Transcript ECVAM - IHCP

Ankara, 12 Novembre 2007 – Alternatives 2007
ECVAM Key Area Topical Toxicity
Summary of ongoing activities
Chantra Eskes
European Centre for the Validation of Alternative Methods - ECVAM
Institute for Health and Consumer Protection
European Commission Joint Research Centre
http://ecvam.jrc.cec.eu.int
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Cosmetics industry
• EU: 2000 companies, 60 billion €
turnover
• EU: 5000 new products per year
• 25% turnover due to products
released within last 6 months
• 2003: EU 7th Amendment to
Cosmetics Directive
 Critical need for alternatives
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7th Amendment to EU Cosmetics Directive
Ban on animal testing
Cosmetic finished products
Ingredients
as soon as alternatives are
a) validated by ECVAM and
b) adopted in EU legislation
2004
2005
2006
Complete ban on
animal testing
Irrespective of availability of
alternatives
2007
2008
2009
Marketing ban
Cosmetic products
and their ingredients
as soon as alternatives are
a) validated by ECVAM,
b) adopted in EU legislation
c) and with due regard to OECD process
Most human health endpoints
Except  2013
■ repeated dose tox.
■ reproductive tox.
■ toxicokinetics
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EU Chemical legislation REACH
(Registration, Evaluation, Authorisation of Chemicals)
30,000 chemicals > 1 t per year to be assessed
billions of Euro, millions of animals,
decades of testing
Major saving impact of in silico and in vitro tests
Base-set testing (~ 20’000 substances)
In vitro testing required for eye & skin irritation
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Topical Toxicity Key Area Activities
 Support to policies
- Cosmetics
- REACH
 Evaluation of advanced in vitro alternatives
- Phototoxicity, Skin Corrosion
- Skin Irritation
- Eye Irritation
 Collaboration
Task Forces, ICCVAM, COLIPA, Industry
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Time estimation for phasing-out animal testing
• 100 scientists & stakeholder representatives
6 DG services
4 Industry associations
3 Animal Welfare NGOs
OECD
• 11 areas of human health effects of concern
• Inventory & status of validation of
most promising alternative
methods
• Recommendations to achieve
validation in a timely manner
Eskes & Zuang (2005)ATLA 33S1
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EC Timetables for
phasing-out animal testing *
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Testing / Marketing ban
Skin Corrosion
Acute Phototoxicity
Skin Absorption / Penetration
Skin Irritation – exp.’08 hazard identification
Photogenotoxicity – exp. ’08
Eye Irritation – exp. ’09
Acute Toxicity
Genotoxicity / Mutagenicity
2009 / 2009
Toxicokinetics / Metabolism
Subacute & Subchronic Toxicity
Skin Sensitisation
Photo-allergy (-sensitisation)
Carcinogenicity
Reproductive & Developmental Toxicity
2009 / 2013
* substantial reduction of animal use could be achieved earlier
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ECVAM
Research
Animal
model
In vitro
model
Optimised
test
chemicals
cosmetics
drugs
ecology
work safety
biomaterials
Prevalidation
Regulation
Validation
ESAC
Peer
Review
OECD
Pharmacopoeia
ECB
DGs
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Skin Corrosion & Acute Phototoxicity
• 1998 and 2000: ECVAM validated
replacement methods
TER, EPISKINTM, EpiDermTM
3T3NRU
• 2000 EU acceptance (B.40, B.41)
2004 OECD adoption
Skin Corrosion TG 430 & 431
Acute Phototoxicity TG 432
• Similar tests submitted to ECVAM
for skin corrosion
 Catch-up validation
SkinEthic: validated in 2006
Cellsystems EST-100: peer review
Straticell
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Skin irritation
1999-2001
Prevalidation study
2002
Optimisation studies
2003-2006 ECVAM Validation Study
Goal
To replace the Draize skin irritation test
performed in albino rabbits
Study design
Three models: EpiDermTM, EPISKINTM,
Mouse, Skin Integrity Function Test SIFT
Phase 1. Protocol optimisation & training
Phase 2. Testing 58 chemicals in 3 labs/test
Sponsor ECVAM
U.S. Observers
Bill Stokes, NICEATM
Karen Hamernik, ICCVAM
Chemical Selection
Sub Committee
Thomas Cole, ECB
Chantra Eskes, ECVAM
Sebastian Hoffmann, ECVAM
Andrew Worth, ECB
Amanda Cockshott, HSE
Ingrid Gerner, BfR
Chemical coding
and distribution
RCC-CCR
Management
Team
Chair: Phil Botham, Syngenta
Co-chair: Julia Fentem, Unilever
Biostatistics
ECVAM
Sebastian Hoffman
Valérie Zuang, ECVAM
Sebastian Hoffmann, ECVAM
Horst Spielmann, BfR-Zebet
Andrew Worth, ECB
Thomas Cole, ECB
Roland Roguet, L’Oréal
Manfred Liebsch, Zebet
Jon Heylings, Syngenta
Bill Stokes, NICEATM
Karen Hamernik, ICCVAM
Contractor
BfR-ZEBET
Wolfgang Völkner
Andreas Röder
Horst Spielmann
EPISKIN
SIFT
L‘ORÉAL
Syngenta CTL
ZEBET
Roland Roguet
José Cotovio
Jon Heylings
Helen Owen
Manfred Liebsch
Helena Kandárová
EpiDerm
Unilever
TNO
BASF
Julia Fentem
Penny Jones
Johannes van de Sandt
W.J.M. Maas
Armin Gamer
Martina Remmele
Sanofi-Synthélabo
E.I. du Pont
IIVS
Catherine Robles
William Fasano
Larue Manning
John Harbell
Hans Raabe
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Skin irritation
 Test Definition
Harmonised exposure: 15 min exposure + 42 hr post-incubation
Endpoints:
- Cell viability - MTT
- Release of IL1 in media (when cell viability > 50%)
Prediction model:
MTT cell viability  50%  skin irritant (R38)
MTT cell viability > 50%  non irritant
Additional: MTT > 50% + IL1 > 60 pg/ml  skin irritant (R38)
 Reproducibility
Within-laboratory (a)
Between-laboratory (b)
EPISKIN (MTT)
93.9 %
89.5 %
EpiDerm (MTT)
96.0 %
88.5 %
Proportion of identical MTT classifications (a) over 3 independent experiments,
(b) based on median classifications
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Skin irritation
 Predictive Capacity
EPISKIN (MTT)
EPISKIN (MTT+IL1-α)
EpiDerm (MTT)*
SENSITIVITY
74.7 %
90.7 %
57.3 %
SPECIFICITY
80.8 %
78.8 %
83.8 %
Concordance
78.2 %
83.0 %
72.4 %
Replacement of the
Draize test.
(EU Annex V B.4.
OECD TG 404)
ESAC
Only specificity acceptable.
No replacement
recommendation, but useful
in testing strategy.
ESAC
* Addition of IL1-α to the EpiDerm protocol gave no improvement to the outcome
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STATEMENT ON THE VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION
At its 26th meeting, held on 26-27th 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)1 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-1α
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reduction, and for being used as a replacement (based on the performance of the
assay as specified in the annex) for the Draize Skin Irritation Test (OECD TG 404 &
Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing
between R38 skin irritating and no-label (non-skin irritating) test substances. At the
present time, the IL-1 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 defined validation criteria;
supplementary submissions made by the Management 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 Commission
Ispra
27 April 2007
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Eye Irritation Background
6 Major international studies took place in the 90’s
EC/HO, COLIPA, CTFA, IRAG, BFA/BMBF, MHW/JCIA
No single assay capable of replacing the Draize rabbit eye test
- In vitro tests only partially model in vivo response
- Animal data of limited quality (low reproducibility across laboratories)
- Insufficiently developed protocols and prediction models
- Limitations in statistical analyses
The usefulness of alternative methods however well
established within industry and EU regulatory agencies
for specific and limited purposes
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ECVAM activities on Eye Irritation
 Evaluation of advanced in vitro alternatives
- Organotypic assays
- Cytotoxicity / cell function based assays
- Human Reconstituted Tissue models
 Identification of testing strategies
 Critical evaluation of Draize rabbit eye test
 Mechanistic developments
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Organotypic assays
Bovine Corneal Opacity and Permeability test (BCOP)
Isolated Chicken Eye (ICE)
Isolated Rabbit Eye (IRE)
Hen`s egg test on the Chorio-Allantoic Membrane assay (HET-CAM)
 Underwent several multi-laboratory studies
 Routinely used within industries & contract laboratories for specific purposes
 2002 Regulatory Acceptance by EU Competent National Authorities: Positive outcom
accepted for C&L of severe irritants (R41), although not validated
 Retrospective validation
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Organotypic assays
2003-2006 ICCVAM Retrospective evaluation with ECVAM coll.
Expert Panel Review
ICCVAM Recommendations
Current status for detecting ocular corrosives / severe irritants
April ’07
ESAC Statement
- BCOP & ICE: endorsed ICCVAM recommendations
- HET-CAM & ICE: further analyses requested
Follow-up activities
- OECD & EU Test Guidelines under preparation
- Further improvements and analyses as recommended by ESAC
- Evaluation for mild ranges of irritancy
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Cytotoxicity- / Cell function- based assays
Neutral Red Release (NRR)
Red Blood Cell Test (RBC)
Fluorescein Leakage (FL)
Cytosensor Microphysiometer (CM)
 Underwent several multi-laboratory studies
 Routinely used within industries & contract laboratories
 Specific purposes, e.g., non irritants versus irritants, surfactants
 ECVAM Retrospective weigh-of-evidence evaluation
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Retrospective evaluation – initiated fall 2005
Modular Approach
Weight-of-evidence Principles
General management
I.
Test Definition
II.
Within Laboratory Variability
III.
Transferability
IV. Between Laboratory Variability
V.
Predictive Capacity
VI. Applicability Domain
VII. Performance Standards
ECVAM WS (May ’04)
Background
Review
Documents
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Retrospective weight-of-evidence evaluation
Sponsor & oversight
Study design
Core Validation Management Group
Data & evidence
collection
Weigh-of-evidence
assessment
Peer review
ECVAM
Data Compilation Group & QC Audit Group
Core Validation Management Group
ESAC
Weight-of-evidence principles - Balls et al. 2006, ATLA 34, 1-19
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Summary Data Collection NRR, RBC, FL, CM
Major protocols
NRR Predisafe, Invittox 54, IIVS protocol
RBC Invittox 37 & 99, Japanese protocols
FL
Invittox 120, 71, 86 & 82
CM IIVS protocol, Invittox 97 & 102
Within Lab. Variability
62 - 130 test substances from 4 to 14 studies
Between Lab. Variability
55 -112 tested substances
2 to 9 laboratories, 1 to 4 major studies
Predictive Capacity
86 - 175 tested substances from 3 to 4 studies
Data Gaps
NRR data on French Official protocol / Predisafe
Raw data & protocol of RBC from Japanese studies
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Human Reconstituted Tissue Models
Submission
to ECVAM
ECVAM TF
recommendations
2005
Protocol
Validation WS
2006
Protocol
optimisation
& COLIPA
feasibility study
Outcome
2007
EpiOcular OCL-200
Corporate validation
Surfactant ingredients
4 laboratories
54 test substances
ECVAM
Workshop
to plan
Validation Study
SkinEthic HCE
Corporate pre-validation
4 laboratories
20 chemicals
Protocol
Optimisation
Prediction Model
Enlarge Appl. Domain
& Validation WS
Protocol
optimisation &
feasibility study
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Test Strategies
Organotypic”
“ assays
Whole organ explants
Cytotoxicity
Cell function
Tissue integrity
Human reconstituted
models
In vitro protein
& mucosal protein
assays
Range of Irritation
Test Methods
Physico-chemical
class/categories
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Test Strategies
Proposed Testing Strategy
ECVAM Expert Meeting, Feb 2005
30 Partners: COLIPA, Industry, CROs, Regulators, Academia, Welfare
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Topical Toxicity - Summary of activities
Status
in 2003
Phototoxicity




Skin Corrosion




Skin Penetration


Skin Irritation

Eye Irritation


*
* Partial replacement for severe irritants
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Topical Toxicity - Summary of activities
Progress
in 2007
Phototoxicity




Skin Corrosion




Skin Penetration


Skin Irritation

Eye Irritation


*
Ongoing
Ongoing


Ongoing
*
Ongoing*
* Partial replacement for severe irritants
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Acknowledgements
Valérie Zuang
Claudius Griesinger
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