29/03/2012
Overview of 3D-human skin
reconstructed models
for irritation and corrosion testing
An Van Rompay
VITO NV & CARDAM, Belgium
29/03/2012 2 Confidential – © 2009, VITO NV – All rights reserved
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 3 Confidential – © 2009, VITO NV – All rights reserved
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
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In vivo
CLP Category 1
Skin Corrosive
CLP Category 2
Skin Irritant
Destruction of skin tissue: Visible necrosis through epidermis
and into dermis in at least 1 tested animal
Reversible serious
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 ≤ 4
Observation ≤ 1h Observation ≤14d
Observation
≤14d
Persistent
inflammation of skin
H314: “causes severe skin burns and eye damage H315: “causes skin
irritation
CLP: Classification, Labelling and Packaging
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Human Skin
Stratum corneum
Stratum spinosum
Stratum basale
Stratum lucidum
Stratum granulosum rhe
FT
3D models
systemic
keratinocytes
melanocytes
langerhans cells
collagen + elastin
blood vessels
nerves
sweat glands
hair roots
fat cells
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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,…
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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
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3-Dimensional Human Skin Reconstructed Models
Stratum
Corneum
Stratum
Granulosum
Stratum
Spinosum
Stratum
Basale
rhe= reconstructed human epidermis
Histological similar
from EST1000
from RHE
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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…
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3D-models skin corrosion + irritation: Experimental layout
sample
µl or mg
Topical application (e.g. 3-15-20-60-42min) +
Rinsing of test item
Culture
medium
Post-Incubation at 37°C
Cytokine release
(IL-1a, IL-8, TNFa, …)
0
10
20
30
40
50
60
70
80
90
Control Pr 1 Pr 2 Pr 3
Tissue viability (MTT)
MN, Comet, Gene expression Morphology
No post-Incubation at 37°C
From SkinEthic Lab.
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Purpose determines protocol
Purpose » 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 assessments
Protocols » Single exposure time protocols: C / NC or I / NI
» Multiple exposure times : I/ SLI / NI
Corrosive Severe Irritant
Moderate Irritant Mild to Non-irritant
3 min 60 min 2 hrs 4 hrs 8 hrs 16 hrs 24 hrs
IIVS
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Purpose determines protocol
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Skin corrosion: 3D - models
OECD TG 431
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Certificate of Analysis
EST1000 of Cellsystems
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2 exposure-times
Viability assessment
Treatment time
Post-treatment incubation period
60 Minutes 0 Hours MTT
3 Minutes MTT
Short
Long
Prediction model: 50 % viability for 3 min
and 15 % viability for 60 min
Skin corrosion: EST1000 of Cellsystems
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Cell-viability with MTT-reaction
Yellow Blue/Purple
MTT = Methylthiazolyldiphenyl-tetrazolium bromide
=Thiazolyl Blue Tetrazolium Bromide
Mitochondria
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Cell-viability with MTT-reaction
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Skin corrosion: other 3D- models
from Dr. Eskes
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Skin irritation: RHE model from Skinethic
OECD TG 439
29/03/2012 20 Confidential – © 2009, VITO NV – All rights reserved
• NC: PBS
• PC: 5% SDS
• TI1: Heptanal
• TI2: Methyl stearate
Skin irritation: RHE from Skinethic
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Skin irritation: Episkin and EST1000
20 min 0 incubation 42 hours
Cytotoxicity
(MTT)
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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
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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
Time (hour)
…+ Histology
1.5 3 4.5 6 7.5 0 h
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Quality Control + Technical issues
Colour interaction add tissues with
medium instead of MTT
Modification of washing Oily or viscous test items
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Quality Control + Technical issues
MTT interaction
add killed tissues
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3D-Models: Conclusions
It is human tissue and truly in vitro
Epidermis
Barrier function, penetration
Wide application in product development, safety and efficacy screening, industrial hygiene and pharmaceutical research
Multiple applications:
Phototoxicity,
Genotox: Micronucleus, Comet;
Toxicogenomics, Transfection,,…
Future perspectives:
Fullthickness skin models,
Skin factory ....
29/03/2012 27 Confidential – © 2009, VITO NV – All rights reserved
Acknowledgements
An Van Rompay, Study Director in Alternative Toxicology
CARDAM - VITO
Industriezone Vlasmeer 7
2400 Mol BELGIUM Tel: +32 (0)14 335246
INVITROM
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A good question beats a good answer!