European Commission
ECVAM, TP 580
JRC Environment Institute21020 Ispra (VA)
Italy

ECVAM

European Centre for the Validation of Alternative Methods

STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST(AN IN VITRO TEST FOR SKIN CORROSIVITY)

At its 10th meeting, held on 31 March 1998 at the European Centre for the Validation of
Alternative Methods (ECVAM), Ispra, Italy, the ECVAM Scientific Advisory Committee(ESAC) 1 unanimously endorsed the following statement:
The results obtained with the EPISKINTM test (involving the use of a reconstructedhuman skin model) in the ECVAM international validation study on in vitro tests
for skin corrosivity were reproducible, both within and between the threelaboratories that performed the test. The EPISKIN test proved applicable totesting a diverse group of chemicals of different physical forms, including organic
acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganicsalts, electrophiles, phenols and soaps/ surfactants. The concordances between theskin corrosivity classifications derived from the in vitro data and from the in vivo
data were very good. The test was able to distinguish between corrosive and noncorrosive chemicals for all of the chemical types studied; it was also able todistinguish between known R35 (UN 2 packing group I) and R34 (UN packinggroups II & III) chemicals. The Committee therefore agrees with the conclusionfrom this formal validation study that the EPISKIN test is scientifically validated
for use as a replacement for the animal test, and that it is ready to be considered
for regulatory acceptance.
The ESAC has been regularly kept informed of the progress of the study, and this endorsement
was based on an assessment of various documents, including, in particular, the report on theresults and evaluation of the validation study by the Management Team, which is to be publishedin Toxicology in Vitro.3
This validation study was conducted in accordance with the general principles laid down in thereport of the CAAT2/ERGATT2 workshop held in 1990,4 guidelines contained in the report of anECVAM/ERGATT workshop held in 1995,5 criteria laid down by ECVAM and the ECB,2,6
criteria recommended at an OECD2 workshop held in 1996,7 and the US ICCVAM2 report onvalidation and regulatory acceptance.8 The outcome of a prevalidation study on in vitro tests forskin corrosivity was published in 1995, as ECVAM workshop report 6.9 A separate report on theselection of the test chemicals for the validation study is to be published alongside theManagement Team’s report in Toxicology in Vitro.10
Michael Balls
Head of Unit
ECVAM
JRC Environment InstituteEuropean Commission
Ispra

Guy Corcelle
Head of Unit
DGXI/E/2
European Commission
Brussels
3 April 1998

1. The ESAC was established by the European Commission, and is composed ofrepresentatives of the EU Member States, industry, academia and animal welfare, together
with representatives of the relevant Commission services. The following members of theESAC were present at the meeting on 31 March 1998:
Dr B Blaauboer (ERGATT)
Professor J Castell (Spain)
Dr B Garthoff (EFPIA)Dr C Hendriksen (The Netherlands)
Professor G Papadopoulos (Greece)
Dr B Rusche (Eurogroup for Animal Welfare)
Professor H Spielmann (Germany)
Professor H. Tritthart (Austria)
Professor E Walum (Sweden)

Dr P Botham (ECETOC)Dr D Clark (UK)
Professor A Guillouzo (France)
Dr R Lorenzini (Italy)
Professor V Rogiers (Belgium)
Dr O de Silva (COLIPA)Dr O Svendsen (Denmark)Dr M Viluksela (Finland)

Professor M Balls (ECVAM)
Dr J Fentem (ECVAM)
Ms S Louhimies (DGXI)Mr A Van Elst (DGXXIV)Mr G Corcelle (DGXI)Dr G Fracchia (DGXII)Dr M Robert (DGIII)

2.

CAAT: Center for Alternatives to Animal Testing, Baltimore, USA; ECB: European
Chemicals Bureau, Ispra, Italy; ERGATT: European Research Group for Alternatives in
Toxicity Testing, Utrecht, The Netherlands; ICCVAM: ad hoc Interagency Coordinating
Committee on the Validation of Alternative Methods, Research Triangle Park, USA; OECD:Organization for Economic Cooperation and Development, Paris, France; UN: United
Nations.

3.

Fentem JH, Archer GEB, Balls M, Botham PA, Curren RD, Earl LK, Esdaile DJ, Holzhütter
H-G & Liebsch M (1998) The ECVAM international validation study on in vitro tests forskin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro, inpress.

4.

Balls M, Blaauboer B, Brusick D, Frazier J, Lamb D, Pemberton M, Reinhardt C, Roberfroid
M, Rosenkranz H, Schmid B, Spielmann H, Stammati AL & Walum E (1990) Report andrecommendations of the CAAT/ERGATT workshop on the validation of toxicity testprocedures. ATLA 18: 303-337.

5.

Balls M, Blaauboer BJ, Fentem JH, Bruner L, Combes RD, Ekwall B, Fielder RJ, Guillouzo
A, Lewis RW, Lovell DP, Reinhardt CA, Repetto G, Sladowski D, Spielmann H & Zucco F(1995) Practical aspects of the validation of toxicity test procedures. The report andrecommendations of ECVAM workshop 5. ATLA 23: 129-147.

6.

Balls M & Karcher W (1995) The validation of alternative test methods. ATLA 23: 884-886.

7.

Anon. (1996) Final Report of the OECD Workshop on Harmonization of Validation andAcceptance Criteria for Alternative Toxicological Test Methods. 60pp. Paris: OECD.

8.

Anon. (1997) Validation and Regulatory Acceptance of Toxicological Test Methods. A
Report of the ad hoc Interagency Coordinating Committee on the Validation of AlternativeMethods. 105 pp. Research Triangle Park, NC: NIEHS.

9.

Botham PA, Chamberlain M, Barratt MD, Curren RD, Esdaile DJ, Gardiner JR, Gordon VC,Hildebrand B, Lewis RW, Liebsch M, Logemann P, Osborne R, Ponec M, Régnier J-F,Steiling W, Walker AP & Balls M (1995) A prevalidation study on in vitro skin corrosivity
testing. The report and recommendations of ECVAM workshop 6. ATLA 23: 219-255.

10. Barratt MD, Brantom PG, Fentem JH, Gerner I, Walker AP & Worth AP (1998) The
ECVAM international validation study on in vitro tests for skin corrosivity. 1. Selection anddistribution of the test chemicals. Toxicology in Vitro, in press.
General information about the ECVAM skin corrosivity validation study:
A. The study was coordinated from ECVAM, and the Management Team (MT) was chaired byDr Julia Fentem (ECVAM). The other four MT members acted as representatives of the“lead laboratories” and each took responsibility for one of the four tests included in thevalidation study: Dr Rodger Curren (Microbiological Associates Inc., USA;
CORROSITEX™), Dr Lesley Earl (Unilever, UK; rat skin TER assay), Mr David Esdaile(Rhône-Poulenc Agro, France; EPISKINTM), and Dr Manfred Liebsch (ZEBET, Germany;
Skin2TM assay). The study was principally funded by ECVAM, under the terms of 14 separate
contracts with the participating organisations. Professor Michael Balls (ECVAM) and Dr
Philip Botham (ESAC; ZENECA CTL, UK) represented the sponsors in any contacts with theMT. In addition to ECVAM, the participating organisations were: Agence du Medicament(France), BASF Aktiengesellschaft (Germany), BIBRA International (UK), COVANCE(UK), Humboldt University (Germany), Huntingdon Life Sciences (UK), INRS (France),
Microbiological Associates Inc. (USA), Microbiological Associates Ltd (UK), RhônePoulenc Agro (France), Sanofi Recherche (France), Unilever Research (UK), ZEBET, BgVV(Germany) and ZENECA CTL (UK).
B. This study began in 1996, as a follow-up to a prevalidation study on in vitro tests for
replacing the in vivo Draize rabbit test for skin corrosivity. The main objectives were to: (a)identify tests capable of discriminating corrosives (C) from non-corrosives (NC) for
selected groups of chemicals (e.g. organic acids, phenols) and/or all chemicals (single
chemical entities only); and (b) determine whether the tests could identify correctly known
R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests
selected for inclusion in the validation study were: (a) the rat skin TER assay; (b)CORROSITEX™; (c) the Skin2TM ZK1350 corrosivity test; and (d) EPISKINTM. Each test
was conducted in three independent laboratories, according to the principles, criteria andprocedures for undertaking validation studies outlined previously by ECVAM in conjunction
with international experts in this area. Prediction models for the four tests were clearly
defined in the test protocols.
C. A test set of 60 chemicals was selected by an independent Chemicals Selection SubCommittee, including organic acids (6C/5NC), organic bases (7C/3NC), neutral organics(9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganicsalts (1C/2NC), electrophiles (3C/5NC) and soaps/surfactants (3NC). The first set of ten
coded chemicals was distributed independently of the MT and participating laboratories inJune 1996. Further to the satisfactory completion of the first phase of the study, the remaining
50 coded chemicals were distributed in September 1996. The results obtained weresubmitted to ECVAM’s statistician, Dr Graeme Archer, for independent analysis in
consultation with Dr Hermann-Georg Holzhütter (Humboldt University, Berlin, Germany).
Data analysis and preparation of the final reports took place between May and October 1997.
D. EPISKINTM is a three-dimensional human skin model comprising a reconstructed epidermis
with a functional stratum corneum. Its use for skin corrosivity testing involves topicalapplication of test materials to the surface of the skin for 3, 60 and 240 min, and thesubsequent assessment of their effects on cell viability by using the MTT assay. An in-houseevaluation and prevalidation of the test was conducted during 1994-96. On the basis of thesestudies, the test protocol was refined prior to its inclusion in this validation study.
EPISKIN Prediction Model:

Treatment time(mins)

Viability (%)

C/NCEU risk phrase

UNgroup33 /6060 /240240< 35≥ 35 / < 35
≥ 35 / < 35
≥ 35

CCCNC

R35
R34
R34
no labelIII
III-packing

E. The prediction model for the EPISKIN test was used to classify the corrosivity potentials ofthe 60 test chemicals on the basis of the in vitro data obtained in the three laboratories
conducting the test. Comparing these in vitro classifications with the in vivo classifications
independently assigned to the chemicals before the blind trial began gave the following key
statistical parameters:
Sensitivity:

CR34/II & III
R35/I

Specificity:

83%
75%
39%
80%

Predictivity:

C
R34/II & III
R35/I

77%
64%
53%

Accuracy:

C/NC
R35/R34/NC

81%
74%

The underprediction and overprediction rates for the EPISKIN test relative to the study
objectives were:
Objective (a): C v NC

underprediction rate
overprediction rate

17%
20%

Objective (b): R35/I v R34/II & III v NC
underprediction rate
R35/I → NC
R34/II & III → NC

17%
18%

overprediction rate
NC → R35/I
NC → R34/II & III
R34/II & III → R35/I

1%
19%
8%

F. In order for the EPISKIN test to be considered for use for legislative and other purposes,
measures will be taken to press for the updating of OECD Testing Guideline 404 and Annex
V method B.4 of Directive 67/548/EEC.
G. A statement on the scientific validity of the rat skin transcutaneous electrical resistance(TER) assay for skin corrosivity testing was also endorsed by the ESAC on 31 March 1998.
The two other methods included in the validation study, CORROSITEX and Skin2, did notmeet all of the criteria for them to be considered acceptable as replacement tests. The
corrosivity potentials of about 40% of the test chemicals could not be assessed with
CORROSITEX, although it may be valid for testing specific classes of chemicals (such asorganic bases and inorganic acids). The Skin2 assay, as conducted in this validation study,
had an unacceptably high underprediction rate (57%), although it had a specificity of 100%.
It is recognised that both of these methods could be useful if they were incorporated into atiered testing strategy for skin corrosivity.