Mr Elian LATI. BIO-EC Laboratory Longjumeau, France

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1 Human living skin explant model as an alternative to animal experimentation for evaluation of cosmetic products, raw materials and finished products, activities and tolerance Mr Elian LATI BIO-EC Laboratory Longjumeau, France The Asian Congress on Alternatives and Animal Use in the Life Science 15 th November 2016 Karatsu, Saga, JAPAN

BIO-EC laboratory Private company created in

since 2007 16 collaborators Paris Research

dermo-cosmetics field MAIN GOAL: Respond to

2 BIO-EC laboratory Private company created in 1998 Situated in Longjumeau (Paris area) since collaborators Paris Research centre dedicated to the companies of dermo-cosmetics field MAIN GOAL: Respond to the needs of our costumers in terms of claim substantiations

3 Alternative methods for the evaluation of dermo-cosmetic products

4 Alternative models EU bans cosmetics tested on animals Alternative methods for the evaluation of dermo-cosmetic products in vitro ex vivo in vivo

5 European cosmetic regulation and animal experimentation.

6 History of the prohibition of the animal experimentation: 1976: First attempt at harmonization of the legislations on the cosmetics with adoption by Europe of directive 76/768/CEE, known as Directive Cosmetics. 1993: Directive 93/35/CEE modifies deeply the Directive Cosmetics. 1996: A report proposes to carry forward of prohibition. 1997: The prohibition envisaged by directive 93/35/CEE is pushed back. 2000: The Prohibition is again pushed back.

7 2003: New directive 2003/15/CE removes and replaces directive 93/35/CEE. It is envisaged two prohibitions: 1) to test on the animals the end products starting from 09/ 2004; 2) execution on the European territory of tests on animals for the cosmetic products and theirs ingredients. September 2004: Prohibition to test on animals the finished cosmetic products and the ingredients if there are alternative methods. March 2009: Coming into effect of prohibition to sell end products and raw materials tested on animals. March 11h, 2013: Total ban to sell in Europe cosmetic products having had recourse to the animal experimentation.

9 in vitro Alternative models

10 in vivo Alternative models

11 3D models Alternative models

12 3D models: reconstructed skin Reconstructed epidermis synthetic support Collagen lattice de-epidermized dead dermis Collagen cellularized sponge These models are widely used for raw materials screening and toxicity assessment But compared to in vivo or ex vivo human skin : The stratum corneum is thicker and lipid synthesis is disturbed The barrier function is not normal The dermal-epidermal junction is more or less present All cellular and tissular structures of the human skin are not present The expression of keratinocyte differentiation markers is modified

13 Human living skin explant Full skin Human living skin explants (epidermis+dermis) Epidermis+ dermis Adipose tissue Separeted epidermis Scalp Isolated hairs

14 Treatments Raw meterials Active ingredients Application in the culture medium Single or chronical treatments

15 Human living skin explant Raw meterials Active ingredients Formulation under developing Finished products Topical application Single or chronical treatments

16 Ex vivo phase Ultraviolets Tensioactifs Injectable products Allergens Delipidation Infrared Lesions Burns Lasers Pollutants. 12 days of survival Genomics Measures Raman Imaging UV Specific culture medium Histology Dosages

17 Human living skin explants and claim substantiation in dermo-cosmetic field

18 Many claims Solar protection Anti-ageing Pigmentation/depigmentation Anti-bacterial Wound healing Anti-glycation Anti-stress Hair growth Hydration Slimming Cellular viability Cutaneous barrier protection And more

19 Histology Observation of histilogical sections after classical colorations or after immunochemistry General morpohology Cellular viability Extracellular matrix: GAGs Epidermal immunity: Langerhans cells Skin pigmentation: melanocyte Skin hydration: AQP3

20 Histology Evaluation of epidermal regeneration Wound healing

21 Histology Observation of histilogical sections after classical colorations or after immunofluorescence Laminin-5 FLG CK14 Blood vessels

22 Histology Transmission electron microscopy Observation of lipid bilayers restructuration in inter-corneocyte spaces on external stratum corneum

23 Histology Scanning electron microscopy Observation of corneocyte cohesion on stratum corneum surface

24 Pollubox Exposure systhem to air pollutants Heavy metals Volatile organic compounds Polycyclic aromatic hydrocarbons Diesel particulate matters Ozone Genomic Measures Raman Platforme Histology

25 IMMUNOSTAININGS Untreated batch Nrf2 Heavy metals treated batch Pollubox 50 µm 50 µm HO-1 50 µm 50 µm

26 Solarbox Système d illumination en lumière visible a: Spectre solaire de référence Lumière bleue Lumière rouge Lumière verte b: Spectre solaire de la Solarbox

27 Solarbox

28 Cosmetogenomic Product activities caracherization Fonctional genomic Extraction and ARNs validation Quantification and data normalisation Microarrays

29 Skin moisturizing Lipids and proteins conformation Antioxydants Skin absorption Raman spectroscopy

30 Distribution of skin lipids, keratins and nucleic acids in the epidermis Raman spectroscopy

20160314_pt_j0_exp1_img_spectrale -40-30

31 Y (µm) Intensity (counts) Raman spectroscopy Skin moisturizing : Distribution of NMF components in the epidermis _pt_j0_exp1_img_spectrale X (µm) 5 µm

32 Antioxydants: Distribution of carotenoids in the epidermis Raman spectroscopy

33 Topography (light interferometry) z step measurement: 20 nm

34 Topography (light interferometry) Skin surface topographie

application 15 minutes after product application Depth

35 Topography (light interferometry) Evaluation of the effect of smoothing foundation Before product application 15 minutes after product application Depth of the fold: 34µm Depth of the fold: 26µm (-23% vs before treatment)

product application Roughness: Sa =12.2 Roughness: Sa =9.

36 Topography (light interferometry) Evaluation of the effect of smoothing foundation Roughness Before product application 15 minutes after product application Roughness: Sa =12.2 Roughness: Sa =9.9 (-19% vs before treatment) Roughness: Sa =11.1 Roughness: Sa =9.6 (-13% vs before treatment)

37 The ex-vivo models Raw materials, end-products, medical devices Activity evaluation Demonstrate mechanism of action Early and predictive Conditions close to reality Iconography Fine and accurate measurements

38 Acknowledgements Thank you for your attention Mr LATI ELIAN contacts telephone BIO-EC laboratory 1 chemin de Saulxier Longjumeau France Visite our web site!

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