Altern. Animal Test. EXperiment. 5, 195-200 (1998)

Original Article

Evaluation of a New Alternative to Primary Draize Skin
Irritation Testing Using the EpiDermTM Skin ModelMichiru GENNO1, Ryohei YAMAMOTO1, Hajime KOJIMA2, Hiroaki
KONISHI2 and Mitch KLAUSNER3

1

Technical Research Laboratory, Kurabo Industries Ltd. 14-5, Shimokida-cho,Neyagawa-shi, Osaka, 572-0823, Japan.
2Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7, Torimi-cho, Nishiku, Nagoya, 451-0071, Japan
3MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA

Abstract
EpiDermTM is a commercially available human skin model consisting of normal human-derived epidermal keratinocytes (NHEK), which have been cultured
to form a multilayered, highly differentiated model of the human epidermis. We
evaluated this model as an alternative to the primary Draize skin irritation test
using rabbits.
Eighteen cosmetic products were tested using a new assay system which utilized the EpiDermTM and a single 24-hour exposure at 100% concentration. The
MTT assay results were compared to the results from skin irritation Draize testing using rabbits. The correlation coefficient for 18 test substances between thein vivo and in vitro results was r= - 0.671. However a much better correlation wasobtained provided that 2 products, both of which contained high ethanol levels(>60% (w/w)), were excluded from the analysis (r= - 0.870). As such, this method
may prove useful in making in vitro skin irritation assays more economical andconvenient.
Introduction
Recently, to reduce the number of animals
used for testing chemicals, drugs, and cosmetics
Correspondence : Michiru Genno, Technical ResearchLaboratory, Kurabo Industries Ltd., 14-5 Shimokidacho Neyagawa-shi, Osaka 572-0823, Japan. Tel.: +(81)
720 23 8800, Fax: +(81) 720 23 7906, E-mail:genno@lab.kurabo.co.jp
TMKey words : skin irritation test, skin model, EpiDerm ,detergents, cosmetic products, MTT% assay, singlepoint assay
Received : April 17, 1998.
Accepted : September 21, 1998.

etc., many alternative in vitro methods have beendeveloped. Regarding prediction of the skin irritation, monolayer cultures and 3-dimensional
tissue models composed of human skin have
Abbreviations:NHEK: normal human epidermal keratinocytes
Skin irritation test: primary Draize skin irritation test
MTT-50: the time of exposure that reduces mitochondrial
activity by 50%
MTT%: cell viability calculated using the following formula, MTT% = 100 x {[A570(sample)A650(sample)]/[A570(negative control)A650(negative control)]}
MDSS: maximal primary Draize rabbit skin irritation score
PBS: phosphate-buffered saline– 195 –

Alternative to Skin Irritation Test using a New Skin Model
Table 1. Alcohol contents, MDSS and MTT% values of 18 final cosmetic products.
SampleAbbreviation

Alcohol content(%)

MDSS**

MTT%(%)***

Shampoo 1

S10

7.0

4.4

Shampoo 2

S20

4.0

5.6

Rinse 1

S30

3.0

67.5

Rinse 2

S40

1.7

51.1

Face wash 1*

S50

2.0

27.8

Face wash 2*

S60

2.0

32.0

Face wash 3

S70

5.7

4.0

Face wash 4*

S80

2.0

27.8

Cleansing cream*

S90

1.8

103.1

Skin lotion 1

S10

6.0

0.8

103.7

Milky lotion*

S110

2.2

114.5

Skin lotion 2

S12

10.0

0.5

61.8

Cream*

S1301.5

111.4

Massage cream*

S1400.7

110.6

Face pack 1*

S158.00.0

113.1

Face pack 2

S160

1.5

96.7

Hair liquid

S1763.0

0.2

14.3

Hair tonic

S1870.0

0.3

13.3*These samples were applied to the skin model as the amount of 100mg. The others applied as the volume of 100ul.
**MDSS : Maximal primary Draize rabbit skin irritation score.
***MTT%(%) = 100x{[A570(sample)-A650(sample)/[A570(negative control)-A650(negative control)]}been used to predict the irritancy of substances(Triglia et al., 1989, Torishima et al., 1990,
Naughton et al., 1989, Bell et al., 1988, Cannon
et al., 1994). In this report, a new method ofusing the 3-dimensional skin model, EpiDermTM
which consists of normal human-derived epidermal keratinocytes (NHEK), was evaluated as analternative to the primary Draize skin irritation
test by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) (Mosmann, 1983).
It is reported that EpiDermTM has a structure
which is very similar to real skin and it contains
many protein markers of mature epidermis-specific differentiation (Cannon et al., 1993). Goodcorrelation with the skin irritation test has also
been reported (Cannon et al., 1994, Doyle et al.,
1994, Perkins et al., 1996). These results aremainly given by calculation of MTT-50 values
that is the time of exposure that reduces mitochondrial activity by 50%. When the toxicity ofsubstances is evaluated, tests are often conducted
at many concentrations or exposure times. Thisis a reasonable strategy to determine the poten-

tial dermal irritancy or toxicity of new cosmetic
products. However, it is also important to put
alternative methods to practical use. If in vitro
tests can be made simpler, more cost effective,
and less labor intensive, more examiners will use
them. With these goals in mind, we evaluatedthe ability of EpiDermTM to predict the skin irritation potential of final cosmetic products using
a single point assay that employed a single concentration and exposure time.
Materials and Methods
Materials
Test Substances
The 18 test substances used for this study werecosmetic products (Table 1). They were chosen
for this study because they had a wide range ofdermal irritancy. They were supplied by Nippon
Menard Cosmetics Co.,Ltd. (Nagoya, Japan). All
substances were applied to the EpiDermTM testat 100% concentration.

– 196 –Fig. 1 A schematic depiction of dosed EpiDermTM.

Skin Model
The EpiDermTM Skin Model (part # EPI-100a)
was imported by Kurabo Industries (Osaka, Japan) from MatTek Corporation (Ashland, MA,USA) for use in this study. Note: EpiDermTM
consists of NHEK, which have been cultured toform a multilayered, highly skin-like structure
on specially prepared permeable cell culture inserts (Fig. 1).
Methods
Draize skin irritation test
Six Japanese white female rabbits weighingfrom 2.9 to 3.5kg were used for the in vivo portion of this study. The rabbits were shaved on
the trunk and lateral areas. Test substances at
100% concentration (0.15 ml) were applied tointact skin under 1.7cm diameter gauze patches.
After 24hr the patches were removed and the skin
response was scored for erythema and oedema
for each rabbit at 1 and 24 hr after the removal(Draize et al., 1944, FDA officials, 1959). The
average value was calculated at each time foreach test substance and the maximum value between two time periods was used as the observed
value of MDSS (maximal primary Draize rabbit
skin irritation score)(Kojima et al.,1995).

MTT% assay (Single point assay)
The cytotoxicity on EpiDermTM skin-model
was evaluated by MTT, which was purchasedfrom Dojindo Laboratories, Kumamoto, Japan.
MTT solution was used at 1mg/ml in Dulbecco’s
modified Eagle’s medium (Gibco BRL, Grand
Island, NY). The test substances were applied tothe apical surface of the skin model for 24 hours
at 37C˚ in a humidified atmosphere (95% air and
5%CO2). For highly viscous test substances thatcannot be pipetted, applicator pins were used toprovide a reproducible dose. They were put on
the head of a pin as the amount of 100mg, andapplied to the skin model. After removing test
substances by washing with 1ml phosphate-buffered saline (PBS) twice, the skin model cups
were transferred to new 24-well multi-plates
filled with 300µl MTT solution. The skin model
cup (Millicell CM, Millipore Corp., Bedford,MA, USA) contacted with the MTT solution onlyat the bottom. After reaction for 3 hours at room
temperature, they were washed with 1ml PBS
twice. Then the tissues were transferred to new
24-well multi-plates and were submerged in 2mlof extraction solution (isopropanol). Extractionof the MTT was allowed to proceed overnight atroom temperature in the dark in 24-well plates
which were sealed in plastic bags. Prior to assaying, the extract was homogenized. Then theabsorbance of the solution was measured at
570nm using spectrophotometer (U-3210,– 197 –Alternative to Skin Irritation Test using a New Skin ModelFig. 2 Correlation between MTT% and MDSS for 18 cosmetic products.
The #S1 to S18 are abbreviations for cosmetic products shown in Table 1.
MTT% was calculated from following formula: 100 x{[A570(sample)-A650(sample)]/[A570(negative control)A650(negative control)]} using skin model EpiDermTM.
MDSS means maximal primary Draize rabbit skin irritation score.

Fig. 3 Effect of ethanol on EpiDermTM.
MTT% was calculated from following formula: 100 x{[A570(sample)-A650(sample)]/[A570(negative control)A650(negative control)]} using skin model EpiDermTM.
MDSS means maximal primary Draize rabbit skin irritation score.

Hitachi Ltd., Tokyo, Japan). The absorbance at
650nm was used as reference. The cell viability(MTT%) was calculated using the following formula:result, the coefficient of correlation between
MTT% values and the MDSS values in 18 test
substances was r= - 0.671. A relatively goodcorrelation between them was observed with exception of the hair liquid (S17) and the hair tonic(S18) (r= - 0.870). S17 and S18 showed relatively strong toxicity to the skin model comparedwith the result from in vivo. Because S17 andS18 contain high concentrations of ethanol(>60%), the cytotoxicity of ethanol on the skinmodel was examined (Fig. 3). It was found thatethanol above 30% decreased skin model viability.

MTT% = 100 x {[A570(sample)-A650(sample)]/[A570(negative control)-A650(negative control)]}Distilled water was used as the negative control. In the case of using applicator pins, thenegative control was determined by applying
water with a pin on the skin model. Each MTT%
value was determined from triplicate assays.

Discussion
Results
MDSS and MTT% assay (Single point assay)
The MDSS values and MTT% values for 18
test substances are shown in Table 1 and the relationship between them is shown in Fig. 2. As a

The 18 test substances which were all cosmetic products, were tested by the MTT% assay(Single point assay) to evaluate the possibilityof using EpiDermTM as an alternative method tothe rabbit skin irritation test.
The single point assay for the cosmetic prod-– 198 –ucts gave a relatively good correlation with thein vivo results (Fig. 2). Good correlation (r= 0.870) with the in vivo results was observed withthe exception of the two cosmetics containing
high concentrations of ethanol. The two cosmetic products (S17 and S18) were relatively
toxic to the skin model in relation to the in vivo
results. In an effort to understand why the skinmodel was overpredicting the irritation potential of these substances, we confirmed the toxicity of ethanol alone on the skin model (Fig. 3).
In a dose-response test, it was found that ethanol concentrations above 30% affected the skinmodel, but had minimal effects in vivo (Fig. 3).
The explanation for this difference is believed
to be due to the higher permeability of ethanol
through the skin model versus true skin. In a
related tissue model produced by MatTek Corporation (EpiOcular TM), ocular irritation is
overpredicted for ethanol containing substancesand requires special handling as a separate groupof substances (Klausner et al., 1997).
Furthermore, some test substances representing approximately score 2 in MDSS, which areestimated as mild toxicants in MDSS, were notclearly discriminated by the MTT% values (e.g.
S4, S5, S6, S8, S9 and S11 in Fig.2). However
moderate or severe toxicants (S1, S2 and S7) andnon toxicants (S10, S12, S14 and S15) were predicted by the MTT% values. These facts showthat this in vitro method can discriminate between non toxicants and moderate or severe ones
except for cosmetic products containing highconcentrations of ethanol. If we try to discriminate mild toxicants, or to predict the toxicity ofReferencesAppa, Y., Asuncion, A.G., Podda, M., Duke, M. A., Rachui,S. R. (1996) An in vitro method to evaluate the antioxidant efficacy of topical formulations, Presented at
American Acad. of Dermatol. Annual Meeting.
Bell E., Parenteau N.L., Haimes H.B., Gay, R.J., Kemp,P.D., Fofonoff, T.W., Mason, V.S., Kagan, D.T. andSwiderek, M. (1988) Testskin: a hybrid organism covered by a living human skin equivalent designed fortoxicity and other testing, In alternative methods intoxicology, vol. 6, 15-25.

cosmetic products containing high concentrations of ethanol, it seems that other detection
methods are needed. We applied only the single
point MTT% assay on the skin model. It is possible that other assay methods will give different results, for example, cytokine release or theleakage of enzymes. Recently, some cytokinesand chemical mediators related to skin inflammation have been analyzed in this skin model(Kubilus et al., 1996). The combination of cell
viability assay and cytokine release assay may
offer a more accurate prediction for skin irritation.
An advantage of skin model cultured at theair-interface is that it presents a useful dosing
surface to apply test substances in a manner similar to topical application to human skin (Osborneet al., 1994). EpiDermTM consists of organizedbasal, spinous, granular, and cornified layers
analogous to those found in vivo (Appa et al.,1996). Creams can be applied directly to theapical surface which has stratum corneum. The
3-dimentional and differentiated structure is adistinct advantage of this in vitro method andmay give a good correlation with in vivo.
Thus, with some exceptions, this single
point assay using EpiDermTM is likely to be applicable for cosmetic products and become agood alternative to the primary Draize skin irritation test because it is simple, cost effective,
and less labor intensive.
We conclude that EpiDermTM appears to bea useful pre-clinical tool which can assure thesafety of volunteers used to test new cosmeticproducts.

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– 200 –