1. Alternative Methods to Animal Testing – A Cosmetic Industry Perspective
Conference on Alternative Approaches to Animal Testing
“EUROPE GOES ALTERNATIVE”
Brussels, 7 November 2005
Odile de Silva
L’Oréal

2. 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)
MEIC Programme and acute toxicity
IRAG: Alternatives to eye irritation (8 methods).
1994 Ring study on the BCOP test
1995 ECVAM/COLIPA Validation study on Phototoxicity
Colipa Validation study on eye irritation (2 methods)
th 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 II
2002 Dendritic cells and the Colipa Research Programme
2004 ECVAM validation study on skin irritation
th FWP - Sensitiv

3. PCOP for liquid and water-soluble materials Prediction of MAS
Statistical analysis on 41 substances
predicted MAS = X DO30 – X DO30²
Concordance : 90 %
Kappa = 0.83 (p<0.01).
R2 = 0.84
95% confidence intervals
too wide
 = overpredicted = underpredicted.

4. «The living epidermis : « The full reconstructed
Reconstructed skin and L’Oréal
RESEARCH
« Tanned epidermis »
« Epidermis mimicking an allergenic response »
The full
reconstructed
human skin
( E. Bell,
D. Asselineau)
The 1rst living
reconstructed
human epidermis
(M. Pruniéras,
M. Régnier)
1983
1986
1994
1997
2001
2002
2005
«The living epidermis :
the Episkin kit  »
«The : HTS 
human
epidermis model»
« The mini living
kit epidermis
EKIN »
« The full reconstructed
human skin kit »
« EPISKIN » Industrial models

5. Replacement of the skin irritancy test
Stratum corneum
Stratum granulosum
Stratum spinosum
Basal layers
Normal Human
Epidermis
Reconstructed Human
Epidermis EpiSkin
The EpiSkin Model
-12 tissues / kit
-QC on all components
-Tissue histology, viability, SDS IC.50
-ISO 9001 norms

6. Replacement of the skin irritancy test
Viability endpoint -Predictive model
Viability
Class. EU
OECD
< 50 %
Irritant
R38
> 50 %
Non Irritant
No Class
No label
Set of 48 chemicals
-20- Irritants
-28- Non Irritants
EpiSkin Optimized Protocol (EOP)
Performance
EpiSkin
Limits*
Sensitivity
85.0%
> 60%
Specificity
78.6%
Accuracy
81.3%
Positive Predictivity
73.9% nd
Negative Predictivity
88.0%
False Pos.
26.1%
<40%
False Neg.
12.0%
Irritants
Non Irritants
* Recommended ECVAM limits

7. Episkin® : ranking compounds according their transcutaneous diffusion potential
Stratum corneum
Viable epidermis 4h
Collagen Matrix
Used in wells
Low penetration
Medium
High penetration
%Dose RF
=100 Q
applied
Analysis performed with LCMSMS
Group 1
Group 2
RF : receptor fluid

8. Comet assay on Episkin : Detection of photogenotoxic compounds
UV-A 15 min
+ Lomefloxacin in a formulation
(topical application)
UV-A 15 min
+ Lomefloxacin in the medium
UV-A 15 min
Quantification

9. Epidermis and Langerhans cells
Physiology of Langerhans cells
Histology
Langerin positive
cells
“Epidermal sheet”
Sensitisation
Photoimmunosuppression and UV
Control
Irritant
Sensitiser
Control
SSR
SSR + UV filters
M. Régnier et al., J.Invest.Dermatol., 1997 –V. Facy et al. , J. Invest. Dermatol. , 2004

10. In vitro identification of contact sensitizers with human cell lines : one component of the battery ?
Naive T
Lymph node
Langerhans cells
Sens T
U937 cell line
Read out system : 48h
U937 / CD86 test: L’Oréal internal validation
67 references tested including 52% sensitizers
Accuracy with human clinic : 95%
Kappa : 0.91

11. The use of the Reconstructed Human Epidermis Model 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
C-16 carbons
C-22 carbons
In vitro:
strong decrease of epidermis viability (50% reduction).
Poor tolerance, dose-effect.
In vivo(clinical data):
Significant increase of irritation values since 0.125%.
Irritant, with dose-effect
In vitro:
no change in epidermis viability.
Good tolerance
In vivo(clinical data):
no significant increase of irritation values.
Well tolerated

12. 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.

13. THE COSMETIC CONTEXT
RISK ASSESSMENT and NOT HAZARD Article 2 of the Cosmetics Directive
NOT ONLY TOLERANCE BUT SYSTEMIC AND SUB-CHRONIC TOXICITY

14. 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

15. Not applicable 2009 2013 Done Genotoxicity Eye irritation
Photomutagenicity
Subacute
Subchronic
Skin irritation
Skin sensitisation
Photosensitisation
Acute toxicity
Toxicokinetics
Phototoxicity
2013
Teratogenicity
Percutaneous
absorption
Reprotoxicity
Finished Products
Skin corrosion
Carcinogenicity
Done

16. THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO 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
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
IDENTIFICATION OF SIGNAL TRANSDUCTION PATHWAYS
INSERM Lyon, LVMH
RESEARCH PROGRAMME
INITIATED 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.
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
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)

17. THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES TO SKIN IRRITATION
EPIDERMAL BIOAVAILABILITY
Univ. Cincinnati, P&G
CHANGES IN CYTOKINES RESPONSES
Henkel
RESEARCH PROGRAMME
INITIATED 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 GENE EXPRESSION IN KERATINOCYTES
Unilever
FURTHER PROJECTS
Under consideration and following the outcome of the ECVAM study

18. IN VITRO DYNAMIC CORNEAL CULTURE ASSAY 3D HUMAN CELL CULTURE MODELS
THE COLIPA STRATEGY FOR THE DEVELOPMENT OF IN VITRO ALTERNATIVES IN EYE IRRITATION
COLLABORATION
With 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.
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
3D HUMAN CELL CULTURE MODELS
INITIATED in January 2002
At the Bristol University (UK)
Dr M. Berry
M. Radburn-Smith
AIM : identify new endpoints
RESEARCH PROGRAMME
INITIATED 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.
DEVELOP NEW OR IMPROVED METHOD READY FOR PREVALIDATION

19. COLLABORATION WITH ECVAM
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.
ASSESSMENT OF THE PREDICTIVE CAPACITY OF THE CURRENT IN VITRO METHODS : THE REASONS FOR FALSE POSITIVES (DR KIRKLAND)
COLLABORATION WITH ECVAM
Through its TF
PROJECT UNDER DISCUSSION TO IDENTIFY MECHANISMS RESPONSIBLE FOR FALSE POSITIVES AND TO DEVELOP A STRATEGY BASED ON THIS NEW KNOWLEDGE
PROJECT UNDER CONSIDERATION TO IMPROVE RISK ASSESSMENT, USING BIOLOGICAL TARGETS RELEVANT FOR SKIN EXPOSURE, BASED ON INTERNAL WORK OF COMPANIES, ON 3D MODELS
DEVELOPMENT OF THE COLIPA STRATEGY

20. 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)

21. THE CHALLENGES Good science : the cutting edge
To attract the best scientists from academia
To start now integrated projects for systemic and sub-chronic toxicity
To combine all these data :
Systems Biology
New and pragmatic thinking
Thresholds of Toxicological Concern (TTC)

22. 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