Ankara, 12 Novembre 2007 – Alternatives 2007 1 ECVAM Key Area Topical Toxicity Summary of ongoing...

Ankara, 12 Novembre 2007 – Alternatives 2007 1

ECVAM Key Area Topical Toxicity ECVAM Key Area Topical Toxicity

Summary of ongoing activities Summary of ongoing activities

Chantra Eskes

European Centre for the Validation of Alternative Methods - ECVAM

Institute for Health and Consumer ProtectionEuropean Commission Joint Research Centre

http://ecvam.jrc.cec.eu.int


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


2004 2005 2006 2007 200920082004

Ingredientsas soon as alternatives are a) validated by ECVAM and b) adopted in EU legislation

Ban on animal testingCosmetic finished products

7th Amendment to EU Cosmetics Directive

Marketing banCosmetic products and their ingredientsas soon as alternatives are a) validated by ECVAM, b) adopted in EU legislationc) and with due regard to OECD process

Complete ban onanimal testingIrrespective of availability of alternatives

Most human health endpointsExcept 2013 ■ repeated dose tox.■ reproductive tox.■ toxicokinetics


Base-set testing (~ 20’000 substances)

In vitro testing required for eye & skin irritation

billions of Euro, millions of animals, decades of testing

EU Chemical legislation REACH

(Registration, Evaluation, Authorisation of Chemicals)

30,000 chemicals > 1 t per year to be assessed

Major saving impact of in silico and in vitro tests


Support to policies- Cosmetics- REACH

Evaluation of advanced in vitro alternatives- Phototoxicity, Skin Corrosion- Skin Irritation- Eye Irritation

Collaboration Task Forces, ICCVAM, COLIPA, Industry

Topical Toxicity Key Area Activities


Time estimation for phasing-out animal testing

• 100 scientists & stakeholder representatives

6 DG services4 Industry associations

3 Animal Welfare NGOsOECD

• 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


Eye Irritation – exp. ’09Photogenotoxicity – exp. ’08Skin Irritation – exp.’08 hazard identificationSkin Absorption / PenetrationAcute PhototoxicitySkin Corrosion

Acute ToxicityGenotoxicity / Mutagenicity

Reproductive & Developmental ToxicityCarcinogenicity

Photo-allergy (-sensitisation)Skin Sensitisation

Subacute & Subchronic ToxicityToxicokinetics / Metabolism

EC Timetables for phasing-out animal testing *

* substantial reduction of animal use could be achieved earlier

2009 / 2009

2009 / 2013

Testing / Marketing ban


ECVAM

Pre-validation

ValidationESACPeer

Review

Research

Animalmodel

In vitro model

OECDPharmacopoeia

ECBDGs

Regulation

chemicalscosmetics

drugsecology

work safetybiomaterials

Optimised test


Skin Corrosion & Acute Phototoxicity

• 1998 and 2000: ECVAM validated replacement methodsTER, 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 2006Cellsystems EST-100: peer review

Straticell


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 & trainingPhase 2. Testing 58 chemicals in 3 labs/test

U.S. Observers

Bill Stokes, NICEATM Karen Hamernik, ICCVAM

Sponsor ECVAM

Biostatistics ECVAM

Sebastian Hoffman

Management Team

Chair: Phil Botham, Syngenta Co-chair: Julia Fentem, Unilever

Valérie Zuang, ECVAMSebastian Hoffmann, ECVAMHorst Spielmann, BfR-Zebet

Andrew Worth, ECBThomas Cole, ECB

Roland Roguet, L’OréalManfred Liebsch, ZebetJon Heylings, Syngenta

Bill Stokes, NICEATMKaren Hamernik, ICCVAM

Chemical SelectionSub Committee

Thomas Cole, ECBChantra Eskes, ECVAM

Sebastian Hoffmann, ECVAMAndrew Worth, ECB

Amanda Cockshott, HSEIngrid Gerner, BfR

ContractorBfR-ZEBET

Horst Spielmann

Chemical codingand distribution

RCC-CCR

Wolfgang VölknerAndreas Röder

EPISKINL‘ORÉAL

Roland RoguetJosé Cotovio

UnileverJulia FentemPenny Jones

Sanofi-Synthélabo Catherine Robles

SIFTSyngenta CTL

Jon HeylingsHelen Owen

TNOJohannes van de Sandt

W.J.M. Maas

E.I. du PontWilliam FasanoLarue Manning

EpiDermZEBET

Manfred LiebschHelena Kandárová

BASFArmin Gamer

Martina Remmele

IIVSJohn HarbellHans Raabe

U.S. Observers

Bill Stokes, NICEATM Karen Hamernik, ICCVAM

Sponsor ECVAM

Biostatistics ECVAM

Sebastian Hoffman

Management Team

Chair: Phil Botham, Syngenta Co-chair: Julia Fentem, Unilever

Valérie Zuang, ECVAMSebastian Hoffmann, ECVAMHorst Spielmann, BfR-Zebet

Andrew Worth, ECBThomas Cole, ECB

Roland Roguet, L’OréalManfred Liebsch, ZebetJon Heylings, Syngenta

Bill Stokes, NICEATMKaren Hamernik, ICCVAM

Chemical SelectionSub Committee

Thomas Cole, ECBChantra Eskes, ECVAM

Sebastian Hoffmann, ECVAMAndrew Worth, ECB

Amanda Cockshott, HSEIngrid Gerner, BfR

ContractorBfR-ZEBET

Horst Spielmann

Chemical codingand distribution

RCC-CCR

Wolfgang VölknerAndreas Röder

EPISKINL‘ORÉAL

Roland RoguetJosé Cotovio

UnileverJulia FentemPenny Jones

Sanofi-Synthélabo Catherine Robles

SIFTSyngenta CTL

Jon HeylingsHelen Owen

TNOJohannes van de Sandt

W.J.M. Maas

E.I. du PontWilliam FasanoLarue Manning

EpiDermZEBET

Manfred LiebschHelena Kandárová

BASFArmin Gamer

Martina Remmele

IIVSJohn HarbellHans Raabe

Skin irritation


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

EPISKIN (MTT) EpiDerm (MTT)Within-laboratory (a) 93.9 % 96.0 %Between-laboratory (b) 89.5 % 88.5 %

Proportion of identical MTT classifications (a) over 3 independent experiments, (b) based on median classifications


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

Skin irritation

* Addition of IL1-α to the EpiDerm protocol gave no improvement to the outcome

Predictive Capacity


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 release); 2. EPISKIN (with MTT reduction and IL-1α 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 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


6 Major international studies took place in the 90’sEC/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 purposesspecific and limited purposes

Eye Irritation Background


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

ECVAM activities on Eye Irritation


Bovine Corneal Opacity and Permeability test (BCOP)Bovine Corneal Opacity and Permeability test (BCOP)

Isolated Chicken Eye (ICE)Isolated Chicken Eye (ICE)

Isolated Rabbit Eye (IRE)Isolated Rabbit Eye (IRE)

Hen`s egg test on the Chorio-Allantoic Membrane Hen`s egg test on the Chorio-Allantoic Membrane assay assay (HET-CAM)(HET-CAM)

Underwent several multi-laboratory studiesUnderwent several multi-laboratory studies

Routinely used within industries & contract laboratories for specific purposesRoutinely used within industries & contract laboratories for specific purposes

2002 Regulatory Acceptance by EU Competent National Authorities: 2002 Regulatory Acceptance by EU Competent National Authorities: Positive outcome accepted for C&L of severe irritants (R41), although not validated

Retrospective validation

Organotypic assays


2003-2006 ICCVAM Retrospective evaluation with ECVAM coll.Expert Panel Review ICCVAM RecommendationsCurrent status for detecting ocular corrosives / severe irritants

April ’07ESAC 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

Organotypic assays


Neutral Red Release (NRR)Neutral Red Release (NRR)

Red Blood Cell Test (RBC) Red Blood Cell Test (RBC)

Fluorescein Leakage (FL)Fluorescein Leakage (FL)

Cytosensor Microphysiometer (CM)Cytosensor Microphysiometer (CM)

Underwent several multi-laboratory studiesUnderwent several multi-laboratory studies

Routinely used within industries & contract laboratories Routinely used within industries & contract laboratories

Specific purposes, e.g., non irritants versus irritants, surfactantsSpecific purposes, e.g., non irritants versus irritants, surfactants

ECVAM Retrospective weigh-of-evidence evaluation

Cytotoxicity- / Cell function- based assays


General management

I. Test Definition

II. Within Laboratory Variability

III. Transferability

IV. Between Laboratory Variability

V. Predictive Capacity

VI. Applicability Domain

VII. Performance Standards

Modular Approach Weight-of-evidence Principles

Background Review

Documents

Retrospective evaluation – initiated fall 2005

ECVAM WS (May ’04)


Retrospective weight-of-evidence evaluation

Sponsor & oversight

Study design

Data & evidence collection

Weigh-of-evidence assessment

Peer review

ECVAM

Core Validation Management Group

Data Compilation Group & QC Audit Group

Core Validation Management Group

ESAC

Weight-of-evidence principles - Balls et al. 2006, ATLA 34, 1-19


Summary Data Collection NRR, RBC, FL, CM

Data Gaps NRR data on French Official protocol / Predisafe

Raw data & protocol of RBC from Japanese studies

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


Outcome

Submissionto ECVAM

ECVAM TF recommendations

Protocol Optimisation

Human Reconstituted Tissue Models

2005 2006 2007

SkinEthic HCESkinEthic HCECorporate pre-validation4 laboratories20 chemicals

ProtocolValidation WS

Prediction ModelEnlarge Appl. Domain& Validation WS

ECVAMWorkshop

to plan Validation Study

EpiOcular OCL-EpiOcular OCL-

200200Corporate validationSurfactant ingredients4 laboratories54 test substances

Protocol optimisation & COLIPA feasibility study

Protocol optimisation & feasibility study


Test Strategies

““Organotypic” assaysWhole organ explants

CytotoxicityCell function

Tissue integrity

In vitro protein& mucosal protein

assays

Human reconstitutedmodels




Tissue integrity


Tissue integrity


assays


assays



Range o

f Ir

rita

tion

Physico-chemical class/ categories

Test Methods

Range o

f Ir

rita

tion

Physico-chemical class/ categories

Test Methods


Test Strategies

Proposed Testing Strategy

ECVAM Expert Meeting, Feb 200530 Partners: COLIPA, Industry, CROs, Regulators, Academia, Welfare


Regula

tory

acce

ptance

Topical Toxicity - Summary of activities

Phototoxicity

Skin Corrosion

Eye Irritation

Skin Irritation

*

Valid

atio

nESAC

stat

emen

t

Preva

lidat

ion

Follow-u

p

regulat

ory

acce

ptance

* Partial replacement for severe irritants

Skin Penetration

Status in 2003


Ongoing*

Regula

tory

acce

ptance

Topical Toxicity - Summary of activities

Phototoxicity

Skin Corrosion

Eye Irritation

Skin Irritation

Valid

atio

nESAC

stat

emen

t

Preva

lidat

ion

Follow-u

p

regulat

ory

acce

ptance

Skin Penetration

*Ongoing

* Partial replacement for severe irritants

Ongoing

Ongoing

*

Progress in 2007


Valérie Zuang

Claudius Griesinger

Acknowledgements

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