-1αconcentrations (between 10pg/ml
and approximately 600pg/ml) was observed. For
the remaining approximately 10% of test articles,
the tissue viability was below 85% and was typi-
cally associated with a high IL-1αrelease (Figure
2b). For the remaining formulations tested, IL-1α
production of ≥ 600pg/ml indicated increased toxic-
ity associated with materials that can be classified
as moderate to severe irritants. Overall, our data
confirmed the initial observations that IL-1α
released from the tissues is a more sensitive end-
point for distinguishing and rank-ordering final
formulations that range in irritancy potential.
Skin irritation assessment of commercial
skincare cleansers by the EpiDerm test
Previous studies reported that different classes of
surfactants have different skin irritation profiles
(12, 23). In our study, 16 individual surfactants
commonly used in personal care formulations were
assessed. As shown in Figure 3a, there was a wide
range of responses (over two orders of magnitude
in IL-1αrelease). At constant active concentra-
tions, non-ionic surfactants (light grey bars) were
assessed by our test system as the mildest (lowest
IL-1αreleased), whereas the anionic surfactants
(black bars) resulted in the greatest IL-1α release.
Amphoteric surfactants (dark grey bars) had inter-
mediate effects. Among the anionic surfactants,
the sulphate and sulphonate head groups were
most irritating, whereas surfactants with alterna-
tive anionic head groups (such as isethionate or
glutamate) were milder. Glucamide non-ionic sur-
factants with longer carbon chains, such as cocoyl
methyl glucamide and lauroyl methyl glucamide
(C16–18), were milder than shorter carbon chains,
such as capryloyl/caproyl methyl glucamide (C8–
10). Independent of charge, surfactants with long
hydrophilic repeat units, such as polyglycerol or
polyethylene oxide, were among the mildest in
their particular class. Polyglycerol-10 laurate and
sodium laureth-13 carboxylate are representative
of this trend.
We used this in vitro system to determine the
surfactant aggressiveness of 46 different commer-
cial skin cleansers. The commercial cleansers were
grouped into three categories based on their stated
marketing segment: baby washes and shampoos
(n= 21), adult facial cleansers (n= 9), and adult
body washes and shampoos (n= 16). As shown in
Figure 3b, each category displayed a range of irri-
tation responses: baby wash and shampoo tended
to induce less IL-1αrelease than adult body wash
and shampoo, which induced a greater release of
Skin irritation potential of cleansers 527
Figure 2: Sensitivity of the test system to 224 different surfactant-based formulations
Graph a) shows the distribution of the tissue viability response (%) for the 224 surfactant-based test articles in bins of
tissue viability. Graph b) shows the distribution of the IL-1
response for the 224 surfactant-based test articles in
bins of IL-1
. Graph c) shows the correlation between tissue viability (%) and IL-1
concentration (pg/ml) for 224
surfactant-based test articles with rinse-off applications. The dotted line indicates 85% viability.
20 30 40 50 60 70 80 90 100 110 120 130
tissue viability (%)
fraction of tests
0 50 150 250 350 450 550 650 750 850 950 1050 1150 1250 1350 1450 1550 1650
Log IL-1α(pg/ml)
fraction of tests
Log IL-1α(pg/ml)
fraction of tests
tissue viability (%)
528 R.M. Walters et al.
Figure 3: Cytokine release in response to cleanser exposure
= Anionic; = amphoteric; = non-ionic.
SH = sodium hydrolysed.
Graph a) shows the individual surfactants typically used in commercial cleanser products.
Graph b) shows the three classes of commercial cleanser products. For each class of cleansers, mean values are
represented by horizontal lines. The * in graph b) represents a liquid castile soap considered to be an outlier for the
class of baby washes and shampoo products. Each set of symbols is slightly offset for ease of visualisation.
Alpha olefin sulphonate
Sodium lauryl sulphate
Sodium lauroyl methyl isethionate
Sodium cocoyl glycinate
Sodium cocoyl glutamate
Sodium cocoyl isethionate
Capryloyl/caproyl methyl glucamide
Lauroyl methyl glucamide and ethanol
Cocamidopropyl betaine
Sodium-laureth-13 carboxylate
Cocoyl methyl glucamide
Lauroyl methyl glucamide
Polyglyceryl-10 laurate
SH potato starch dodecenylsuccinate
0baby wash
and shampoo
adult facial
adult body
wash and
Skin irritation potential of cleansers 529
the cytokine. The adult facial cleanser group had
the widest range of irritation responses; this was
expected, as this group contained mixed categories
of cleansers with some intended for use on sensi-
tive skin, described as mild products, and some
more aggressive products, such as acne cleansers.
In the baby wash and shampoo class, although
most commercial formulations induced little IL-1α
release (median 61pg/ml), one of the commercial
baby cleansers appeared to be an outlier, inducing
the release of 504pg/ml of IL-1α(depicted as an
asterisk in Figure 3b). This outlier product was a
liquid castile soap with a high pH of 9.6. Castile
soaps and other high-pH cleansers are well known
to be harsh, and thus are not recommended for
babies by the Association of Women’s Health,
Obstetric and Neonatal Nurses (AWHONN).
AWHONN guidelines for neonatal skin care (24) rec-
ommend neutral to slightly acidic pH (5.5 to 7.0)
cleansers for babies. Although this outlier product is
marketed as a baby cleanser, it would not be consid-
ered by AWHONN as an appropriate baby cleanser
product, and the high level of IL-1αrelease identifies
the product as a potential irritant.
Correlation of preclinical and clinical test
Twenty-eight different personal care cleansers
were evaluated in the in vitro IL-1α release test
and in a human clinical study that tested the
effects of the cleansers on the skin barrier. Figure
4 shows the correlation between IL-1αrelease from
the EpiDerm reconstructed tissues and barrier dis-
ruption (assessed by change in TEWL) in the clin-
ical study. The range of IL-1αcytokine release
spanned the full range of cleanser aggressiveness
that was observed in the commercial cleanser set
in Figure 3b. Over this wide range of surfactant-
based skin cleansers, a positive correlation could
be established between the results generated by
the in vitro test system and in the human clinical
study. For example, increased TEWL corresponded
to high IL-1αproduction, whereas minimal
changes in TEWL corresponded to a lower cytokine
response. These results suggested that the in vitro
test successfully predicted the change in TEWL, a
known human clinical response to surfactant-
based personal care cleansers. A calculation based
on this data set showed an R2= 0.66, which indi-
cates a strong correlation (P < 0.01) when consider-
ing the variation that is typically present in
human test models.
Reproducible in vitro test systems can be used to
accurately assess the irritation potential of raw
materials and formulated products, thus avoiding
the use of animals for testing. The test system pre-
sented here, based on the 3-D EpiDerm model
(MatTek Corporation), and employing tissue viabil-
ity and IL-1αrelease as the endpoints, has proven to
be a useful tool for new formulation development
and provided a testing platform for Johnson &
Johnson’s skin irritation assessment programme.
During our testing programme, two representa-
tive benchmark materials were qualified for use as
reference materials in the evaluation of skin irrita-
tion of formulations containing new surfactant
ingredients. We have developed a database and
established a range of potential irritation
responses to the benchmarks, by using the IL-1α
release endpoint (Figures 1a and 1b). Comparison
of the dermal irritation potential of new formula-
tions with that of existing mild formulations can
guide the formulation development of new mild
cleansing products.
We also report on a large in vitro data set of indi-
vidual surfactants and typical skincare cleansing
systems. Although the tissue viability endpoint
could identify relatively harsh personal care
cleansers, IL-1αwas found to be the more sensitive
endpoint in discriminating mild cleansers (Figures
2 and 3). Values of tissue viability of ≤ 80% are
Figure 4: The correlation between cytokine
release in vitro and clinical TEWL
R2= coefficient of determination = 0.66;
TEWL = transepidermal water loss.
The graph shows the correlation between in vitro IL-1
concentration (pg/ml) and the change in TEWL after
4 days’ treatment in the exaggerated patch clinical model.
in vitro: IL-1α(pg/ml)
clinical: TEWL (mg/cm2/h)
530 R.M. Walters et al.
generally associated with unpredictable, unreli-
able, and difficult-to-interpret levels of IL-1α
released by the affected tissues (3). Within our
testing programme, the model was valuable for the
comparison of internal products with products pro-
duced by others, and was adaptable to a variety of
rinse-off products with a range of mildness pro-
files. Additionally, we found a promising correla-
tion between our large set of in vitro results and
the corresponding human clinical results that
assessed changes in skin barrier function by using
TEWL as an endpoint, demonstrating the value of
the in vitro testing platform in predicting clinical
results (Figure 4).
It is well known that surfactant concentration
and surfactant combinations in fully formulated
cleansers affect the overall dermal irritation poten-
tial (12, 14, 23). Thus, the irritation assessment of
individual surfactants is typically used as a cur-
sory pre-screening technique before formulation
development. It is necessary to also evaluate the
irritation potential of fully formulated prototypes
or products. In addition to providing a measure of
dermal irritation potential of individual surfac-
tants for formulation guidance, the test system
based on the EpiDerm model has been a useful
research tool for evaluating the dermal irritation
potential of internal products and prototypes.
Overall, our data showed that the in vitro der-
mal irritation test specifically designed to address
the testing needs for the classes of raw materials
and formulated products, described here, has util-
ity for developing an understanding of individual
ingredient skin irritation potential, and for pre-
dicting clinical irritation profiles, as well as for
guiding the formulation development of non-irri-
tating rinse-off products.
This work was supported in full by Johnson &
Johnson Consumer Inc., Skillman, NJ, USA.
The authors would like to acknowledge the clin-
ical subjects participating in these studies. Medical
writing and editorial assistance was provided by
Alex Loeb, PhD, CMPP, of Evidence Scientific
Solutions (Philadelphia, PA, USA), and was
funded by Johnson & Johnson Consumer Inc.
Received 05.05.16; received in final form 25.10.16;
accepted for publication 27.10.16.
1. Draize, J.H., Woodard, G. & Calvery, H.O. (1944).
Methods for the study of irritation and toxicity of
substances applied topically to the skin and mucous
membranes. Journal of Pharmacology & Experi -
mental Therapeutics 82, 377–390.
2. Bernhofer, L.P., Barkovic, S., Appa, Y. & Martin,
K.M. (1999). IL-1αand IL-1ra secretion from epi-
dermal equivalents and the prediction of the irrita-
tion potential of mild soap and surfactant-based
consumer products. Toxicology in Vitro 13, 231–
3. Bernhofer, L.P., Seiberg, M. & Martin, K.M. (1999).
The influence of the response of skin equivalent sys-
tems to topically applied consumer products by
epithelial–mesenchymal interactions. Toxicology in
Vitro 13, 219–229.
4. Cottin, M. & Zanvit, A. (1997). Fluorescein leakage
test: A useful tool in ocular safety assessment.
Toxicology in Vitro 11, 399–405.
5. Deo, N., Jockusch, S., Turro, N.J. & Somasundaran,
P. (2003). Surfactant interactions with Zein protein.
Langmuir 19, 5083–5088.
6. Gotte, E. (1964). Skin Compatibility of Tensides
Measured by Their Capacity for Dissolving Zein
Protein. [Presentation at Proceedings of the 4th Inter -
national Congress of Surface Active Substances,
Brussels, Belgium, 7–12 September 1964].
7. Lee, J.K., Kim, D.B., Kim, J.I. & Kim, P.Y. (2000).
In vitro cytotoxicity tests on cultured human skin
fibroblasts to predict skin irritation potential of sur-
factants. Toxicology in Vitro 14, 345–349.
8. Martin, K. & Stott, C. (1992). The Trans-Epithelial
Permeability Assay as an In Vitro Assay for
Predicting Ocular Irritation of Surfactant Form -
ulations. [Presentation at World Congress on Cell
and Tissue Culture, Washington, DC, USA, 20–25
June 1992].
9. Tchao, R. (1988). Trans-epithelial permeability of
fluorescein in vitro as an assay to determine eye
irritants. In Alternative Methods in Toxicology (ed.
A.M. Goldberg), pp. 271–283. New York, NY, USA:
Mary Ann Liebert, Inc.
10. Vinardell, M.P. & Mitjans, M. (2008). Alternative
methods for eye and skin irritation tests: An over -
view. Journal of Pharmaceutical Sciences 97, 46–
11. Ghosh, S. & Blankschtein, D. (2007). The role of
sodium dodecyl sulfate (SDS) micelles in inducing
skin barrier perturbation in the presence of glyc-
erol. Journal of Cosmetic Science 58, 109–133.
12. LiBrizzi, J.J., Walters, R.M., Fevola, M. & Tamar -
selvy, K. (2010). Low-irritation compositions and
methods of making the same. United States Patent
7,803,403, 28 September 2010.
13. Moore, P.N., Shiloach, A., Puvvada, S. & Blank -
schtein, D. (2003). Penetration of mixed micelles
into the epidermis: Effect of mixing sodium dodecyl
sulfate with dodecyl hexa(ethylene oxide). Journal
of Cosmetic Science 54, 143–159.
14. Moore, P.N., Puvvada, S. & Blankschtein, D. (2003).
Challenging the surfactant monomer skin penetra-
tion model: Penetration of sodium dodecyl sulfate
micelles into the epidermis. Journal of Cosmetic
Science 54, 29–46.
15. Mao, G., Flach, C.R., Mendelsohn, R. & Walters, R.M.
(2012). Imaging the distribution of sodium dodecyl
sulfate in skin by confocal Raman and infra red
microspectroscopy. Pharmaceutical Research 29,
16. Saad, P., Flach, C.R., Walters, R.M. & Mendelsohn,
R. (2012). Infrared spectroscopic studies of sodium
dodecyl sulphate permeation and interaction with
stratum corneum lipids in skin. International
Journal of Cosmetic Science 34, 36–43.
Skin irritation potential of cleansers 531
17. de Jongh, C.M., Verberk, M.M., Spiekstra, S.W.,
Gibbs, S. & Kezic, S. (2007). Cytokines at different
stratum corneum levels in normal and sodium lau-
ryl sulphate-irritated skin. Skin Research &
Technology 13, 390–398.
18. Lévêque, J.L., de Rigal, J., Saint-Léger, D. & Billy,
D. (1993). How does sodium lauryl sulfate alter the
skin barrier function in man? A multiparametric
approach. Skin Pharmacology 6, 111–115.
19. van der Valk, P.G., Nater, J.P. & Bleumink, E.
(1984). Skin irritancy of surfactants as assessed by
water vapor loss measurements. Journal of Invest -
igative Dermatology 82, 291–293.
20. Cannon, C.L., Neal, P.J., Southee, J.A., Kubilus, J. &
Klausner, M. (1994). New epidermal model for der-
mal irritancy testing. Toxicology in Vitro 8, 889–891.
21. Berridge, M.V., Tan, A.S., McCoy, K.D. & Wang, R.
(1996). The biochemical and cellular basis of cell
proliferation assays that use tetrazolium salts.
Biochemica 4, 14–19.
22. Pappas, A., Fantasia, J. & Chen, T. (2013). Age and
ethnic variations in sebaceous lipids. Dermato-
Endocrinology 5, 319–324.
23. Walters, R.M., Fevola, M.J., LiBrizzi, J.J. & Martin,
K. (2008). Designing cleansers for the unique needs
of baby skin. Cosmetics & Toiletries 123, 53–60.
24. Association of Women’s Health Obstetric and Neo -
natal Nurses (2013). Neonatal Skin Care: Evidence-
based Clinical Practice Guideline, 3rd edn, 81pp.
Washington, DC, USA: Association of Women’s
Health, Obstetric and Neonatal Nurses.
532 R.M. Walters et al.
... There are currently three commercially available RhS and a number of in-house models ( Table 2). The EpidermFT™ has been used to determine the skin irritation potential of surfactants by assessment of the release of the primary cytokine interleukin IL-1α after exposure to 46 commercial skin cleansers (containing 224 nonionic or anionic surfactant-containing formulations) (Walters et al. 2016). The IL-1α release measured in vitro was compared to clinical TEWL (transepidermal water loss) measurements and showed good correlation (R 2 = 0,66). ...
Full-text available
Contact with the skin is inevitable or desirable for daily life products such as cosmetics, hair dyes, perfumes, drugs, household products, and industrial and agricultural products. Whereas the majority of these products are harmless, a number can become metabolized and/or activate the immunological defense via innate and adaptive mechanisms resulting in sensitization and allergic contact dermatitis upon following exposures to the same substance. Therefore, strict safety (hazard) assessment of actives and ingredients in products and drugs applied to the skin is essential to determine I) whether the chemical is a potential sensitizer and if so II) what is the safe concentration for human exposure to prevent sensitization from occurring. Ex vivo skin is a valuable model for skin penetration studies but due to logistical and viability limitations the development of in vitro alternatives is required. The aim of this review is to give a clear overview of the organotypic in vitro skin models (reconstructed human epidermis, reconstructed human skin, immune competent skin models incorporating Langerhans Cells and T-cells, skin-on-chip) that are currently commercially available or which are being used in a laboratory research setting for hazard assessment of potential sensitizers and for investigating the mechanisms (sensitization key events 1–4) related to allergic contact dermatitis. The limitations of the models, their current applications, and their future potential in replacing animals in allergy-related science are discussed.
... Although the antioxidant, anti-inflammatory and antimicrobial activities of the aqueous and ethanolic extracts were as high as expected, at this stage, it is also important to test the irritation potential of the formulations at the site of application (Walters et al., 2016). Therefore, testing the final product to evaluate its combability to the skin is essential before further in vivo preclinical analyses (Okur et al., 2018). ...
Ethnopharmacological relevance Okra fruit (Abelmoschus esculentus (L.) Moench) has been extensively used for the treatment of skin damage and subcutaneous tissue abscess for many years in Turkish folk medicine. Aim of study In this study, we aimed to investigate the wound healing potential of okra fruit by in vitro and in vivo experimental models in detail. Furthermore, based on the results of experiments, a wound healing formulation was developed and its activity profile was studied. Materials and methods For this purpose, the phenolic, flavonoid and proanthocyanidin contents and chemical profile of aqueous and ethanolic extracts prepared from okra fruits cultivated in two different locations of Turkey, i.e. Aegean and Kilis regions, were comparatively determined and the tryptophan levels, which is known to be an influential factor in wound healing, were measured. Antioxidant activity of the okra fruit extracts was determined by DPPH test, ABTS radical scavenger activity, iron-binding capacity, total antioxidant capacity and copper reduction capacity assays. Moreover, antibacterial activity potentials of the aqueous and ethanolic extracts of okra fruits were determined. The protective effect of the extracts against H2O2-induced oxidative stress and anti-inflammatory activity were assessed in HDF (human dermal fibroblast) cells and in RAW 264.7 murine macrophages, respectively. The biocompatibility of the gel formulations prepared with the best performing extract were evaluated by human Epiderm™ reconstituted skin irritation test model. Wound-healing activity was investigated in rats by in vivo excision model and, histopathological examination of tissues and gene expression levels of inflammation markers were also determined. Results According to our findings, the aqueous and ethanolic extracts of okra fruits were found to possess a rich in phenolic content. Besides, isoquercitrin was found to be a marker component in ethanolic extracts of okra fruits. Both extracts exhibited antioxidant activity with significant protective effect against H2O2-induced damage in HDF cells by diminishing the MDA level. Also, the highest dose of ethanolic extracts has displayed a potent anti-inflammatory activity on LPS-induced RAW264.7 cells. Besides, both water and ethanolic extracts were shown to possess antimicrobial activity. On the other hand, the formulations prepared from the extracts were found non-irritant on in vitro Epiderm™ -SIT. In vivo excision assay showed that tissue TGF-β and IL-1β levels were significantly decreased by the 5% okra ethanolic gel formulation. The histopathological analysis also demonstrated that collagenisation and granulation tissue maturation were found higher in 5% (w/v) okra ethanolic extract-treated group. Conclusion 5% of okra ethanolic extract might be suggested as a potent wound healing agent based on the antimicrobial, antioxidant and anti-inflammatory tests. The proposed activity was also confirmed by the histopathological findings and gene expression analysis.
Introduction: Harmful algal blooms are becoming a serious issue in the Mediterranean Sea (MS); dinoflagellate blooms are among the most worrying, as some representatives of this group are capable of producing potent marine toxins. Among these, the Ostreopsis genus are well known for the production of palytoxin-like compounds. Blooms of Ostreopsis cf. ovata have caused health issues, and damages to the economy and the environment. Ostreopsis cf. ovata often co-occurs with other benthic dinoflagellates such as Prorocentrum lima and Coolia monotis, in bloom events in the MS. Algae from the genus Prorocentrum are able to produce diverse toxins responsible for severe diarrhetic shellfish poisoning, while C. monotis is not included in the UNESCO-Intergovernmental Oceanographic Commission toxic species list. Materials and Methods: In this study, an integration of in vitro techniques has been applied for the first time to investigate the potential toxicity of the natural mixture of toxins produced by each of three dinoflagellates mentioned above. The proposed approach allowed to evaluate (1) skin and eye irritation potential on human three-dimensional reconstructed tissues; (2) alteration of neuronal activity by means of microelectrode array (MEA) electrophysiology on mouse neuronal networks; and (3) environmental toxicity by lethal toxicity test on Artemia franciscana. Results: Results revealed no significant effect on human skin and eye irritation tests for all the tested species. Interestingly, MEA analyses on mean firing rate and mean bursting rate revealed strong inhibition of functional activity by Ostreopsis cf. ovata and P. lima. The same species showed an important ecotoxicological effect after 48 hours of exposure to A. franciscana. Conclusion: Our approach was found to be suitable for the assessment of the whole algal toxicity potential, also accounting for the potential synergic effects of the mixture of toxins produced by each species.
Face à la demande croissante d’utiliser une chimie plus respectueuse de l’environnement, les tensioactifs dérivés de sucres se sont progressivement positionnés comme des candidats à la substitution des tensioactifs conventionnels issus de la pétrochimie. La thèse se place dans le contexte d’une stratégie de valorisation optimale et raisonnée des biomolécules amphiphiles issues de la bioraffinerie. L’objectif est d’acquérir une meilleure compréhension des relations entre structures moléculaires, propriétés physicochimiques, et propriétés fonctionnelles, afin de mieux cibler les molécules pouvant répondre à des besoins exprimés par différents secteurs applicatifs, tout en limitant les étapes de criblage expérimental. Plus particulièrement, les travaux se concentrent sur les propriétés de mousse. Différents axes de réflexion ont été employés dans cette thèse. Les glycolipides non ioniques ont été étudiés, ainsi que des glycolipides anioniques, largement moins étudiés dans la littérature. La détermination expérimentale rigoureuse de propriétés et l’analyse des données collectées ont permis l’identification de relations entre structures moléculaires, propriétés physicochimiques et propriétés fonctionnelles, pour les tensioactifs non ioniques. Notamment, pour ces molécules, les résultats obtenus permettent d’anticiper la quantité de mousse formée, selon un mode opératoire bien défini. La comparaison de nouveaux glycolipides anioniques avec les tensioactifs préoccupants de type SLES (Sodium Lauryl Ether Sulfate) en termes de propriétés moussantes et irritantes ont mis en évidence le potentiel de substitution de ces tensioactifs, pour des applications de détergence ou soin personnels (savon, shampoing ou autres). L’aspect du procédé de moussage et l’effet des conditions opératoires sur l’obtention de la mousse ont également été abordés. Les analyses dimensionnelles, opérées sur différents procédés, ont permis d’appréhender la complexité des procédés de moussage. Les résultats obtenus sont prometteurs et entrevoient la poursuite d’études supplémentaires, dans le but d’améliorer l’anticipation des propriétés moussantes.
Background At present, it is no longer possible to use animal testing for ingredients and cosmetic products in the Organisation for Economic Co‐operation and Development (OECD) member states. However, in vitro tests are widely used to determine the safety and efficacy of ingredients and medicines. Objective Obviously, 3D skin models with natural human features can be used to analyze cosmetic ingredients and formulations. Skin irritation by cosmetic products is studied less than ingredients on 3D skin models. Therefore, it is necessary for us to explore using 3D skin models to detect skin irritation with resident and cleaning cosmetic products. Methods We used HE staining to observe the structure of reconstructed skin models, the MTT assay to analyze tissue activity, and the ELISA to detect the relative expression of IL‐1α release to evaluate skin irritation with cosmetic products. Results We found that 0.3% SLS treatment and 1% Triton X‐100 in 3D skin models resulted in a tissue activity of <20% and increased IL‐1α release. We suggest that 0.3% SLS be used as a positive control for resident cosmetics and 1% Triton X‐100 be used for cleaning products. After a comprehensive analysis of the relative expression of tissue activity and IL‐1α, we found that 4 cosmetic products were skin irritants. Compared with multiple skin irritation tests using rabbit irritancy evaluation, we find that skin models can objectively respond to skin irritation with reliability. Conclusion We may redefine the exposure method time for cosmetics. For resident cosmetic products, the exposure time is 18 hours. For cosmetic cleaning products, the exposure time is 1 hour, with 10% dilution. We suggest that skin irritation evaluation in 3D skin models have a tissue activity of <50% and, at the same time, have a relative expression of IL‐1α that is 3‐fold greater than baseline.
Surface-active compounds derived from biomass, especially sugar-based amphiphiles, have received wide attention regarding their biodegradability, low toxicity and ecological acceptability. Compared to nonionic sugar-based surfactants, the anionic ones show significantly better solubility, higher surface activity and foaming performance. Thus they are largely used in personal care formulations and many technological applications. However, anionic surfactants are well known to induce skin and eye irritation. In this study, three sugar-based anionic surfactants, bearing a lipidic chain grafted to the anomeric position of a monosaccharide (glucose or xylose) and a sulfate group on the primary hydroxyl, were synthesized: 6-O-sulfo-N-(β-D-glucopyranosyl) dodecanamide (GlcNC12S), N-dodecyl-6-O-sulfo-D-gluconamide (GlcCC12S) and N-dodecyl-6-O-sulfo-D-xylonamide (XylCC12S). These molecules were investigated in details for their self-assembling behavior, foaming properties and biological effects. All their properties were compared to those of two commercially available anionic surfactants, sodium laureth sulfate (SLES) and sodium dodecylsulfate (SDS). Results revealed that the three anionic glycolipids show surface properties and foaming behavior comparable to those of SDS. Furthermore, their cytotoxic and irritation potentials are significantly lower compared to commercial molecules, which make these renewable molecules potential candidates for replacement of petroleum-based compounds.
In recent years, in-vitro skin models for chemical hazard identification have been developed. Most of them consist only of human keratinocytes, neglecting the contribution of other skin constituents. Cultures containing the dermal and epidermal component provide an attractive system to investigate, in a more realistic model, toxicological responses, which represents a distinct advantage over keratinocytes-based models that do not mimic faithfully the in vivo environment. This study aimed to validate dermo-epidermal organotypic cultures (ORGs) as a platform to perform irritation and corrosion tests. Skin models were constructed by seeding keratinocytes on fibroblast-containing fibrin gels. After 21 days, the ORGs were evaluated histologically, and the irritant and corrosion potential was determined by means of viability measurements (MTT assay) and cytokine release, according to 431 and 439 OECD tests guidelines. Skin models showed similar histological characteristics to native skin and were able to classify different substances with high accuracy, showing their applicability to skin irritation and corrosion tests. Although cytokines release seems to be chemical-dependent, a tendency was observed, leading to the improvement of the prediction capacity. Nevertheless, further studies should be done to reduce variability in order to increase prediction capacity.
Full-text available
This study was conducted to compare lipid components of sebum from persons from three ethnic backgrounds-Caucasian, African American and Northern Asian. Men and women with no acne in two age groups (18‒25 y and 35‒45 y) were recruited. Skin surface hydration (SkiCon 200EX and NovaMeter), barrier function (Delfin VapoMeter), high-resolution clinical imaging, self-assessments and two pairs of sebutapes on the forehead that extracted the lipids on the surface of their skin were used. Significant differences (p < 0.05) in skin hydration between African Americans and Caucasians in both age groups were noted, with the order from highest to lowest absolute values: African American > Northern Asian > Caucasian. Transepidermal water loss (TEWL) measurements demonstrated that African Americans and Caucasians were significantly different (p < 0.05), with the trend being the inverse of the hydration trend-Caucasian > Northern Asian > African American, which would indicate better barrier function for African Americans with a lower TEWL. African American women had more total lipid production than Northern Asian or Caucasian women. When analyzing the three lipid classes (free fatty acids, triglycerides and wax esters), the trend became significant (p < 0.05) in the wax ester fraction when directly comparing African Americans with Caucasians. Additionally, six lipids were identified in the wax ester fractions that were significantly different in quantity (p < 0.05) between African Americans and Caucasians. These results identified significant differences in sebaceous lipid profiles across ethnic groups and determined that the differences correlated with skin barrier function.
Full-text available
Baby cleansing is important for hygiene, but water alone is insufficient. Additionally, because baby skin is still developing and different from adult skin, adult cleansers are inappropriate. Here, the authors review how baby cleansers containing mild surfactants are milder than and differentfrom adult cleansers.
Full-text available
To image SDS distribution across different skin regions, to compare the permeability difference between porcine and human skin, and to evaluate the interaction between SDS and skin. Full thickness porcine and human skin was treated with acyl chain perdeuterated SDS (SDS-d(25)) at room temperature and at 34 °C for 3, 24 and 40 h. SDS distribution in skin was monitored by confocal Raman and IR microspectroscopic imaging. Permeation profiles of SDS-d(25) in skin were derived from the band intensities of the CD(2) stretching vibrations. The interaction between SDS and skin was monitored through the CH(2) and CD(2) stretching frequencies and the Amide I and II spectral region. SDS-d(25) penetrates both porcine and human skin in a time and temperature-dependent manner, with slightly higher permeability through the stratum corneum (SC) in porcine skin. When SDS permeates into the SC, its chains are more ordered compared to SDS micelles. The secondary structure of keratin in the SC is not affected by SDS-d(25). The spatial distribution of SDS-d(25) in skin was obtained for the first time. Infrared microscopic imaging provides unique opportunities to measure concentration profiles of exogenous materials in skin and offers insights to interaction between permeants and skin.
Full-text available
The barrier function of skin is primarily provided by the lamellar lipid matrix of the stratum corneum (SC), which has been shown in previous infrared (IR) and related studies to consist predominantly of ordered lipids packed in orthorhombic and hexagonal domains. In the current work, we investigate the effects of the anionic surfactant, sodium dodecyl sulphate (SDS), on SC lipid packing and phase behaviour, using FT-IR spectroscopy. The use of acyl chain perdeuterated SDS allows unequivocal spectroscopic detection of both endogenous lipid and exogenous material in intact tissue. IR spectra were acquired as a function of temperature from isolated human SC exposed to SDS for various incubation periods at 34°C. SDS is found to enter the SC and is observed to be in a more ordered state in the SC than in solution, indicating that the SDS interacts with the ordered SC lipids. The results reveal that SDS reduces the amount of orthorhombic phase in the SC and increases the amount of hexagonally packed lipid at physiologically relevant temperatures. In addition, a small decrease in the lipid T(m) (acyl chain melting temperature) is observed. Furthermore, these SDS-induced changes were found to be strongly dependent on the time of exposure.
Interactions of a model surfactant, sodium dodecyl sulfate (SDS), with a water-insoluble model protein, zein, were investigated to gain an understanding of the effects, such as skin irritation and protein denaturation, of surfactants that are common in personal-care products. To elucidate the mechanisms of such effects, the zein protein interaction with SDS in aqueous solutions was investigated using a multipronged approach involving a range of techniques, such as UV−visible and fluorescence spectroscopy, TOC (total organic carbon analysis), light scattering, and viscosimetry. The zein protein solubilization increases with an increase in the SDS concentration. Solubilization of zein occurs in two distinct stages followed by a complete unfolding of the protein. In the first stage ([SDS] 4 mM; critical complexation concentration), SDS is incorporated into the globular zein structure, forming small hydrophobic microdomains. From the pyrene fluorescence lifetime decay measurements, the aggregation number of SDS in such hydrophobic microdomains was found to be markedly lower than the aggregation number of pure SDS micelles in the bulk solution. The vibrational fine structure of pyrene fluorescence, however, showed the core of SDS−zein complex micelles to be more hydrophobic than that of the SDS micelles. In the second stage ([SDS] 200 mM; unfolding concentration), the protein unfolds, as is evidenced by viscosity and dynamic light scattering measurements.
An interlaboratory comparison of a new model of the human epidermis (EpiDerm) was conducted using a range of anionic and non-ionic surfactants and surfactant-containing final formulations. The toxicity of the materials was estimated by MTT conversion, using both concentration (EC(50)) and time (ET(50)) protocols. A range of 16 compounds was tested on different production lots of EpiDerm following storage periods of 1 and 2 days (after shipping) at MatTek and at two independent testing laboratories, Microbiological Associates (MA), USA and Scotland, UK. The EC(50) and ET(50) values were compared and the least squares fit lines with resulting correlation coefficients (r) calculated. Correlation of in vitro results to human clinical chamber irritation and repeat handwash testing gave r values ranging from 0.977 to 0.993 and comparison of the results obtained in the independent laboratories with the site of manufacture was good (MA, USA, r = 0.84; MA, UK, r = 0.74). The model appears to have utility in predicting clinically observed dermal irritation in vitro which is reproducible in different laboratories and after transatlantic shipping, such that it is worthy of further investigation.
We have previously evaluated the measurement of viability and cytokine release from skin equivalents, for predicting the skin irritation potential of topically applied surfactants and demonstrated that IL-1alpha and interleukin-1 receptor antagonist (IL-1ra) release from epidermal skin equivalents correlates with skin irritation potential. In this study, the utility of the model was confirmed by the evaluation of cleansing bars and cleansing lotions that exhibited varying degrees of irritation potential as determined by exaggerated arm wash human clinical studies. Epidermal equivalents were exposed to increasing concentrations of the cleansing bars and cleansing liquids and viability and release of IL-1alpha and IL-1ra were measured. Loss of viability was used to identify the stronger irritants of the products tested. A linear correlation was demonstrated between IL-1alpha and IL-1ra secretion and human irritation data, demonstrating that the model can correctly predict the irritation potential of soap and surfactant products. These results show that this in vitro model is useful for rank ordering the irritation potential of mild consumer products and for demonstrating enhanced mildness in products with minor differences.
A number of diverse in vitro model systems have been employed for the prediction of irritation potential of test articles. Monolayer systems have proven to be useful for preliminary screening but are not always capable of distinguishing mild effects or adaptable to fully formulated product. Three-dimensional reconstructed skin equivalents integrate cellular toxicity with the kinetics of exposure and absorption, serving as more realistic models; however, it is not obvious which of the three-dimensional models will give the most predictive response, and which biomarker should be used for an endpoint measurement for different groups of irritants. While evaluating these variables, we have shown that different irritants modulate various cytokine mRNA levels and secretion patterns in a time- and concentration-dependent manner that is unique to each product category. These profiles are also dependent on keratinocyte-fibroblast interactions. The most predictive combinations of model systems and biomarkers for each product category were identified following comparison to preclinical data and human in vivo skin responses. Using a panel of representative consumer products, we identified IL-1alpha, IL-1ra, IL-8 and GM-CSF release from skin equivalents as being the best indicators of irritation.