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 RAT SKIN
TRANSCUTANEOUS ELECTRICAL RESISTANCE (TER) TEST(AN IN VITRO TEST FOR SKIN CORROSIVITY)
At its 10th meeting, held on 31 March 1998 at the European Centre for the Validation ofAlternative Methods (ECVAM), Ispra, Italy, the ECVAM Scientific Advisory Committee(ESAC) 1 unanimously endorsed the following statement:
The results obtained with the rat skin transcutaneous electrical resistance (TER)test in the ECVAM international validation study on in vitro tests for skincorrosivity were reproducible, both within and between the three laboratories that
performed the test. The rat skin TER test proved applicable to testing a diverse
group of chemicals of different physical forms, including organic acids, organicbases, neutral organics, inorganic acids, inorganic bases, inorganic salts,electrophiles, phenols and soaps/surfactants. The concordances between the skincorrosivity 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. The Committee
therefore agrees with the conclusion from this formal validation study that the rat
skin TER test is scientifically validated for use as a replacement for the animal
test for distinguishing between corrosive and non-corrosive chemicals, and that
this test 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, togetherwith 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: UnitedNations.

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. 105pp. 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 by
Dr 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;
CORROSITEXTM) Dr Lesley Earl (Unilever, UK; rat skin TER assay), Mr David Esdaile(Rhône-Poulenc Agro, France; EPISKINTM), and Dr Manfred Liebsch (ZEBET, Germany;Skin2™ 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 the MT. In addition to ECVAM, the participating organisations were: Agence duMedicament (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),
Rhône-Poulenc 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)CORROSITEXTM; (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 inJune 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 inconsultation 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. The rat skin TER assay has been used successfully as a routine in-house test for several
years. When used in screening mode, the TER method is employed to predict corrosivity
potential rather than the degree of corrosive effect (i.e. potency), and it has been usedprimarily to guide humane in vivo skin testing. The TER assay has been evaluated in several
intralaboratory and interlaboratory studies, and it performed creditably in the prevalidation
study conducted during 1993 and 1994. The test protocol evaluated in this validation study
had been refined on the basis of recommendations from the prevalidation study, to include adye binding procedure for reducing the number of false positive predictions obtained
previously with test materials containing surfactants and solvents. In outline, test materials
are applied for up to 24 hr to the epidermal surfaces of skin discs taken from the pelts ofhumanely killed young rats. Corrosive materials are identified by their ability to produce aloss of normal stratum corneum integrity and barrier function, which is measured as areduction in the inherent TER below a predetermined threshold level (5kΩ).
Rat Skin TER Assay Prediction Model:
TER (kΩΩ)Treatmenttime(hours)

Mean discdye content

C/NC

EU risk phrase

UN packing
group>52 & 24

NMa

NC

no label

-

≤5

224-

C
C

R35
R34

I
II/III≥ +ve control< +ve control

CNC

R34
no label

II/III-

Surfactants/neutral organics:≤5a2424

NM = not measured

E. The prediction model for the rat skin TER test was used to classify the corrosivity potentialsof the 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:

C
R34/II & III
R35/I

Specificity:
Predictivity:

88%
18%
88%
72%

C
R34/II & III
R35/I

72%
40%
22%

Accuracy:

C/NC
R35/R34/NC

79%
55%

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

12%
28%

R35/I v R34/II & III v NC
underprediction rate
R35/I → NC
R34/II & III → NC

6%
14%

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

12%
16%
69%*

*unacceptable according to the criteria defined by the MT before undertaking the data analysis
F.

In order for the rat skin TER 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 EPISKINTM assay for skin corrosivity testing wasalso endorsed by the ESAC on 31 March 1998. The two other methods included in thevalidation study, CORROSITEX and Skin2, did not meet all of the criteria for them to beconsidered acceptable as replacement tests. The corrosivity potentials of about 40% of thetest chemicals could not be assessed with CORROSITEX, although it may be valid fortesting specific classes of chemicals (such as organic 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 a tiered testing strategy for skin corrosivity.