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NCn NC PCn PC IC

Normal Negative Control tissue (NCn)

Injured Negative Control tissue (NC)

Injured Positive Control tissue (PC)

Injured Internal Control tissue (IC)Normal RHE

Injured RHE

NCn NC PCn PC IC0

20

40

60

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VIA

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ITY

%

100%

79% 79% 76%

27%

MTT testFigure1 - Cells viability

*p<0.06 (T test)

TEER variations at 24h versus t0

Decrease barrier functionalityIncrease permeability

Figure2 - TEER measurement

RHE

30µl of product / tissue

x3Injured RHE

t024h of

contact

M E A A n a l y s i sA n a l y s i s

y In vitro reconstructed human tissue models are recognized as being sensitive and reliable models to replace or reduce laboratory animal use in preclinical studies [1,2]. y To target medical device and dermo-pharmaceutical applications, the status of the skin barrier is a key parameter [3]. y The study objective was to design a new 3-D Reconstructed human Epidermis models (RHE) with a physically impaired barrier function in order to challenge the

tolerance of dedicated ingredients at usual dosage through a Multiple Endpoints Analysis (MEA) [4].

• Two types of ingredient functionalities: emulsifier and rheology modifier.• Ingredients tested in simple dilutions at effective dosages.• Experimental surfactant at irritant dosage to check the model sensitivity (Internal Control=IC).• Negative Controls= Phosphate-Bufferered Saline (PBS) on normal RHE (NCn) and on Injured RHE (NC).• Positive Controls= Sodium Dodecyl Sulfate (SDS 0.15%) on normal RHE (PCn) and on Injured RHE (PC).

Functionality Chemical nature Code Dose VehicleMedical device

Dermo- Pharmacy

Emulsifier (Non ionic surfactant)

Octadecanoic acid, 12-hydroxy-, homopolymer, ester with α-hydro-ω-hydroxypoly(oxy-1,2 –ethanediyl) SE1 1%

paraffin oil •

Acetalization products between glucose and C20/22 alcohol SE2 2% water •Acetalization products between glucose and C16/18 alcohol SE3 2% water • •

Rheology Modifier (Anionic polymer)

Ammonium 2 acrylamido 2 methyl propane sulfonate copolymer RM1 1% water •2-proprenoic acid, 2-hydroxyethyl ester, polymer with 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid monosodium salt

RM2 2% water • •

2-proprenoic acid, 2-hydroxyethyl ester, polymer with 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid monosodium salt & 2,6,10,15,19,23-hexamethyltetracosane & Sorbitan stearate ethoxylated

RM3 3% water •

Acrylamide/ 2-acrylamido-2-methylpropanesulphonic acid, sodium salt copolymer & i 2,2,4,4,6,8,8- heptamethylnonane & Sorbitan monooleate,ethoxylated

RM4 5% water • •

Experimental Surfactant

Amphiphilic experimental structure with C12 chain IC 0.8% water Internal Control

Trans-Epithelial-Electrical-resistance (TEER) measurement y Physical parameter of skin barrier functionality (overall epidermis). yMeasure of the movement of ions across the paracellular pathway regulated by polarized plasma membrane surfaces and by cell-to-cell tight junctions (Millicell-ERS=Electrical Resistance System).

Histo-Morphological (H&E) analysis (for controls) y Paraffin embedded tissues / Haematoxylin and eosin staining=H&E / Light microscopy analysis (Leica DM 2500 FLUO). y Scoring from 0 to 4 related to increasing toxicity:

0=Standard=no significant modification1=Slight=modification to SC2=Moderate=modification to SC & granular layer3=Strong=modification at basal layer (necrotic cells, intercellular, holes, oedema)4=Severe=loose structure ( connexions, tissue detachment, necrotic cells)

TEER measurement

Histo-Morphological analysis

Cellular viability (MTT test / Model prediction: Non irritant when viability≥50%).

Biotin permeability assay y Specific barrier integrity assessment of living epidermis (Tight Junctions / structural level) in an inside-out permeation model. y Biotin treatment from the basal chamber / Paraffin embedded tissues / Secondary antibody Texas Red Streptavidin Conjugate staining + DAPI for nuclei / Fluorescent microscopy (Leica DM 2500 FLUO).

Normal Skinethic™Reconstructed Human Epidermis (RHE)Morphology reference

Injured RHE

In v

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ysis

mechanical abrasion of the Stratum Corneum (SC)

The different controls: injured versus normal RHE as well as the Internal Control were used to challenge the model response using the so-called MEA approach parameters.

Biotin staining in the granular layer.

Visible biotin within the whole tissue pointed out a reduced tissue integrity and cell to cell cohesion.

Biotin passage.

Skin barrier integrity: Biotin fluorescence signal practically absent in extracellular matrix at granular layer.

Ingredient Code

Cells Viability (% ± SD%)

H&E score (0 to 4)

TEER ∆% versus t0*p<0.06 (T test)

Biotin passage

NC 100 ± 19 0-1 -36 Granular layer / low passage

SE1 99 ± 20-1

Restored structure & morphology compared to NCIncreased keratohyalin granules

-68* Not done

SE2 120 ± 11

Thicker & Swollen SCIncreased keratohyalin granules

-32 Granular layer / low passage ≈ NC

SE3 130 ± 9 0-1 -13 Granular layer / low passage ≈ NC

RM1 99 ± 3 1 -46* Not done

RM2 99 ± 0 1 -35* Not done

RM3 114 ± 2 0-1Thicker SC

-72* Lower passage < NC / low passage ≈ NCn

RM4 106 ± 11

Swollen SC & slightly modified granular layer morphology

-46 Granular layer / low passage ≈ NC

• The high cells viability level indicated the absence of toxic effect at basal level for all the ingredients.

• Despite a statistically overall barrier impairment versus t0, RM1 and RM2 were not considered different from the Negative Control. SE1 and RM3 didn’t directly affect the viable tissue morphology and barrier structure as demonstrated by H&E score and non-increased biotin passage compared to the injured Negative Control.

• Results were consistent to conclude as potential good tolerance for all the emulsifying and rheolo-gy modifying ingredients tested at recommended use levels. Larger dataset are required to draw finer relationship with the ingredient structure.

References

[1] Alépée et al., 2014 State-of-the-Art of 3D Cultures

(Organs-on-a-Chip) in Safety Testing and Pathophysiology Altex 31, 4/14, 441-477.

[2] Gordon S. et al., 2015 Non-Animal Models of Epithelial Barriers (Skin, Intestine and Lung) in

Research, Industrial Applications and Regulatory Toxicology Altex 32(4), 327-378.

[3] Casas J.W. et al. 2013 In vitro human skin irritation test for evaluation of medical devices extracts, Toxicology in vitro Vol 27(8), 2175-2183.

[4] Meloni, M. et al., 2002 The importance of multiple endpoint analysis (MEA) using reconstituted

human tissue models for irritation and biocompatibility assay. Invitox Congress

Proceedings 4, 7.

y The Multiple Endpoints Analysis (MEA) approach permitted to assess ingredients potential effects on epidermis at cellular, morphological and functional level.

y The injured RHE model opens perspectives to better discriminate the overall biocompatibility of composi-tions applied directly on skin with impaired barrier functions such as medical devices or Dermo-Phar-maceutical products. The higher tissue sensitivity could allow to early identify toxicity mechanisms that correlate to infra-clinical reactions.

y Moreover, the methodology offers a promising tool to better understand the interactions of the compositions with the living epidermis, helping thus to optimized the tolerance of ingredients and formulations at early development stages.

Cells viability (Figure 1): • Epidermis abrasion reduced

viability from 100% (NCn) to 79% (NC), indicating that the applied damage occurred

mainly at the SC level without significantly affecting the viable layers.

• Positive Controls (PCn & PC), expected to be slightly irritant at this dosage (selected whith the aim to avoid total tissue destruction and get relevant results on the other parameters), didn’t actually affect viability, both on normal and injured RHE. • Internal Control (IC), considered as non irritant

on normal RHE (86% viability at the same concentration, data not shown), induced a

strong viability decrease on injured RHE (viability=26%), highlighting the

model sensitivity.

TEER (Figure 2): • Abrasion of RHE

induced a significant TEER decrease versus t0 contrary to

normal RHE (NC compared to NCn).• Positive Controls (PCn & PC), as well as Internal Control (IC) resulted in a severe impairment of the epidermal

barrier with around 90% TEER decrease after 24h.

H&E:• Injured Negative

Control tissue (NC) displayed modified SC lamellar structure with

reduced cohesion between the layers (score=0–1) compared to the normal RHE

(score=0).• Positive Control (PC) exhibited significant damage

on injured SC: amorphous appearance + collapsed lamellar structure + clear toxicity at the basal level, with decreasing numbers of layers and keratohyalin granules as well as detachment from the poly-carbonate support (score=2).

• Internal Control (IC) induced marked damage: SC lamellar regular morphology loss + viable

layers detached from polycarbonate + increased cell vacuolization and

picnotic condensed nuclei (score=3).

Biotin staining (Figure 3): • Biotin staining of the Injured

Negative Control tissue (NC) in the granular layer > normal RHE control

(NCn). • Distribution of biotin within the whole injured tissue for the Positive Control (PC), confirming the damage observed in TEER and H&E parameters.

• Huge increase of biotin passage with the Internal Control (IC), in agree-

ment with MTT, TEER and H&E results.

Discussion:• Mechanical abrasion

resulted in loose of skin barrier fonctions at SC level and to a lesser

extend in the viable layer of the epidermis.• Short chain surfactants treated tissues (PC & IC)

presented expected adverse effects, consistent with barrier disruption (decreased TEER + increased biotin

passage) as well as morphological disturbances.• Positive controls (PCn & PC) showed a significant and comparable skin barrier function impairment with or wit-hout SC abrasion, assumed to be linked to the fluctuating irritant status of the SDS depending on the dosage.� Considering this MEA approach, the injured

RHE confirmed higher sensitivity, validating its interest for the next step: tolerance investi-

gation of ingredients dedicated to spe-cific dermal applications.

Figure 3 - Biotin staining(x 40 Magnification)

Materials & Methods

Introduction

Results & DiscussionInjured RHE model validation

Ingredient on injured tissue: Conclusion

Skin Irritation Potential on RHE with Impaired BarrierMickael PUGINIER1, Marisa MELONI2, Laura CERIOTTI2, Mathilde BERGAL3 and Alicia ROSO1

1 SEPPIC, 127 Chemin de la Poudrerie - 81100 Castres, France2 VitroScreen, Via Mosè Bianchi, 103 - 20149 Milano, Italy3 SEPPIC, 22 Terrasse Bellini - Paris La Défense, 92800 Puteaux, France

Keywords: In vitro • Skin • Barrier Impairment • Tolerance • Biocompatibility

Tested ingredients