ECVAM News & Views

305

ESAC STATEMENT ON THE CONCLUSIONS OF THE ICCVAM RETROSPECTIVE STUDYON ORGANOTYPIC IN VITRO ASSAYS AS SCREENING TESTS TO IDENTIFYPOTENTIAL OCULAR CORROSIVES AND SEVERE IRRITANTS AS DETERMINED BY US
EPA, EU(R41) AND UN GHS CLASSIFICATIONS IN A TIERED TESTING STRATEGY, AS
PART OF A WEIGHT OF EVIDENCE APPROACH
At its 26th Meeting, held on 26-27 April 2007 at the European Centre for the Validation of Alternative
Methods (ECVAM), lspra, Italy, the non-Commission members of the ECVAM Scientific Advisory
Committee (ESAC)l unanimously endorsed the following statement:
1. With regard to the results and conclusions from the ICCVAM retrospective study2,3 on:"Organotypic in vitro assays as screening tests to identify potential ocular corrosives and severe irritants
as determined by US EPA, EU(R41) and UN GHS classifications in a tiered testing strategy, as part of aweight of evidence approach"
the ESAC endorses the following conclusion:
There are sufficient data to support the use of the Bovine Corneal Opacity and Permeability (BCOP) test ··method, and the Isolated Chicken Eye (ICE) test method in appropriate circumstances and with certain
limitations, a as screening tests to identifY substances as ocular corrosives and severe irritants in a tiered­testing strategy, as part of a weight-of-evidence approach.
2. With regard to the Isolated Rabbit Eye assay (IRE) and the Hen's Egg Test-Chorio-Allantoic Membrane
assay (HET-CAM), the ESAC recommends that further work should be performed before a statement on
their validity can be made.
It should be noted, however, that European authorities previously stated that while all four tests were
not yet validated, positive outcomes from these tests could be used as the basis for classifYing andlabelling substances as severe eye irritants (R41).4
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra
27 April 2007
1. The ESAC was established by the European
Commission, and is composed of nominees from the
EU Members States, industry, academia and animal
welfare, together with representatives of the relevantCommission services.

This statement was endorsed by the following
Members of the ESAC:
Ms Sonja Beken (Belgium)
Ms Dagmar Jirova (Czech Republic)
Mr Tonu Piissa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)

Ms Argelia Castano (Spain)
Mr Patrie Amcoff (Sweden).
Mr Jon Richmond (UK)Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)
The following Commission Services and Observer
Organisations were involved in the consultation
process, but not in the endorsement process itself:

Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Ms Elke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)

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306

Ms Grace Patlewicz (ECB, DG JRC)Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACVAM)
Ms Laurence Musset (OECD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)
2. "Background Review Documents on in vitro test meth­ods for detecting ocular corrosives and severe irritants":- Bovine Corneal Opacity and Permeability test;- Isolated Rabbit Eye test;- Hen's Egg Test-Chorio-Allantoic Membrane test;- Isolated Chicken Eye test.
Website: http://iccvam.niehs.nih.gov/methods/ocutox/

ivocutox/ocu_brd.htm
3. The "!CCVAM Test Method Evaluation Report on invitro test methods for detecting ocular corrosives andsevere irritants". Website: http://iccvam.niehs.nih.
gov/methods/ocutox/ivocutox/ocu_tmer .htm
4. EC (2004). Manual of Decisions for Implementation of
the 6th and 7th Amendments to Directive 67/548/EEC
on Dangerous Substances. Updated version of July
2004, 189pp. lspra, Italy: European Chemicals Bureau,

European Commission JRC. Website: http://ecbjrc.it/
DOCUMENTS/New-Chemicals/Manual_of_decisions.
pdf

ESAC STATEMENT ON THE REDUCED LOCAL LYMPH NODE ASSAY (rLLNA)
At its 26th Meeting, held on 26-27 April 2007 at the European Centre for the Validation of Alternative
Methods (ECVAM), lspra, Italy, the non-Commission members of the ECVAM Scientific Advisory
Committee (ESAC)l unanimously endorsed the following statement:
Skin sensitisation is an important toxicological endpoint with respect to human safety. Having reviewed
the fmal report of the independent peer review evaluation co-ordinated by ICCVAM and NICEATM, 2 thereport by the EMEA,3 the pre-report of the SCCNFP,4 and evidence made available since the original sub­missions to ICCVAM in March 2000, the 14th meeting of ESAC stated:"Following a review of the scientific report and publications on the local lymph node ·assay (LLNA), it is con­cluded that the LLNA is a scientifically validated test which can be used to assess the skin sensitisation
potential of chemicals. The LLNA should be the preferred method, as it uses fewer animals and causes lesspain and distress than the conventional guinea-pig methods. In some instances and for scientific reasons,the conventional methods can be used."
Since its acceptance for regulatory purposes, the LLNA has proved suitable for the purposes of satisfying a
range of EU and other regulatory requirements.5 The developers of the LLNA have now undertaken a ret­rospective analysis of published data obtained with the LLNA.6 They conclude that, within a tiered testing
strategy in the context of REACH, a "reduced" version of the LLNA (rLLNA), using only a negative control
group and the equivalent of the high-dose group from the full LLNA, can be used as a screening test to dis­tinguish between sensitisers and non-sensitisers.
The ESAC established a peer review panel to evaluate whether there was the potential to minimise animaluse, by employing the rLLNA as a screening test as part of a tiered-testing strategy for chemicals. Mindfulthat with the rLLNA:-When compared with the full LLNA, the rLLNA cannot and will not result in additional false-positives.
-When compared with the full LLNA, the rLLNA may produce a few false-negatives (3:169 in the refer­ence document, reducing to 2:169 when negative results obtained with concentrations of < 10% are con­sidered invalid).
-

The test results provided by the rLLNA do not allow the determination of the potency of a sensitisingchemical.

The ESAC states that the peer reviewed and published information is of a quality and nature to support theuse of the rLLNA within tiered-testing strategies, to reliably distinguish between chemicals that are skinsensitisers and non-sensitisers, and that animal use can be minimised, providing that:-

The concentration used to evaluate sensitisation potential is the maximum consistent with solubility andthe need to avoid local and other systemic adverse eff~s, and that this principle, rather than strict
adherence to the specific recommended absolute concentrations as in OECD TG 429, should be used.

-

Negative test results associated with testing using concentrations of less than 10%, should undergo fur­ther evaluation.

-

Positive and negative (vehicle) control groups are used, as appropriate, as per OECD TG 429.

ECVAM News & Views-

307

The full LLNA should be performed, when it is known that an assessment of sensitisation potency isrequired.

The ESAC recommends that further work should be undertaken to determine whether the 10% concentra­tion threshold referenced above is optimal.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra
27 April 2007
1. The ESAC was established by the European
Commission, and is composed of nominees from the
EU Members States, industry, academia and animal
welfare, together with representatives of the relevantCommission services. ·.Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACVAM)
Ms Laurence Musset (OECD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)I

This statement was endorsed by the following
Members of the ESAC:
Ms Sonja Beken (Belgium)
Ms Dagmar Jirova (Czech Republic)
Mr Tonu Piissa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)
Ms Argelia Castano (Spain)
Mr Patrie Amcoff (Sweden)
Mr Jon Richmond (UK)Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)

The following Commission Services and Observer
Organisations were involved in the consultation
process, but not in the endorsement process itself:

Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Ms Elke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)
Ms Grace Patlewicz (ECB, DG JRC)

2. NIH (1999). TheMurineLocalLymphNodeAssay. The
Results of an Independent Peer Review Evaluation Co­
ordinated by the Interagency Coordinoting Committee
on the Validation ofAlternative Methods aCCVAM) aru1
the National Toxicology Program Center for the
Evaluation of Alternative Toxicological Methods(NICEATM). NIH Publication n.99-4494. (http://
iccvam.niehs.nih.gov/methods(unmunotox{unmunotox.
htm)
3. EMEA (2000). Report from the Ad-hoc Expert Meetingon Testing for Immunohypersensitivity (11/01/2000).
London, UK: European Agency for the Evaluation of
Medicinal Products.
4. SCCNFP (2000). Opinion Adopted by the SCCNFP
during the 11th Plenary Meeting, 17 February 2000.
(http://ec.europa.eu/health/ph_risk/committees/sccp/d
ocshtml/sccp_out114_en.htm)
5. Cockshott, A, Evans, P., Ryan, C.A, Gerberick, G.F.,
Betts, C.J., Dearman, R.J., Kimber, I. & Basketter,
D.A (2006). The local lymph node assay in practice: acurrent regulatory perspective. Human & Experi­mental Toxicology 26, 387--394.
6. Kimber, 1., Dearman, R.J., Betts, C.J., Gerberick, G.F.,
Ryan, C.A, Kern, P.S., Patlewicz, G.Y. & Basketter,
D.A (2006). The local lymph node assay and skin sen­sitisation: a cut-down screen to reduce animal require­ments? Contact Dermatitis 64, 181-185.

ECVAM News &Views

308

ESAC STATEMENT ON THE VALIDITY OF IN VITRO TESTS FOR SKIN IRRITATION
At its 26th meeting, held on 26--27 April 2007 at the European Centre for the Validation of Alternative
Methods (ECVAM), Ispra, Italy, the non-Commission members of the ECVAM Scientific Advisory
Committee (ESAC)l unanimously endorsed the following statement:
After a review of scientific reports and peer reviewed publications on the following range of in vitro tests,
which had been subjected to a full validation study:
1. EpiDerm (with MTT reduction and IL-la. release); and2. EPISKIN (with MTT reduction and IL-la. release);of these, the EPISKIN method showed evidence of being a reliable and relevant stand-alone test for pre­dicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction, and for being used as areplacement (based on the performance of the assay as specified in the annex) for the Draize Skin Irritation
Test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguish­
ing between R38 skin irritating and no-label (non-skin irritating) test substances. At the present time, theIL-la. endpoint should be regarded as a useful adjunct to the MTT assay, as it has the potential to increase
the sensitivity of the test, without reducing its specificity. This endpoint could be used to confirm negatives
obtained with the MTT endpoint.
At this time, due to its high specificity, the EpiDerm model reliably identifies skin irritants, but negative
results may require further testing (e.g. according to the tiered strategy, as described in. the OECD TG 404).
Improvement of the EpiDerm protocol should be made to increase the level of sensitivity.
This endorsement takes account of the dossiers prepared for peer review; the views of independent experts
who evaluated the dossiers against defmed validation criteria; supplementary submissions made by theManagement Team; and the considered view of the Peer Review Panel appointed to oversee the process.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commissionlspra
27 April 2007

1. The ESAC was established by the European
Commission, and is composed of nominees from the
EU Members States, industry, academia and animal
welfare, together with representatives of the relevantCommission services.
This statement was endorsed by the following mem­bers of the ESAC:
Ms Sonja Bek.en (Belgium)
Ms Dagmar Jirova (Czech Republic)
Mr Tonu Piissa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)
Ms Argelia Castano (Spain)
Mr Patrie Amcoff (Sweden)
Mr Jon Richmond (UK)Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)
The following Commission Services and Observer
Organisations were involved in the consultation
process, but not in the endorsement process itself:
Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Ms Elke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)
Ms Grace Patlewicz (ECB, DG JRC)Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACVAM)
Ms Laurence Musset (OECD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)!

ECVAM News & Views

Annex
General information on the ECVAM skinirritation validation study
After extensive optimisation and prevalidation
activities (see background to the SIVS here below),
ECVAM launched a formal validation study on
three in vitro test systems in 2003. Two of theassays employed reconstituted human epidermis
models (EPISKIN, EpiDerm) and one, the skinintegrity function test (SIFT), employed ex vivo
mouse skin. The aim of the study was to replace
the regulatory Draize skin irritation test (EU B.4
method; OECD TG 404) currently performed on
albino rabbits by assessing the relevance (predic­
tive capacity) and reliability (reproducibility
within and between laboratories) of these test sys­
tems with a set of 58 cooed test chemicals. The
goal of the study was to evaluate whether the invitro tests would predict in vivo classificationaccording to the EU classification system, using
the risk phrase R38 for skin irritants and no clas­
sification for non-irritants. In addition, the chemi­
cal selection was representative for the three
categories (strong [category 2], mild [category 3]
and non-irritants [no category]) of the Globally
Harmonised Classification System (GHS) for per­mitting a post-hoc evaluation of the results accord­
ing to the GHS. The validation study was
conducted according to the principles and criteria
documented in the OECD Guidance Document on
the Validation and International Acceptance of
New or Updated Test Methods for Hazard Assess­ment (No. 34). Furthermore, to ensure a high qual­
ity of the commercially produced human skinmodels, the facilities of the producers of thehuman skin models EPISKIN and EpiDerm were
evaluated by independent auditors at the begin­ning of the ECVAM Skin Irritation Validation
Study (SIVS). The study was sponsored by
ECVAM, coordinated by a main contractor(ZEBET-BfR, Germany) and managed by aManagement Team (MT; see Table 1 for the com­position of MT).
A Chemicals Selection Sub-Committee (CSSC)
was appointed to identify test chemicals to be usedin the SIVS, having high quality existing in vivo
data with which to correlate the in vitro measure­ments. Since chemicals from the European Centre
for the Ecotoxicology and Toxicology of Chemicals(ECETOC) database of reference chemicals for skinirritation/skin corrosion had been extensively usedin the preceding studies, the esse was requested tomake use of novel sources for potential test chemi­cals. For this purpose, chemicals were selected fromthe New Chemicals Database (NCD), which is thecentral archive within the EU notification scheme
for new commercial chemicals. In addition, existing309

chemicals readily available from major manufactur­
ing and/or distribution sources were selected fromalternative databases such as the Toxic SubstanceControl Act (TSCA) database maintained by the US
Environmental Protection Agency (EPA) and the
ECETOC database, excluding those chemicals
which had been used in the previous optimisationand prevalidation phases.
Table 1: Composition of the Management
Team of the SIVS

Chair (Dr Phil Botham)
Co-chair (Dr Julia Fentem)
Sponsor representative (Dr Valerie Zuang, alternate: Dr
Chantra Eskes)
Independent biostatistician (Dr Sebastian Hoffmann)
Representative of the main contractor (Dr Horst
Spielmann)
Representative of the CSSC (Dr Andrew Worth)
ECB customer (Dr Thomas Cole)
Representatives of the test systems:

EPISKIN (Dr Roland Roguet)
EpiDerm (Dr Manfred Liebsch)
SIFT (Dr Jon Heylings)
Obseroers from the USA:!CCVAM (Dr Karen Hamernik; alternate: Dr Abby
Jacobs)NICEATM (Dr William Stokes; alternate: Dr Ray Tice)

A total set of 58 chemicals, comprising a set of 25
existing chemicals and 33 chemicals from the NCD,
were selected and tested in the SIVS. The selectedchemicals: a) represented statistically justified
sample sizes for distinguishing R38 from non-clas­sified chemicals, b) provided a balanced representa­tion of the three GHS categories, to allow forpost-hoc evaluation of the performance of the assays
for that classification system, and c) accounted as
far as possible for the large prevalence known toexist for chemicals which .have oedema and ery­thema scores of 0. These chemicals were independ­
ently coded and distributed to the participating
laboratories. The selected chemicals presented avariety of molecular structures, functional chemicalgroups, and effect and use categories, as well as awide range of physical-chemical properties. They
represented a challenging set of chemicals relevant
to current industrial commerce for the alternative
methods being validated.
In phase 1 of the ECVAM SIVS, 20 chemicals (9irritant, 11 non-irritant) from the NCD were tested
under blind conditions in the lead laboratories(EPISKI~ - L'Oreal,; EpiDerm - ZEBET; SIFT
- Syngenta). The methods applied (with Standard

ECVAM News & Views

310

Operating Procedures [SOPs]) were the refined,
optimised protocols developed after the ECVAM
prevalidation study. Fqr the human skin model
assays, this consisted of applying the test chemicals
to the surface of the skin for 15 minutes, followed
by a post-treatment incubation period of 42 hours,
and the subsequent assessment of their effects oncell viability by using the MTT assay.
The prediction model related to MTT used inPhases 1 and 2 was the following:"The test substance is considered to be irritating toskin (R38), if the tissue viability after exposure andpost incubation is less than or equal (:5:) to 50%,"
When cell viability (MTT reduction) was used as theendpoint, the two skin models met the acceptancecriteria set by the MT of the study. While the speci­
ficity (correct prediction of non-irritants) of boththe EPISKIN and EpiDerm assays was 91%, their
sensitivities (correct prediction of R38 irritants)
were 67% and 56%, respectively. However, sincealmost all of the misclassified chemicals were lying
at the threshold between irritant and non-irritantchemicals according to the EU classification
scheme, the MT concluded that the predictive
capacity of both Epiderm a.ll.d EPISKIN was suffi­
cient to justifY that they proceed to Phase 2. On theother hand, the predictive capacity of the SIFT
method was considered inadequate. For SIFT, it
was suggested that the lead laboratory should re­evaluate the test protocol and prediction model,
particularly in relation to the manner in which
solids and non-surfactant materials are handled.
In Phase 2, all 58 chemicals were assessed inthree different laboratories for each of the tworeconstituted human skin methods. The EpiDermtest was conducted in the following labora~ories:
ZEBET (lead laboratory), Germany; Institute for In
Vitro Sciences (IIVS), USA; and BASF, Germany.
The EPISKIN test was conducted in the following
laboratories: L'Oreal (lead laboratory), France;
Unilever, UK; and Sanofi-Synthelabo, France.
Chemicals were re-coded from Phase 1 to ensure
blind testing. The main endpoint measured for both
Epiderm and EPISKIN was cell viability, measured
by MTT reduction, as used in all previous testing.
However, a second endpoint, interleukin-1a
release, was added for those chemicals which did
not reduce cell viability below the threshold for pre­dicting irritancy, to determine whether it could be
used to improve the sensitivity of the assays. Thissecond endpoint was used in all three laboratories
a8sessing EPISKIN and by the lead laboratory for
Epiderm. The prediction model related to the com­bined use ofMTT and IL-1a release in Phase 2, wasthe following:"The test substance is considered to be irritant toskin, if the viability after 15 minutes of exposureand 42 hours of post-treatment incubation is more(>)than 50%, and the amount of IL-1a release ismore ( >) than 60pg/ml.""The test substance is considered to be non-irritantto skin, if the viability after 15 minutes of exposureand 42 hours of post-treatment incubation is more(>) than 50%, and the amount of IL-1a release isless than or equal (:5:) to 60pg/ml."
The predictive capacities of the assays in thissecond phase are shown in Table 2. The within-lab­oratory reproducibility of classifications over three
independent experiments meeting the acceptancecriteria was 93.9% for EPISKIN (MTT) and 96.0%
for EpiDerm (MTT). The between-laboratory repro­
ducibility, measured as the proportion of identical
median classifications between laboratories, was
89.5% for EPISKIN (MTT) and 88.5% for EpiDerm.
Table 2: Predictive capacities of EPISKIN and
EpiDerm

EPISKIN
EPISKIN
EpiDerm(MTI')(MTI'+IL-la) (MTI')*
Sensitivity
Specificity

Concordance/
Accuracy

74.7%
80.8%
78.2%

90.7%
78.8%
83.0%

57.3%
83.8%
72.4%

MTT: based on the median classification per laboratory;
MTT+IL-la: based on the classification derived from themean viability of the independent experiments per chemi­
cal and laboratory. *The addition ofiL-lato the EpiDerm·protocol gave no improvement to the outcome.

The study was forwarded to the ECVAM Scientific
Advisory Committee (ESAC), with a proposal that
EPISKIN could be considered as a replacement for
the rabbit skin irritation method, and Epiderm as aconstituent of a testing strategy.

Background to the ECVAM skin irritation
validation study
In 1998, the ECVAM Skin Irritation Task Forcepublished a report on the actual status of in vitro
skin irritation testing, and proposed 10 "challenge
chemicals" for which promising, concordant in vivo
data from the rabbit test, in vivo data from the 4­
hour human patch test, and in vitro data from thehuman skin model, EpiDerm, were available.
Proponents of new in vitro test systems wereencouraged to submit data obtained with new invitro skin irritation test protocols for these chemi­

ECVAM News & Views

cals (1), for assessment as to whether these tests
could be considered in an ECVAM prevalidation
study. At the same time, the suitability of various
endpoints for prediction of human skin irritation
was evaluated in an EU 4th Framework
Programme collaborative project on several humanreconstructed skin models, revealing cell viability
reduction (MTT reduction) and IL-la release to be
the most promising endpoints. Because MTT reduc­tion and IL-la release showed a high inter-correla­tion, and IL-la release was more variable, MTT
reduction was proposed to be the best endpoint forhuman skin models (2).
Of the test systems for which data were submit­
ted to the ECVAM TF, five tests (perfused pig-ear,
Prediskin, SIFT, EPISKIN, EpiDerm) had been
considered promising for participation in the
ECVAM prevalidation study. However, during theprevalidation study, two tests failed in phase 2 due
to insufficient reproducibility, whereas the othertests (SIFT, EPISKIN and EpiDerm) showed a suf­
ficient intra-laboratory and inter-laboratory repro­
ducibility, but failed in their ability to correctlypredict the skin irritation potential of 20 chemicals
that were tested in phase 3 of the ECVAM prevali­
dation study (3). The ECVAM Management Teamof the study therefore proposed the refinement andoptimisation of these three tests before considering
them for formal validation.
In 2001, the ECVAM Skin Irritation Task Forceand the laboratories responsible for the refinementof the tests met again, and discussed ways forward
to approach formal validation. In addition, since a
post-hoc analysis of prevalidation data for MTT
reduction for EPISKIN and EpiDerm revealed sim­
ilar sensitivities, it was recommended to develop acommon test protocol for both skin models before
the start of a formal validation study (4).
In November 2002, the ECVAM Skin Irritation
Task Force (TF) discussed the refinements of the
SIFT (5) and the skin model tests (6), and came tothe conclusion that processing the tests to formal
validation could be recommended. However,
because all the refinements were made by using the
20 chemicals from the prevalidation study, the TF
recommended that the SIVS be performed in twophases: a first phase (phase 1) for the confirmationof the refinements made by the lead laboratories(Syngenta [SIFT], L'Oreal [EPISKIN], and ZEBET[EpiDerm]), by testing new chemicals in a con­trolled way under blind conditions. If the outcomeof phase 1 were still promising, the tests would thenproceed to a second phase (phase 2), i.e. to a blind
trial involving three laboratories per test.
During 2003, the EPISKIN test was further
refined by L'Oreal, by extending the post-incuba­tion period of the tissues (after a 15-minute chem­
ical exposure) to 42 hours, which allowed
significant effects to increase, and recovery fromweak effects.

311

In May 2003, an ECVAM Stakeholder Workshop
recommended that a formal validation study be
conducted, and that work should concentrate on
the predictions of the EU classification system(R38 vs. no label), because the tests were devel­
oped and optimised for this classification scheme.
L'Oreal and ZEBET then collaborated in develop­
ing a common test protocol to be used in the
ECVAM SIVS, and evaluated it first with the 20"challenge" chemicals of the ECVAM prevalida­tion study. In 2004, at the request of the ECVAMSIVS Management Team and in parallel with per­forming phase 1 of the SIVS, the database was fur­ther increased by testing all the non-corrosive
chemicals recommended in the ECETOC reference
data base (ECETOC Report No. 66). The dataobtained in both skin models with the optimised
common protocol were very promising, and were
published back-to-hack in 2005 (7, 8). The BfR wascontracted in November 2003, further to the pub­lication of a call for tender for the ECVAM SIVS by
the European Commission in June 2003. The
study started formally with the 1st Meeting of the
SIVS Management Team (MT), on 17-18 Novem­
ber 2003.
Manuscripts on the outcome of the skin irritation
validation study, and on the chemicals selection, arecurrently being finalised for publication.
References
1. Botham, P.A., Lesley, KE., Fentem, J.H., Roguet, R.
& van de Sandt, J.J.M. (1998). Altemative methods

for skin irritation testing: the current status. ECVAM
Skin Irritation Task Force Report 1. ATLA 26,
195-211.
2. Faller, C., Bracher, M., Dami, N. & Roguet, R. (2002).
Predictive ability of reconstructed human epidermis
equivalents for assessment of skin irritation of cos­
metics. Toxicology in Vitro 16, 557-572.
3. Fentem, J.H., Briggs, D., Chesne, C., Elliott, G.R.,
Barbell, J.W., Heylings, J.R., Portes, P., van de Sandt,
J.J.M. & Botham, P.A. (2001). A prevalidation study
on in vitro tests for acute skin irritation: results andevaluation by the Management Team. Toxicology in
Vitro 15, 57-93.
4. Zuang, V., Balls, M., Botham, P .A., Coquette, A.,
Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H.,
Heylings, J.R., Liebsch, M., Medina, J., Roguet, R.,
van de Sandt, J.J.M., Wiemann, C. & Worth, A.P.
(2002). Follow-up to the ECVAM prevalidation study
on the in vitro tests for acute skin irritation. ECVAM
Skin Irritation Task Force Report 2. ATLA 30,
109-129.
5. Heylings, J.R., Diot, S., Esdaile, D.J., Fasano, W.J.,
Manning, L.A. & Owen, H.M. (2003). A prevalidation
study on the in vitro irritation function test (SIFT) for
precUction of acute skin irritation in vivo: results andevahiation of ECVAM phase III. Toxicology in Vitro
17, 123-138.
6. Kandarova, H., Liebsch, M., Genschow, E., Gemer, 1.,
Traue, D., Slawik, B. & Spielmann, H. (2004). Optimi­
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ESAC INTERIM STATEMENT ON THE RETox ASSAY FOR EMBRYOTOXICITY TESTING
At its 26th Meeting, held on 26-27 April 2007 at the European Centre for the Validation of Alternative
Methods (ECVAM), lspra, Italy, the non-Commission members of the ECVAM Scientific Advisory
Committee (ESAC)l unanimously endorsed the following statement:
On the basis of the available data submitted for peer review, the ESAC has not endorsed the scientific valid­
ity of the RETox assay.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commissionlspra
27 April 2007

The ESAC was established by the European Com­mission, and is composed of nominees from the EU
Members States, industry, academia and animal wel­
fare, together with representatives of the relevantCommission services.

Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)

This statement was endorsed by the following membersof the ESAC:

The following Commission Services and Observer
Organisations were involved in the consultation process,but not in the endorsement process itself:

Ms Sonja Beken (Belgium)

Ms Dagmar Jirova (Czech Republic)
Mr Tonu Piissa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)
Ms Argelia Castano (Spain)
Mr Patrie Amcoff (Sweden)
Mr Jon Richmond (UK)Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Ms Elke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)
Ms Grace Patlewicz (ECB, DG JRC)Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACVAM)
Ms Laurence Musset (OECD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)