Alternative Methods to Animal Testing

– A Cosmetic Industry PerspectiveConference on Alternative Approaches to Animal Testing

“EUROPE GOES ALTERNATIVE”Brussels, 7 November 2005

Odile de Silva L’Oréal

L’OREAL contribution to Validation Studies and International Programmes

on Alternative Methods

1989 L’Oréal ends tests on cosmetic finished products

1992 EC/HO: Validation study on eye irritation (3 methods)1993 MEIC Programme and acute toxicity

IRAG: Alternatives to eye irritation (8 methods).1994 Ring study on the BCOP test1995 ECVAM/COLIPA Validation study on Phototoxicity

1996 Colipa Validation study on eye irritation (2 methods)1997 4th FWP – Langerhans cells in reconstructed skin

- Human skin models 1999 Colipa Guidelines on in vitro percutaneous absorption

2000 ECVAM pre validation study on skin irritation 2001 BIOMED II2002 Dendritic cells and the Colipa Research Programme2004 ECVAM validation study on skin irritation2005 6th FWP - Sensitiv

0

10

20

30

40

50

0 10 20 30 40 50 predicted MAS

in v

ivo

MA

SPCOP for liquid and water-

soluble materials Prediction of MAS

Statistical analysis on 41 substances

predicted MAS = 8.08 + 26.16 X DO30 – 5.47 X DO30²

= overpredicted = underpredicted.

Concordance : 90 %

Kappa = 0.83 (p<0.01).

R2 = 0.84

95% confidence intervals

too wide

Reconstructed skin and L’Oréal Reconstructed skin and L’Oréal

«The living epidermis :the Episkin kit  »

«The : HTS human

epidermis model»

« The mini living« The mini living kit epidermis kit epidermis

EKIN »EKIN »« The full reconstructed

human skin kit »

« EPISKIN » Industrial models

« Epidermis mimicking an allergenic « Epidermis mimicking an allergenic response »response »

198

3

199

4

199

7

200

2

200

5

« Tanned epidermis »« Tanned epidermis »

200

1

The 1rst living reconstructed

human epidermis (M. Pruniéras,M. Régnier)

198

6The full

reconstructed human skin

( E. Bell,D. Asselineau)

RESEARCH

Replacement of the skin irritancy test

The EpiSkin ModelThe EpiSkin Model

Stratum corneum

Stratum granulosum

Stratum spinosum

Basal layers

Normal Human

Epidermis

Reconstructed Human

Epidermis EpiSkin

-12 tissues / kit

-QC on all components

-Tissue histology, viability, SDS IC.50

-ISO 9001 norms

Replacement of the skin irritancy test

EpiSkin Limits*

Sensitivity 85.0% > 60%

Specificity 78.6% > 60%

Accuracy 81.3% > 60%

Positive Predictivity

73.9% nd

Negative Predictivity

88.0% nd

False Pos. 26.1% <40%

False Neg.

12.0% <40%

Viability endpoint -Predictive modelViability endpoint -Predictive model

* Recommended ECVAM limits

Viability Class. EU OECD

< 50 % Irritant R38 Irritant

> 50 % Non Irritant No Class No label

PerformancePerformance

Set of 48 chemicals-20- Irritants-28- Non Irritants

EpiSkin Optimized Protocol (EOP)EpiSkin Optimized Protocol (EOP)

0

50

100

CHEMICALS

Viab

ility

%

Irritants

Non Irritants

EpiskinEpiskin®® : ranking compounds according their : ranking compounds according their transcutaneous diffusion potentialtranscutaneous diffusion potential

Used in wells

Stratum corneum

Viable epidermis

Collagen Matrix

4h

ER935

ER4017

2411ER2947

ER3090

Melatonin G6055691P

CaffeineSalicylic Acid Kojic Acid

0,01

0,1

1

10

100

0,1 1 10 100

% RF dose (Episkin)

% R

F d

ose

(ex

viv

o) Group 1

Group 2

%DoseRF

=100RFQ

appliedQ

Analysis performed with LCMSMS

RF : receptor fluid

Low penetration

Medium

High penetration

Comet assay on Episkin :Comet assay on Episkin :Detection of photogenotoxic Detection of photogenotoxic

compoundscompounds

UV-A 15 minUV-A 15 minUV-A 15 min UV-A 15 min

+ Lomefloxacin in a formulation + Lomefloxacin in a formulation (topical application)(topical application)

UV-A 15 min UV-A 15 min + Lomefloxacin in the medium+ Lomefloxacin in the medium

QuantificationQuantification 13,26,9 10,6

15,5

32,1

57,2

0

10

20

30

40

50

60

Mea

nTai

l Mom

ent

unexposed

UVA

Epidermis and Langerhans Epidermis and Langerhans cellscells

HistologyHistology Langerin positiveLangerin positive cellscells

““Epidermal sheet”Epidermal sheet”

ControlControl SSRSSR SSR + UV filtersSSR + UV filters

SensitisationSensitisation

ControlControl IrritantIrritant SensitiserSensitiser

Physiology of Langerhans cellsPhysiology of Langerhans cells

Photoimmunosuppression and UVPhotoimmunosuppression and UV

M. Régnier et al., J.Invest.Dermatol., 1997 –V. Facy et al. , J. Invest. Dermatol. , M. Régnier et al., J.Invest.Dermatol., 1997 –V. Facy et al. , J. Invest. Dermatol. , 20042004

Naive TLymph node

Langerhans cellsSens T

Sens T

U937 cell line

Read out system : 48h

In vitro identification of contact sensitizers with human cell lines : one component of the

battery ?

U937 / CD86 test: L’Oréal internal validation67 references tested including 52% sensitizers

Accuracy with human clinic : 95%Kappa : 0.91

The use of the Reconstructed Human Epidermis Model EPISKIN®EPISKIN®as a predictive in vitro irritation model for cosmetic ingredients.

Comparison of 2 cationic surfactants differing only by their carbon chain length (C-22 and C-16).Similarity between human clinical irritation scores and

in vitro results

In vitro:In vitro:no change in epidermis viability. Good toleranceIn vivo(clinical data):In vivo(clinical data):no significant increase of irritation values. Well tolerated

C-22 carbonsC-22 carbons

In vitro:In vitro:strong decrease of epidermis viability (50% reduction). Poor tolerance, dose-effect.In vivo(clinical data):In vivo(clinical data):Significant increase of irritation values since 0.125%. Irritant, with dose-effect

C-16 carbonsC-16 carbons

0

20

40

60

80

100

120

0 0,125 0,25 0,375 0,5

cationic [C22] surfactant concentration (% w/v)

ep

ide

rmis

via

bili

ty %

of

co

ntr

ol

0

0,20,4

0,6

0,8

11,2

1,4

1,61,8

2

clin

ica

l irr

ita

tio

n v

alu

es

viability (% control) irritation value

0

20

40

60

80

100

0 0,125 0,25 0,375 0,5

cationic [C16] surfactant concentration (% w/v)

ep

ide

rmis

via

bili

ty %

of

co

ntr

ol

0

0,5

1

1,5

2

clin

ica

l irr

ita

tio

n v

alu

es

viability (% control) irritation values

-

The COLIPA research and development programme

(since 1992)

To develop novel approaches in the cosmetics fields of expertise for product

safety assessment that do not involve any new animal testing.

THE COSMETIC CONTEXT

• RISK ASSESSMENT and NOT HAZARD Article 2 of the Cosmetics Directive

• NOT ONLY TOLERANCE BUT SYSTEMIC AND SUB-CHRONIC TOXICITY

COLIPA-SCAAT ACTIVITIES

The Steering Committee for Alternatives to Animal Testing (SCAAT) has provided a focal point for the cosmetics industry’s efforts in the EU to develop alternative approaches for over 10 years

The COLIPA R&D programme is directed towards identifying novel cellular and molecular endpoints for incorporation into new / improved alternative methods

It is the intention that these alternative methods and strategies will be developed and evaluated to the stage that they are ready for prevalidation

Acute toxicity

Not applicableNot applicable

Carcin

og

enicity

Teratogenicity

Toxicokinetics R

eprotoxicityPhotosensitisation

Toxicity

Subacute

Subchronic

DoneDone

Phototoxicity

Fin

ish

ed P

rod

uct

s

Ski

n c

orro

sio

n

Percu

taneo

us

abso

rptio

n

20092009

20132013

Pho

tom

utag

enic

ity

Pho

tom

utag

enic

ity

Skin irritation

Eye irritationG

eno

toxicity

Skin sensitisation

COLLABORATION With ECVAM in their respective COLIPA & ECVAM TFs & ECVAM Workshops

• With other industry sectors : chemical & pharmaceutical companies

• With academia : through COLIPA & EU sponsored projects

RESEARCH PROGRAMMEINITIATED in 2001

OBJECTIVE : Understanding key mechanisms in order to build a battery of methods able to replace the current in vivo test for hazard

evaluation and risk assessment purposes.

IDENTIFICATION OF SIGNAL TRANSDUCTION

PATHWAYS

INSERM Lyon, LVMH

PENETRATION AND REACTION WITH ENDOGENOUS

PROTEINS

-Toxicokinetic model

Univ. Cincinnati and P&G

-Peptide reactivity assay

Univ of Strasbourg and P&G

-Covalent binding assay

Wella, Cosmital

SENS-IT-IV

IP – 6th FWP

Novel in vitro approaches for skin & lung sensitisation

COLIPA, ECVAM, Academia, Novozymes, Pharmaceutical, & Chemical companies, ECOPA, IVTIP. (30 partners)

LANGERHANS CELLS & DENDRITRIC CELL

LINES

-Changes in gene expression

P&G – Syngenta

-Examination of Markers

Wella and Cosmital

-Interlaboratory ring trials

Shiseido, KAO, L’OREAL, HENKEL, P& G, LVMH, Wella

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN SENSITISATION

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN IRRITATION

RESEARCH PROGRAMMEINITIATED in 2001

OBJECTIVE : To identify new markers of Skin Irritation in order to address risk assessment and not

only hazard, the latter being investigated in the current ECVAM validation study

CHANGES IN CYTOKINES RESPONSES

Henkel

EPIDERMAL BIOAVAILABILITY

Univ. Cincinnati, P&G

FURTHER PROJECTSUnder consideration and following the outcome of the ECVAM study

CHANGES IN GENE EXPRESSION IN

KERATINOCYTES

Unilever

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES IN EYE IRRITATION

COLLABORATIONWith academia through Colipa projects

With ECVAM :

Participation in respective COLIPA & ECVAM TFs & in ECVAM Workshops

With ICCVAM / NIEHS & ECVAM :

Contribution to expert reviews & workshop on Mechanisms.

RESEARCH PROGRAMMEINITIATED in 2001

OBJECTIVE : Understanding mechanisms of eye irritation with a focus on injury and recovery of the CORNEA following an expert

workshop and internal company research

DEVELOPMENT OF GENE EXPRESSION FINGERPRINTS TO IDENTIFY DAMAGE TO THE

CORNEA

INITIATED in summer 2005 at Cardiff University (UK)

Prof. Mike Boulton.

3D HUMAN CELL CULTURE MODELS

INITIATED in January 2002

At the Bristol University (UK)

Dr M. Berry

M. Radburn-Smith

AIM : identify new endpoints

IN VITRO DYNAMIC CORNEAL CULTURE

ASSAY

INITIATED in January 2002

At the Aachen University, Germany

Pr N. Schrage

M. Frenz

AIM : identify new signals & end points

DEVELOP NEW OR IMPROVED METHOD READY FOR PREVALIDATION

THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES IN

GENOTOXICITY

OBJECTIVES : To develop new approaches in order to optimize the predictive capacity of current in vitro

methods , or develop new methods able to predict genotoxic potential without the use of animal tests. Develop a battery in order to perform risk assessment and not only

hazard.

COLLABORATION WITH ECVAM

Through its TF

ASSESSMENT OF THE PREDICTIVE CAPACITY

OF THE CURRENT IN VITRO METHODS : THE REASONS FOR FALSE

POSITIVES (DR KIRKLAND)

PROJECT UNDER CONSIDERATION TO IMPROVE

RISK ASSESSMENT, USING BIOLOGICAL TARGETS RELEVANT FOR SKIN

EXPOSURE, BASED ON INTERNAL WORK OF

COMPANIES, ON 3D MODELS

PROJECT UNDER DISCUSSION TO IDENTIFY MECHANISMS RESPONSIBLE FOR FALSE

POSITIVES AND TO DEVELOP A STRATEGY BASED ON THIS

NEW KNOWLEDGE

DEVELOPMENT OF THE COLIPA STRATEGY

OTHER ENDPOINTS R&D NEEDS

COLIPA current focus/priorities cover the fields where we have expertise : eye and skin irritation, skin sensitisation, and genotoxicity.

There is a clear need to develop alternative approaches that cover all toxicological endpoints

The cosmetic industry scientists have no specific expertise for developing systemic and chronic toxicity alternative approaches.

Other partners : national government labs, ECVAM, and other industry sectors are very active in some of these areas (e.g. reproductive toxicity, acute toxicity)

THE CHALLENGES

Good science : the cutting edge To attract the best scientists from academiaTo start now integrated projects for

systemic and sub-chronic toxicityTo combine all these data :

Systems Biology New and pragmatic thinking

Thresholds of Toxicological Concern (TTC)

FACING UP TO THE CHALLENGES

The cosmetics industry has taken a leading role in its areas of expertise

The cosmetics industry is ready to work in partnership with other stakeholders for the remaining challenges and to respond in a positive way to the EU political agenda in relation to developing alternative approaches to animal testing for assessing safety

For the cosmetics industry , the SAFETY of its products is, and must always remain, the number one priority