Download Surfactant irritation: in vitro corneosurfametry and in vivo

Skin Research and Technology 2001; 7: 49–55
Printed in Denmark. All rights reserved
Copyright C Munksgaard 2001
Skin Research and Technology
ISSN 0909-752X
Surfactant irritation: in vitro corneosurfametry and in vivo
bioengineering
Bernard Gabard, Eric Chatelain, Elsbeth Bieli and Stephan Haas
Biopharmacy Department, Spirig Pharma Ltd., Egerkingen, Switzerland
Background/aims: Irritant reactions to surfactants, cleansing
products, soaps and detergents are common in clinical and occupational dermatology. Mildness has become a major benefit
claimed, and testing for mildness now ranks among the first concerns of the manufacturing industry. A wealth of publications
deals with this problem, trying to improve the methodology, reduce the costs of testing and facilitate decision-making. Differences in vivo can be measured clinically and/or instrumentally.
This is difficult, as commercially available products are generally
safe to use and none are harsh in the absolute sense.
Methods: Nineteen different products (syndets, shampoos, personal cleansers), all claiming to be mild, were tested in vitro by
a newly introduced method, corneosurfametry. For evaluating the
aggressiveness of the products, the calculation of an index of
irritation (IOI) was proposed. A concentration-effect curve of sodium lauryl sulfate (SLS) as standard and model surfactant was
obtained. Some of the products were further tested in vivo with
a flex wash test and with a soap chamber test and compared to
SLS. Bioengineering methods (transepidermal water loss TEWL,
skin color) were used to evaluate the results.
Results and Conclusions: The results of the corneosurfametry allowed us to classify the products in three categories, with
increasing aggressiveness towards the stratum corneum, according to their IOIs. The in vivo tests were not able to discriminate between the products, but ranks from the results of
the bioengineering measurements showed a good correlation
between TEWL changes, but not between colour changes, and
IOIs from corneosurfametry. Corneosurfametry emerged as a
simple, low-cost and fast method for ranking commercial products according to their mildness. However, the skin bioengineering techniques showed that some products could lead to
skin reactions, such as erythema, that could not be detected
by the in vitro technique.
I
irritation and to detect differences possibly not apparent to the eye (4–7).
Ideally, an irritation test for evaluating the mildness
of cleansing products should be representative of the
conditions of every day use (2). Thus, exposure
should be open and repeated (usually during relatively short time periods). That is not the case for the
soap-chamber test conducted under occlusive conditions for a prolonged period of time. For these reasons, repeated wash or immersion tests should be preferred (2, 8–11). However, repeated wash or immersion tests are time-consuming, only a few products
can be tested simultaneously and, in addition, they
are influenced by factors such as season, environmental conditions, age and race, and thus require many
subjects, given the known variability of the individual
responses.
In comparison, in vitro tests can be better standardised. Bovine red blood cell hemolysis, pH rise of bovine serum albumin, collagen swelling and zein solu-
reactions are common and important in
clinical and occupational dermatology. Detergents,
surfactants, soaps, cleansing products and dish-washing products can damage the skin, and are considered
an important risk factor in the development of irritant
contact dermatitis. Mildness has become a major
benefit claimed, especially for surfactants and soaps,
and testing for mildness now ranks among the first
concerns of the manufacturing industry (1). A vast
amount of literature deals with the problems of
cleansing product skin irritation potential on human
skin (2). A major step was the introduction, by
Frosch & Kligman, of the so-called soap chamber test,
in which occlusive daily exposure for 5 days made it
possible to separate soaps into classes of mild, moderate and severe irritancy potential (3). Further development included the use of non-invasive bioengineering
measurement methods, such as electrical conductance, transepidermal water loss, colorimetry and
Doppler velocimetry, to quantify the damage due to
RRITANT
Key words: cleansing products – detergents – skin surface biopsy – mildness testing
c Munksgaard, 2001
Accepted for publication 28 July 2000
49
Gabard et al.
bilization are used for evaluating surfactant mildness
(12–14). However, data obtained from these tests may
not always correlate with in vivo observations (13,
14). Thus the need remains for a reliable, practical,
low-cost and fast laboratory test that can assess and
predict the irritation potential of cleansing agents.
The first step, and a key feature in the irritation process, is the interaction of the surfactant(s) with the
stratum corneum. It is now believed that this constitutes a significant contribution to the ensuing barrier
damage (15–17). Corneosurfametry (18) was introduced in 1994 by Piérard as a predictive bioassay for
evaluating the alterations induced by surfactants on
human stratum corneum. A good correlation between
the results of this in vitro assay and evaluation of
mildness by a modified soap-chamber in vivo test
could already be demonstrated (19). It was the purpose of our experiments to investigate in more detail
the interaction of cleansing products with human stratum corneum using this in vitro technique and to relate these results to those of two other well-known
repeated in vivo assays.
Material and Methods
Corneosurfametry
This assay was conducted exactly as described by
Piérard et al. (18). Briefly, skin surface biopsies (SSBs)
(19) were taken with a microscopic slide and cyanoacrylate glue. Aqueous solutions of the test products
(0.5/1/5/10%) or sodium lauryl sulfate (SLS; 0.01–
4%) were sprayed uniformly on the SSBs. After incubation at 25 æC for 2 h, the SSBs were thoroughly
rinsed with tap water, dried, and stained for 3 min
with basic fuchsin–toluidine blue dye solution. The
colour changes were evaluated with a tristimulus
Chromameter CR 300 (Minolta, Osaka, Japan) after
placing the SSBs on a white reference tile. The corneosurfametric index of mildness (CIMΩL* – Chroma C*)
was calculated as described elsewhere (17). To investigate a possible dose dependency and to eliminate the
small but always present variations in CIMs between
repeated experiments, an index of irritation (IOI) was
calculated:
IOIΩ1- (CIMtest product/CIMwater)
The IOI varies between 0 for water (or very mild test
products: CIMtest productΩCIMwater) and a maximum
of 1 for very harsh products (low CIMtest product).
Flex wash test
The flex wash test was conducted using the published
methodology (9) with slight modifications and additional instrumental evaluation of the skin surface.
Six healthy volunteers with intact skin in the antecubital fossae participated in the test. Products (0.5 ml)
were pipetted onto the flex area of one arm. This area
was washed by gently rubbing the product on the
skin for exactly 60 s with a fleecy sponge-like towel
previously moistened in warm tap water. The washing procedure was standardised as an elliptical motion with approximately 120 strokes per min. The
washed area was then rinsed under running tap water
until the lather was removed and gently patted dry
with a soft paper towel. The same procedure was repeated on the contralateral arm with another product
to be tested or with water for control purposes. These
1-min washing procedures were repeated 3 times
daily (9 a.m.; 1 and 4 p.m.) for 4 days. SLS (2%) was
used as standard, and water as a control substance.
Bioengineering measurements were conducted daily
1 h before the first washing procedure and at 8 a.m.
on day 5.
Repeated closed patch test (modified soap chamber
test)
The original Frosch & Kligman test (3) was modified
slightly: test products were used undiluted and the
TABLE 1. Test products and their claims
Test products
Code
Claims
Shampoos
Commercial
Experimental
CSH1-CSH6
ESH1, ESH2
Mild, extra-mild, very mild, for frequent use, perfectly tolerated, care of sensitive hair
Specially designed for dry and fragile hairs
Antidandruff shampoos
Commercial
Experimental
CASH1-CASH4
EASH1-EASH3
(No particular claim)
(No particular claim)
Syndets
Commercial
Experimental
CSY1, CSY2
ESY1, ESY2
Mild, cleans the skin naturally
Specially designed as baby syndets
Experimental: proprietary products from Spirig Pharma Ltd.
50
Surfactant irritation
chambers were left on the skin for 6 h from day 1
to day 5. Briefly, 100 ml of the test products were
pipetted on filter paper disks fitted in 12 mm aluminium Finn-chambers. Five chambers were positioned on each forearm of six healthy volunteers
and fixed with surgical wrap. After 6 h, the
chambers were removed and the skin was gently
cleaned with a soft paper towel to remove product
residues. This procedure was repeated for 5 days.
Every other day bioengineering measurements were
performed before patch application and 1 h after removal of the patch. SLS solutions (0.05% and 0.5%)
were used as standards, and water was used as a
control substance.
Fig. 1. Corneosurfametry: concentration-effect curve of SLS.
Test products
Bioengineering measurements
The following bioengineeering techniques were used:
transepidermal water loss (TEWL; Evaporimeter EP2;
Servomed, Kinna, Sweden) and skin colour
(Chromameter CR300, Minolta, Osaka, Japan; a* –
hue). Measurements were performed in a controlled
environment (T: 22∫0.1 æC; RH: 45∫5%) after acclimatisation of the volunteers for 15 min and following published guidelines (21, 22).
Test products were 19 shampoos and syndets available on the Swiss and/or German market, and/or
proprietary developments from Spirig Company
(Table 1). Most of the products were claimed to be
mild, very mild, extra mild or for babies. Sodium lauryl sulfate (SLS; biochemical grade ⬎98% purity; Fluka, Buchs, Switzerland) was used as standard and
model substance. Distilled water was used throughout as control.
Fig. 2. Corneosurfametry: concentration-effect curves of SLS (H) and of 19 different test products. Classification in Group 1 (mild), Group 2
(intermediate) and Group 3 (aggressive). For classification details see Table 2.
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Gabard et al.
Statistical analysis
The results of corneosurfametry (IOIs) were analysed
by the Kruskal-Wallis test. In case of statistical significance, an analysis of variance (ANOVA) with a
Student-Newman-Keuls post-hoc multiple comparison test followed. The statistical analysis (ANOVA) of
the results of the in vivo tests was performed on the
sums of the changes in baseline TEWL and a* hue between days 1 and 5 after logarithmical transformation
(data were not normally distributed). Correlations between product ranking from IOIs and from in vivo
data were analysed by Spearman’s rank correlation
procedure. All statistical procedures were performed
with SPSS for Windows V9.0 software.
Results
Corneosurfametry
SLS-IOIs were linearly dependent on log[concentration] in the range investigated (Fig. 1). This dose-effect
curve may be handled as a pharmacological dose-response curve, with an Emax (Emax: maximum possible
effect, height of the plateau of the curve) of 0.670∫0.189
at 2% SLS and a D50% (D50%: dose which gives EΩ1/2
Emax), calculated at about 0.17%, just under the critical
micelle concentration (0.2%) (2, 16). The test products
could be classified in three categories (Fig. 2): Group 1
or mild products showed maximal IOIs at about 0.1 and
no dose-effect curve. Group 2 or intermediate products
were characterised by IOIs from 0.1 to 0.2 at 1% and
more pronounced dose-effect curves. Group 3 products, the most aggressive products under our test conditions, showed IOIs ⬎0.2 at 1% and significant doseeffect curves (Table 2).
In vivo tests
In both tests, the well-known irritation reaction to SLS
was noticed (Figs. 3 & 4). In the repeated patch test,
one volunteer interrupted the test on day 3, two
further attained a score of 3 on day 4 with SLS 0.5%
and no reapplication was done. With SLS (0.05%), all
volunteers completed the trial. This was also the case
in the the flex wash test.
Eight different products belonging to the three categories defined from the corneosurfametry results
were tested in both in vivo tests (Table 3). In the flex
wash test, a statistically significant deterioration of the
skin barrier (as TEWL changes) and erythemal reaction (a* hue) was obvious with product no. 5. Sporadic
erythemal reactions were noticed with other products.
In the repeated patch test, no statistically significant
changes were seen.
Correlation between corneosurfametry and in vivo
tests
The test products were ranked on the basis of the
sums of changes from baseline between days 1 and 5
(data used for the statistical analysis). This ranking
TABLE 2. Classification of the test products from IOIs
Test product class
Mean IOI (95% CI)
Group 1: Mild
Group 2: Intermediate
Group 3: Aggressive
Dose-effect curve
Concentration 1%
Concentration 5%
⬍0.09
0.14 (0.10–0.17)
0.22 (0.20–0.23)
⬍0.09
0.22 (0.16–0.27)
0.36 (0.33–0.38)
Absent
Sometimes flat
Significant
TABLE 3. Statistical evaluation of the sums of the changes from baseline between first and last measurements
Test product
rank (group)
Flex wash
1: SLS (conc. %)
S* (2%)
2:
3:
4:
5:
6:
7:
8:
9:
NS
NS
NS
S*
NS
NS
NS
NS
CSH4 (Gr. 3)
CSH1 (Gr. 2)
CSY1 (Gr. 2)
CASH3 (Gr. 2)
CSH3 (Gr. 2)
EASH3 (Gr. 1)
ESH1 (Gr. 1)
ESY2 (Gr. 1)
Colour a*
TEWL
Repeated patch
S* (0.5%)
NS (0.05%)
NS
NS
NS
NS
NS
NS
NS
NS
Flex wash
Repeated patch
S* (2%)
S* (0.5%)
NS (0.05%)
NS
NS
NS
NS
NS
S*
NS
NS
S*
NS
NS
S*
S*
S*
NS
NS
NS: not statistically different from water-induced changes; S*: statistically different from water-induced changes (P⬍0.05).
52
Surfactant irritation
Fig. 3. Repeated closed patch test: bioengineering measurements (transepidermal water loss, left and skin colour a*-hue, right) after application of
distilled water (P), SLS 0.05% (h) and SLS 0.5% (g). (Means∫standard deviation)
Fig. 4. Flex wash test: bioengineering measurements [transepidermal water loss (left) and skin colour a*-hue (right)] after application of distilled
water (P) and SLS 2.0% (g). (Means∫standard deviations)
was compared with the one obtained from the corneosurfametry (IOIs, Table 4). High, statistically significant rank-correlation coefficients were found between
the changes in TEWL in both in vivo tests and the
corneosurfametry. Lower or statistically not significant correlations were observed between color
changes and corneosurfametry.
Discussion
Claims of mild cleansing products are a major concern
of industry, research laboratories and, last but not
least, dermatologists. Consumers also have become
concerned about this particular point, and claims of
mildness sometimes override the primary purpose of
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Gabard et al.
TABLE 4. Correlation (Spearman rank correlation) between corneosurfametry ranking (IOI at 1% test concentration) and ranking from TEWL
and/or colour a* (sums of the baseline between first and last measurements)
Corneosurfametry
rank (group)
1:
2:
3:
4:
5:
6:
7:
8:
9:
SLS (conc. %)
CSH4 (Gr. 3)
CSH1 (Gr. 2)
CSY1 (Gr. 2)
CASH3 (Gr. 2)
CSH3 (Gr. 2)
EASH3 (Gr. 1)
ESH1 (Gr. 1)
ESY2 (Gr. 1)
Spearman rank sum
IOI (1%)
0.59
0.25
0.20
0.19
0.16
0.15
0.018
0.014
0.011
Colour a*
TEWL
Flex wash
Repeated patch
Flex wash
Repeated patch
1:
5:
6:
2:
4:
9:
8:
3:
7:
1:
2:
5:
4:
6:
3:
7:
9:
8:
1:
5:
2:
6:
7:
8:
4:
3:
9:
1:
7:
5:
3:
8:
2:
6:
9:
4:
SLS
CASH3
CSH3
CSH4
CSY1
ESY2
ESH1
CSH1
EASH3
0.867 (S*)
SLS
CSH4
CASH3
CSY1
CSH3
CSH1
EASH3
ESY2
ESH1
0.867 (S*)
SLS
CASH3
CSH4
CSH3
EASH3
ESH1
CSY1
CSH1
ESY2
0.767 (S*)
SLS
EASH3
CASH3
CSH1
ESH1
CSH4
CSH3
ESY2
CSY1
0.317 (NS)
NS: not statistically significant; S*: statistically significant (P⬍0.05).
the products, the cleansing benefit. A wealth of publications are dealing with this problem, trying to improve the methodology, reduce the costs and facilitate
and speed up decision-making, for example (1, 2, 11,
14, 16). Among the main problems the tests deal with
are the influence of the test conditions on product
mildness classification, the use of human volunteers
with different skin types, the necessity of using a huge
number of volunteers due to the variations of the results, and the difficulties of classification due to the
general relationship between aggressiveness of the
test method and its ability to discriminate among the
products (1, 2).
This last point was adressed, in particular, in our
experiments, as the products tested were all claimed
to be mild. Mildness was confirmed throughout in
two in vivo tests. It is known that mild methodologies
tend to distinguish harsh materials from the remainder unless experimental conditions are adapted (e.g.,
self-assessment and bioengineering measurements) or
the sample size is greatly increased (1). On the other
side, aggressive procedures such as the repeated
patch test tend to distinguish mild products from the
rest. Thus, SLS was easily recognized as harsh, but as
the cleansers were of low irritancy, they could not be
properly classified by a classical analysis of the test
results. Moving the test procedure down the aggressiveness axis to the flex wash test allowed, besides the
higher SLS-concentration, one product (no. 5, Table 3)
to be separated from the others. But this test, as well
as the repeated patch test, is not easy to perform. Both
are time-consuming, and the flex wash test allows
testing of only two products at one time. Both, in addition, require the use of human volunteers with their
cohort of constraints, such as standardised measurement conditions, influence of season and skin type.
54
On the other hand corneosurfametry has emerged
as a simple, fast, low-cost in vitro test for ranking
mildness of cleansing products. The concentration-effect curve using IOIs as the effect measurement
showed a D50% at the critical micelle concentration
(CMC). Rhein already discussed the relationship between the CMC and the interactions of surfactants
with the stratum corneum, with relevance to skin irritation (16). She pointed out that SLS irritation still increases at doses above the CMC, a fact that was already known but that appears to contradict the hypothesis that the surfactant monomer is the irritative
species. Our findings are in accordance with her statements. Moreover, the concentration-effect curve of the
IOIs showed a maximum at about 2% SLS, which is
the maximal concentration recommended by the recently published guidelines on SLS irritation testing
(23). This shows that the concentration-effect curve of
SLS obtained by the technique of corneosurfametry is
meaningful for the in vivo effects of surfactants.
Using corneosurfametry, we were able to rank the
products tested and to propose a classification. The
test was simple to perform, fast and reproducible. We
also could show that the classification found in this
test ranking corresponded to the one obtained from
the changes in the barrier function in both in vivo
tests, although these changes in vivo were not significantly different statistically from those of distilled
water. However, a limit of the corneosurfametry is
that dermal irritation noticed as erythema could not
be foreseen. The complex relationship between erythema and barrier changes has been the subject of several publications and is not fully understood (2).
A recent publication by Tupker deals with the problem of ranking detergents using several tests and of
the correlation between the results of these different
Surfactant irritation
tests (2). As he pointed out, literature on the comparison of the irritancy of detergents tested by several
techniques, such as the patch test and the wash test,
is sparse. Corneosurfametry could be a valuable
addtion to help solve this problem.
References
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Address:
Bernard Gabard
Biopharmacy Department
Spirig Pharma Ltd.
CH-4622 Egerkingen
Switzerland
Tel: π41 62 387 87 26
Fax: π41 62 398 24 68
e-mail: bernard.gabard/spirig.ch
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