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

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


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


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


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


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,…


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


3-Dimensional Human Skin Reconstructed Models

Stratum

Corneum

Stratum

Granulosum

Stratum

Spinosum

Stratum

Basale

rhe= reconstructed human epidermis

Histological similar

from EST1000

from RHE


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…


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.


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


Purpose determines protocol


Skin corrosion: 3D - models

OECD TG 431


Certificate of Analysis

EST1000 of Cellsystems


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


Cell-viability with MTT-reaction

Yellow Blue/Purple

MTT = Methylthiazolyldiphenyl-tetrazolium bromide

=Thiazolyl Blue Tetrazolium Bromide

Mitochondria


Cell-viability with MTT-reaction


Skin corrosion: other 3D- models

from Dr. Eskes


Skin irritation: RHE model from Skinethic

OECD TG 439


• NC: PBS

• PC: 5% SDS

• TI1: Heptanal

• TI2: Methyl stearate

Skin irritation: RHE from Skinethic


Skin irritation: Episkin and EST1000

20 min 0 incubation 42 hours

Cytotoxicity

(MTT)


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


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



Colour interaction add tissues with

medium instead of MTT

Modification of washing Oily or viscous test items



MTT interaction

add killed tissues


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 ....


Acknowledgements

An Van Rompay, Study Director in Alternative Toxicology

CARDAM - VITO

Industriezone Vlasmeer 7

2400 Mol BELGIUM Tel: +32 (0)14 335246

[email protected]

INVITROM

mailto:[email protected]




A good question beats a good answer!

Documents

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