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

1. Overview of 3D-human skin
reconstructed models
for irritation and corrosion testing
An Van Rompay
VITO NV & CARDAM, Belgium
29/03/2012

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
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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
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4. In vivo
CLP: Classification, Labelling and Packaging
CLP Category 1 CLP Category 2
Skin Corrosive Skin Irritant
Destruction of skin tissue: Visible necrosis through epidermis Reversible serious
and 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 ≤ 4
Observation ≤ 1h Observation ≤14d Observation Persistent
≤14d inflammation of skin
H314: “causes severe skin burns and eye damage H315: “causes skin
irritation
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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
systemic
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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,…
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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
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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
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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…
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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
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11. 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
IIVS
3 min 60 min 2 hrs 4 hrs 8 hrs 16 hrs 24 hrs
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12. Purpose determines protocol
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13. Skin corrosion: 3D - models
OECD TG 431
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14. Certificate of Analysis
EST1000 of Cellsystems
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15. Skin corrosion: EST1000 of Cellsystems
2 exposure-times
3 Minutes MTT
Short
Treatment Post-treatment Viability
time incubation period assessment
60 Minutes 0 Hours MTT
Long
Prediction model: 50 % viability for 3 min
and 15 % viability for 60 min
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16. Cell-viability with MTT-reaction
Mitochondria
Yellow Blue/Purple
MTT = Methylthiazolyldiphenyl-tetrazolium bromide
=Thiazolyl Blue Tetrazolium Bromide
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17. Cell-viability with MTT-reaction
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18. Skin corrosion: other 3D- models
from Dr. Eskes
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19. Skin irritation: RHE model from Skinethic
OECD TG 439
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20. Skin irritation: RHE from Skinethic
• NC: PBS
• PC: 5% SDS
• TI1: Heptanal
• TI2: Methyl stearate
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21. Skin irritation: Episkin and EST1000
0 20 min incubation 42 hours
Cytotoxicity
(MTT)
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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
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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
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24. 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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25. Quality Control + Technical issues
MTT interaction
add killed tissues
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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, industrial
hygiene and pharmaceutical research
 Multiple applications:
Phototoxicity,
Genotox: Micronucleus, Comet;
Toxicogenomics, Transfection,,…
Future perspectives:
Fullthickness skin models,
Skin factory
....
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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.be
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28. A good question beats a good answer!
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