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					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
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 test
1995   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 II
2002   Dendritic cells and the Colipa Research Programme
2004   ECVAM validation study on skin irritation
2005   6th FWP - Sensitiv
                                      PCOP for liquid and water-soluble
                                                  materials
                                             Prediction of MAS
                                        Statistical analysis on 41 substances
              50                predicted MAS = 8.08 + 26.16 X DO30 – 5.47 X DO30²

              40
in vivo MAS




              30                                                Concordance : 90 %

              20
                                                               Kappa = 0.83 (p<0.01).
                                                                    R2 = 0.84
              10


               0                                              95% confidence intervals
                   0     10     20      30     40     50
                                                                     too wide
                               predicted MAS
                        = overpredicted = underpredicted.
                       Reconstructed skin and L’Oréal

                                                                                            RESEARCH
                                                    « Tanned epidermis »



 The 1rst living    The full
 reconstructed   reconstructed                                    « Epidermis mimicking an allergenic
human epidermis human skin                                                   response »
                           ( E. Bell,
   (M. Pruniéras,
                        D. Asselineau)
    M. Régnier)




                                              « The mini living          «The : HTS
                    «The living epidermis :     kit epidermis              human            « The full reconstructed
                      the Episkin kit »             EKIN »            epidermis model»          human skin kit »


  « EPISKIN » Industrial models
Replacement of the skin irritancy test

Normal Human                         Reconstructed Human
                 The EpiSkin Model    Epidermis EpiSkin
  Epidermis

                Stratum corneum
               Stratum granulosum
                Stratum spinosum
                  Basal layers



                -12 tissues / kit
                -QC on all components
                -Tissue histology, viability, SDS IC.50
                -ISO 9001 norms
   Replacement of the skin irritancy test
Viability endpoint -Predictive model
 Viability       Class.        EU          OECD       Set of 48 chemicals
 < 50 %          Irritant      R38        Irritant   -20- Irritants
 > 50 %       Non Irritant   No Class    No label    -28- Non Irritants

    EpiSkin Optimized Protocol (EOP)                                Performance
                                                                      EpiSkin   Limits*
                               Non Irritants
                                                     Sensitivity       85.0%    > 60%

                                                     Specificity       78.6%    > 60%

                                                     Accuracy          81.3%    > 60%

                                                     Positive          73.9%       nd
                                                     Predictivity
             Irritants
                                                     Negative          88.0%       nd
                                                     Predictivity

                                                     False Pos.        26.1%      <40%

                                                     False Neg.        12.0%      <40%


                                                      * Recommended ECVAM limits
          Episkin® : ranking compounds according their
               transcutaneous diffusion potential
                      Stratum corneum


                      Viable epidermis

                      Collagen Matrix

     Used in wells




                                                       4h
                                                                                                                  Medium
                                                                                         Low penetration                           High penetration
                                                                                                 100
                                                                                                                                                   Group 1



                                                            % RF dose (ex vivo )
                                                                                                                                       Caffeine
                                                                                                  10              Salicylic Acid        Kojic Acid 691P
                                                                                                                                                 G6055
                                                                                                                              Melatonin

                                         Q    RF                                                   1

              %Dose    RF
                            =100                                                   0,1        ER3090 1        ER4017         10                     100

                                   Q         applied                                             0,1
                                                                                              2411
                                                                                                         ER2947   ER935




                Analysis performed with LCMSMS                                                  0,01              Group 2
                                                                                                % RF dose (Episkin)
RF : receptor fluid
                 Comet assay on Episkin :
        Detection of photogenotoxic compounds




                                               UV-A 15 min                   UV-A 15 min
      UV-A 15 min                     + Lomefloxacin in a formulation + Lomefloxacin in the medium
                                           (topical application)
                                                                                                                                          57,2
                                       60                               unexposed
                                       50
                                                                        UVA
                                       40                                                                               32,1
                    MeanTail Moment




                                       30
                                                    13,2                                                       15,5
Quantification                         20
                                                                        6,9
                                                                                          10,6
                                       10
                                        0
                                                   l1              l2                l3                   in             n
                                           n   tr o        n   tr o          n   tr o              15
                                                                                                      m              tio            ium
                                        co              co                co                  -A                  ula            ed
                                                                                            UV                 rm              nM
                                                                                                            fo              e.i
                                                                                                         in              om
                                                                                                    o me              +L
                                                                                                 +L               -A
                                                                                               A               UV
                                                                                          U V-
                           Epidermis and Langerhans cells
                                                    Physiology of Langerhans cells




                                                          Histology   Langerin positive “Epidermal sheet”
                                                                            cells




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


        Langerhans cells
                           Sens T                   U937 cell line
                                                      Read out
                                                    system : 48h
                            Sens T

                       Naive T
                  Lymph node



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

                 Accuracy with human clinic : 95%
                          Kappa : 0.91
               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
                                       viability (% control)     irritation value                                          viability (% control)                         irritation values
                               120                                                  2
                                                                                                                                                    100                                            2




                                                                                                                         epidermis viability % of




                                                                                                                                                                                                         clinical irritation values
                                                                                    1,8
    epidermis viability % of




                                                                                          clinical irritation values
                               100                                                  1,6                                                              80
                                                                                                                                                                                                   1,5
                                                                                    1,4
                                80




                                                                                                                                control
                                                                                    1,2                                                              60
           control




                                                                                                                                                                                                   1
                                60                                                  1
                                                                                                                                                     40
                                                                                    0,8
                                40                                                                                                                                                                 0,5
                                                                                    0,6                                                              20
                                20                                                  0,4
                                                                                    0,2                                                               0                                            0
                                 0                                                  0                                                                       0    0,125     0,25    0,375     0,5
                                       0      0,125     0,25   0,375     0,5                                                                        cationic [C16] surfactant concentration (% w/v)
                                 cationic [C22] surfactant concentration (% w/v)



                                           C-22 carbons                                                                                                           C-16 carbons

In vitro:                                                                                                              In vitro:
no change in epidermis viability.                                                                                      strong decrease of epidermis viability (50% reduction).
Good tolerance                                                                                                         Poor tolerance, dose-effect.
In vivo(clinical data):                                                                                                In vivo(clinical data):
no significant increase of irritation values.                                                                          Significant increase of irritation values since 0.125%.
Well tolerated                                                                                                         Irritant, with dose-effect
    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
                           Not applicable


2009


                        Photosensitisation
       Acute toxicity


                                             2013


  Done
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
   PENETRATION AND
                                           • With academia : through COLIPA & EU sponsored projects
    REACTION WITH
     ENDOGENOUS
       PROTEINS
-Toxicokinetic model                                                                                                                IDENTIFICATION OF
                                                                                                                                  SIGNAL TRANSDUCTION
Univ. Cincinnati and P&G                                                                                                                PATHWAYS
-Peptide reactivity assay                                      RESEARCH PROGRAMME
Univ of Strasbourg and P&G                                                INITIATED in 2001                                       INSERM Lyon, LVMH
-Covalent binding assay                           OBJECTIVE : Understanding key mechanisms in order to build a
Wella, Cosmital                                  battery of methods able to replace the current in vivo test for hazard
                                                              evaluation and risk assessment purposes.



                  LANGERHANS CELLS &
                    DENDRITRIC CELL                                                                                       SENS-IT-IV
                        LINES
                                                                                                               IP – 6th FWP
                  -Changes in gene expression
                                                                                                               Novel in vitro approaches for
                  P&G – Syngenta                                                                               skin & lung sensitisation
                  -Examination of Markers                                                                      COLIPA, ECVAM, Academia,
                  Wella and Cosmital                                                                           Novozymes, Pharmaceutical, &
                                                                                                               Chemical companies, ECOPA,
                  -Interlaboratory ring trials                                                                 IVTIP. (30 partners)
                  Shiseido, KAO, L’OREAL,
                  HENKEL, P& G, LVMH, Wella
THE COLIPA STRATEGY FOR THE DEVELOPMENT OF
 IN VITRO ALTERNATIVES TO SKIN IRRITATION

   EPIDERMAL
 BIOAVAILABILITY                                                                   CHANGES IN CYTOKINES
 Univ. Cincinnati, P&G                                                                 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                                                                  FURTHER PROJECTS
  EXPRESSION IN
  KERATINOCYTES                                                                   Under consideration and following
                                                                                  the outcome of the ECVAM study
        Unilever
 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                                                                                               3D HUMAN CELL
        ASSAY                                                                                                  CULTURE MODELS
  INITIATED in January 2002
                                                        RESEARCH PROGRAMME
   At the Aachen University,                                      INITIATED in 2001                           INITIATED in January 2002
           Germany                          OBJECTIVE : Understanding mechanisms of eye irritation with a     At the Bristol University (UK)
        Pr N. Schrage                       focus on injury and recovery of the CORNEA following an expert            Dr M. Berry
                                                        workshop and internal company research
            M. Frenz                                                                                                M. Radburn-Smith
AIM : identify new signals & end                                                                              AIM : identify new endpoints
              points

                                                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
   THE COLIPA STRATEGY FOR THE DEVELOPMENT
   OF IN VITRO ALTERNATIVES IN GENOTOXICITY


                                     OBJECTIVES : To develop new
ASSESSMENT OF THE                  approaches in order to optimize the
PREDICTIVE CAPACITY                predictive capacity of current in vitro
 OF THE CURRENT IN
                                                                                     COLLABORATION WITH
                               methods , or develop new methods able to
VITRO METHODS : THE                                                                        ECVAM
                               predict genotoxic potential without the use
REASONS FOR FALSE             of animal tests. Develop a battery in order to              Through its TF
    POSITIVES (DR
     KIRKLAND)                    perform risk assessment and not only
                                                 hazard.




                                                                                     PROJECT UNDER
 PROJECT UNDER DISCUSSION                                                      CONSIDERATION TO IMPROVE
  TO IDENTIFY MECHANISMS            DEVELOPMENT OF THE
                                                                                RISK ASSESSMENT, USING
   RESPONSIBLE FOR FALSE              COLIPA STRATEGY                             BIOLOGICAL TARGETS
 POSITIVES AND TO DEVELOP A                                                        RELEVANT FOR SKIN
  STRATEGY BASED ON THIS                                                          EXPOSURE, BASED ON
      NEW KNOWLEDGE                                                                INTERNAL WORK OF
                                                                               COMPANIES, ON 3D MODELS
               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 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)
 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

				
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