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Overview of 3D-human skin      reconstructed modelsfor irritation and corrosion testing            An Van Rompay       VIT...
OUTLINE• Importance of alternatives• Skin…largest human organ• 3D-human skin models• 3D-skin corrosion models• 3D-skin irr...
In vitro toxicology for safety assessment3R’s declaration: Reduction, Refinement & Replacement of animal tests»  EU - Dir...
In vivo                   CLP: Classification, Labelling and Packaging                      CLP Category 1                ...
Human Skin                                                                            3D models   Stratum corneum   Stratu...
Barrier function of Skin                  Protective layer + Variability•   Physical barrier against toxic chemicals and m...
in vitro models for assessing action on the Skin• Monolayer Cell culture systems e.g. 3T3   • Measure potential hazard/eff...
3-Dimensional Human Skin Reconstructed Models    rhe= reconstructed human epidermis                                       ...
Skin corrosion/irritation rhe modelsRHE from SkinEthic Laboratories / L’Oréal recherche                                  ...
3D-models skin corrosion + irritation: Experimental layout                                                                ...
Purpose determines protocolPurpose»   Transport of dangerous goods like industrial chemicals, pesticides, mixtures»   Haza...
Purpose determines protocol29/03/2012                                             12Confidential – © 2009, VITO NV – All r...
Skin corrosion: 3D - models                                                        OECD TG 431 29/03/2012                 ...
Certificate of Analysis                                                   EST1000 of Cellsystems29/03/2012                ...
Skin corrosion: EST1000 of Cellsystems                     2 exposure-times        3 Minutes                              ...
Cell-viability with MTT-reaction                                                                                     Mitoc...
Cell-viability with MTT-reaction   29/03/2012                                             17   Confidential – © 2009, VITO...
Skin corrosion: other 3D- models                                                             from Dr. Eskes      29/03/201...
Skin irritation: RHE model from Skinethic                                                                     OECD TG 439 ...
Skin irritation: RHE from Skinethic                               • NC: PBS                               • PC: 5% SDS    ...
Skin irritation: Episkin and EST10000       20 min                 incubation                            42 hours         ...
Skin irritation: Open Source Epidermal Model from Henkel    =“Namur” Epidermal Model    Standard protocol from Prof. Y P...
Quality Control + Technical issues Barrier function with Triton-X-100 1% Application Trition-X-100: for 1.5 – 3 - 4.5 - 6...
Quality Control + Technical issues                                                    Colour interaction                  ...
Quality Control + Technical issues                                                              MTT interaction           ...
3D-Models: Conclusions It is human tissue and truly in vitro    Epidermis    Barrier function, penetrationWide applica...
Acknowledgements                                                 INVITROM                           An Van Rompay,        ...
A good question beats a good answer!           29/03/2012                                             28           Confide...
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Overview of 3D-human skin reconstructed models for irritation and corrosion testing by An Van Rompay

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Overview of 3D-human skin reconstructed models for irritation and corrosion testing
An Van Rompay
VITO NV & CARDAM, Belgium
from: Invitrom meeting, December 2011

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Transcript of "Overview of 3D-human skin reconstructed models for irritation and corrosion testing by An Van Rompay"

  1. 1. Overview of 3D-human skin reconstructed modelsfor irritation and corrosion testing An Van Rompay VITO NV & CARDAM, Belgium 29/03/2012
  2. 2. OUTLINE• Importance of alternatives• Skin…largest human organ• 3D-human skin models• 3D-skin corrosion models• 3D-skin irritation models• Conclusions and future perspectives  Disclaimer: my personal view based on my expertise and experience 29/03/2012 2 Confidential – © 2009, VITO NV – All rights reserved
  3. 3. In vitro toxicology for safety assessment3R’s declaration: Reduction, Refinement & Replacement of animal tests» EU - Directive 86/609/EEC (update Sept 2010)Cosmetics directive» 7th Amend. to EU Cosmetics Directive (76/768/EEC): marketing ban for finished products and ingredients if animals tests are used REACH: Registration, Evaluation and Authorisation of Chemicals» The new EU Chemicals legislation (COM (2003) 644)» Chantra Eskes: Application of alternative method in regulatory assessment of chemical safety related to human skin corrosion and irritation 2010» ECHA: The Use of Alternatives to Testing on Animals for REACH regulation 2011 29/03/2012 3 Confidential – © 2009, VITO NV – All rights reserved
  4. 4. In vivo CLP: Classification, Labelling and Packaging CLP Category 1 CLP Category 2 Skin Corrosive Skin IrritantDestruction of skin tissue: Visible necrosis through epidermis Reversible seriousand into dermis in at least 1 tested animal effects on skin (Exposure ≤ 4 h) Category 1A Category 1B Category 1C Draize values (mean value at least 2 or 3 rabbits)Exposure ≤ 3 min Exposure ≤ 1 h Exposure ≤ 4 h 2.3 ≤ Erythema/eschar or oedema scores ≤ 4Observation ≤ 1h Observation ≤14d Observation Persistent ≤14d inflammation of skinH314: “causes severe skin burns and eye damage H315: “causes skin irritation 29/03/2012 4 Confidential – © 2009, VITO NV – All rights reserved
  5. 5. Human Skin 3D models Stratum corneum Stratum lucidum keratinocytes Stratum granulosum Stratum spinosum melanocytes rhe Stratum basale langerhans cells collagen + elastin blood vessels FT nerves sweat glands hair roots fat cells systemic29/03/2012 5Confidential – © 2009, VITO NV – All rights reserved
  6. 6. Barrier function of Skin Protective layer + Variability• Physical barrier against toxic chemicals and microbes• Insulates living tissue from chemical and physical trauma• Keratins and lipids prevent trans-epithelial water loss• Protect against solar exposure (melanocytes)• Respond to mechanical forces (elastic and cushion)The differentiation of the skin into a complex lipid based protective membrane makes skin unique• Variable over range of skin sites: hands vs. eyelid• Human to human variability: age, race, …• Skin disorders: acne, eczema, psoriasis, auto-immune disorders, infections,… 29/03/2012 6 Confidential – © 2009, VITO NV – All rights reserved
  7. 7. in vitro models for assessing action on the Skin• Monolayer Cell culture systems e.g. 3T3 • Measure potential hazard/efficacy • Must assure exposure to target cell • More for ingredients/chemicals rather than formulations no penetration (fate of skin exposed chemical?) • What if ingredients insoluble in medium • Applications: Phototoxicity, inflammatory response• 3D skin models • OECD TG 431 and 439 29/03/2012 7 Confidential – © 2009, VITO NV – All rights reserved
  8. 8. 3-Dimensional Human Skin Reconstructed Models rhe= reconstructed human epidermis Stratum Corneum Stratum Granulosum Stratum Spinosum Stratum Basale from EST1000 Histological similar from RHE 29/03/2012 8 Confidential – © 2009, VITO NV – All rights reserved
  9. 9. Skin corrosion/irritation rhe modelsRHE from SkinEthic Laboratories / L’Oréal recherche FranceEpiSkin from L’Oréal recherche / SkinEthic Laboratories FranceEpiderm from MatTek Corporation USAEST1000 from Cellsystems/Advanced Cell Systems GermanyOS-REP from no supplier but e.g. Henkel GermanyStratiCELL® Reconstituted Human Epidermis from Straticell Belgium LHE The NetherlandsHenkel (Phenion) Full Thickness Skin Model from Henkel GermanyStrataTest from Stratatech USAAsia: Gunze, J-Tec, Tego Science, Labcyte… 29/03/2012 9 Confidential – © 2009, VITO NV – All rights reserved
  10. 10. 3D-models skin corrosion + irritation: Experimental layout From SkinEthic Lab.Topical application (e.g. 3-15-20-60-42min) + Rinsing of test item µl or mg sample Post-Incubation at 37°C Tissue viability (MTT) 90 No post-Incubation at 37°C 80 70 60 Culture 50 medium 40 30 20 10 0 Control Pr 1 Pr 2 Pr 3 Cytokine release (IL-1a, IL-8, TNFa, …)MN, Comet, Gene expression Morphology 29/03/2012 10 Confidential – © 2009, VITO NV – All rights reserved
  11. 11. Purpose determines protocolPurpose» Transport of dangerous goods like industrial chemicals, pesticides, mixtures» Hazard identification and labelling of chemicals (Corrosion, Irritation)» Labelling of finished products e.g. cleaning agent, wash conditioner» Product development and candidate formulation assessmentsProtocols» Single exposure time protocols: C / NC or I / NI» Multiple exposure times : I/ SLI / NI Corrosive Severe Irritant Moderate Irritant Mild to Non-irritant IIVS 3 min 60 min 2 hrs 4 hrs 8 hrs 16 hrs 24 hrs 29/03/2012 11 Confidential – © 2009, VITO NV – All rights reserved
  12. 12. Purpose determines protocol29/03/2012 12Confidential – © 2009, VITO NV – All rights reserved
  13. 13. Skin corrosion: 3D - models OECD TG 431 29/03/2012 13 Confidential – © 2009, VITO NV – All rights reserved
  14. 14. Certificate of Analysis EST1000 of Cellsystems29/03/2012 14Confidential – © 2009, VITO NV – All rights reserved
  15. 15. Skin corrosion: EST1000 of Cellsystems 2 exposure-times 3 Minutes MTTShort Treatment Post-treatment Viability time incubation period assessment 60 Minutes 0 Hours MTTLong Prediction model: 50 % viability for 3 min and 15 % viability for 60 min 29/03/2012 15 Confidential – © 2009, VITO NV – All rights reserved
  16. 16. Cell-viability with MTT-reaction Mitochondria Yellow Blue/PurpleMTT = Methylthiazolyldiphenyl-tetrazolium bromide =Thiazolyl Blue Tetrazolium Bromide 29/03/2012 16 Confidential – © 2009, VITO NV – All rights reserved
  17. 17. Cell-viability with MTT-reaction 29/03/2012 17 Confidential – © 2009, VITO NV – All rights reserved
  18. 18. Skin corrosion: other 3D- models from Dr. Eskes 29/03/2012 18 Confidential – © 2009, VITO NV – All rights reserved
  19. 19. Skin irritation: RHE model from Skinethic OECD TG 439 29/03/2012 19 Confidential – © 2009, VITO NV – All rights reserved
  20. 20. Skin irritation: RHE from Skinethic • NC: PBS • PC: 5% SDS • TI1: Heptanal • TI2: Methyl stearate 29/03/2012 20 Confidential – © 2009, VITO NV – All rights reserved
  21. 21. Skin irritation: Episkin and EST10000 20 min incubation 42 hours Cytotoxicity (MTT) 29/03/2012 21 Confidential – © 2009, VITO NV – All rights reserved
  22. 22. Skin irritation: Open Source Epidermal Model from Henkel  =“Namur” Epidermal Model  Standard protocol from Prof. Y Poumay (2004) : ”easy”  No supplier….FREEDOM  OS-REP irritation validation + ECVAM submission 29/03/2012 22 Confidential – © 2009, VITO NV – All rights reserved
  23. 23. Quality Control + Technical issues Barrier function with Triton-X-100 1% Application Trition-X-100: for 1.5 – 3 - 4.5 - 6 and 7.5 h 3 tissues untreated (0h) 3h MTT ON isopropanol Viability 50% viability ?h 1.5 3 4.5 6 7.5 0h Time (hour) …+ Histology 29/03/2012 23 Confidential – © 2009, VITO NV – All rights reserved
  24. 24. Quality Control + Technical issues Colour interaction add tissues with medium instead of MTT Modification of washing Oily or viscous test items 29/03/2012 24 Confidential – © 2009, VITO NV – All rights reserved
  25. 25. Quality Control + Technical issues MTT interaction add killed tissues 29/03/2012 25 Confidential – © 2009, VITO NV – All rights reserved
  26. 26. 3D-Models: Conclusions It is human tissue and truly in vitro Epidermis Barrier function, penetrationWide application in product development, safety and efficacy screening, industrialhygiene and pharmaceutical research Multiple applications: Phototoxicity, Genotox: Micronucleus, Comet; Toxicogenomics, Transfection,,…Future perspectives: Fullthickness skin models, Skin factory .... 29/03/2012 26 Confidential – © 2009, VITO NV – All rights reserved
  27. 27. Acknowledgements INVITROM An Van Rompay, Study Director in Alternative Toxicology CARDAM - VITO Industriezone Vlasmeer 7 2400 Mol BELGIUM Tel: +32 (0)14 335246 an.vanrompay@vito.be29/03/2012 27Confidential – © 2009, VITO NV – All rights reserved
  28. 28. A good question beats a good answer! 29/03/2012 28 Confidential – © 2009, VITO NV – All rights reserved
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