SEPAWA 2011 - Gematria Test Lab
SEPAWA 2011 - Gematria Test Lab
SEPAWA 2011 - Gematria Test Lab
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58 <strong>SEPAWA</strong> Congress 12-14 October, Fulda, Germany<br />
The Radical Status:<br />
a new method for the determination<br />
of the barrier function of skin<br />
Katinka Jung, Marietta Seifert, Thomas Herrling<br />
<strong>Gematria</strong> <strong>Test</strong> <strong>Lab</strong> GmbH, Berlin, Germany<br />
Wolfgang Pittermann, Bettina Blume<br />
Simred GmbH, Großburgwedel, Germany<br />
<strong>SEPAWA</strong> <strong>2011</strong><br />
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Introduction – skin irritants and skin protection<br />
SUN CHEMICALS DETERGENTS ACIDS/ALCALINE<br />
• Several chemical substances / physical influences may damage<br />
the skin.<br />
• Due to the different irritants, the universal protectant does not exist.<br />
• The skin protection should not only isolate the skin‘s surface, but<br />
should be based on a synergistic interaction between the barrier<br />
and the reservoir function of the skin and the protectant.<br />
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How to measure skin irritation and skin protection?<br />
20 % of the occupational diseases are skin diseases caused by<br />
chemicals (BG Chemie 2009)<br />
Due to the variety of noxa and the variety of individual<br />
sensitivities, the quantitative determination of the skin damage<br />
in vivo is difficult.<br />
Skin models should be used.<br />
Significant parameters should be found.<br />
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CONTENT<br />
Parameters for skin damage/irritation<br />
Free Radical Status of the skin<br />
Different skin models<br />
Correlation between different parameters<br />
Antioxidative Power of the skin<br />
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Introduction – skin irritants and skin protection<br />
Free Radical Status and skin irritation<br />
Radical formation in<br />
the control skin<br />
(treated with<br />
protector)<br />
Radical-boosting<br />
due to irritants<br />
Skin Protector and<br />
irritant<br />
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Generation and propagation of free radicals inside the skin:<br />
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Radicals are formed where the chemical or physical noxa are present<br />
Source of oxidative stress<br />
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Example 1: Dihydroxyacetone<br />
Induces the Maillard reaction<br />
Radical intermediates are formed inside the epidermis/dermis<br />
(AGE-products)<br />
UV-radiation induces more free radicals in the DHA-treated skin<br />
Maillard reaction<br />
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Example 2 : Detergents<br />
Induce membrane disorder and dehydration<br />
Radical intermediates are formed inside the sc /epidermis<br />
UV-radiation induces more free radicals in the SDS-treated skin<br />
detergents<br />
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Example 3: UV radiation<br />
Induces oxygen free radicals and lipid peroxides inside the skin.<br />
UV radiation<br />
spectrum<br />
wave length λ<br />
energy ε<br />
UV C<br />
UV B<br />
UV A<br />
C<br />
B<br />
A<br />
100 nm – 280 nm<br />
280 nm – 320 nm<br />
320 nm – 400 nm<br />
12.3 – 9.8 eV<br />
9.8 – 9.2 eV<br />
9.2 – 8.1 eV<br />
IR<br />
760 nm – 1 mm<br />
1.6 – 10 -3 eV<br />
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4. RSF - principle<br />
UV<br />
O 2<br />
.- .<br />
OH L . LOO . R .<br />
Direct<br />
measurement (ESR)<br />
inflammation<br />
pigmentation<br />
DNA-damage<br />
Collagen disorder<br />
Protein disorder<br />
Apoptosis ...<br />
Measurements of<br />
biological endpoints (MED,<br />
PPD,...)<br />
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The Radical Status - principle<br />
Skin biopsy<br />
(pig ear, cow udder,<br />
Human skin, 3D skin models,…)<br />
Application of protective<br />
formulations<br />
- Sunscreens<br />
- anti-ageing products<br />
- antioxidants<br />
Application of noxa<br />
- coolants, acids, detergents<br />
- electromagnetic radiation<br />
- temperature<br />
<strong>Lab</strong>elling with a radical indicator<br />
Induction of free radicals via UV-radiation<br />
Quantification of these FR by ESR spectrometry directly in the skin<br />
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Electron Spin Resonance spectroscopy<br />
Quantitative and qualitative analysis of FR / ROS.<br />
Quantitative and qualitative detection of antioxidative reactions.<br />
Inpedendence from physical – chemical properties of the sample.<br />
Rapid and covenient analysis.<br />
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Choice of the skin model<br />
Pig skin<br />
BUS model<br />
No aerobic metabolism<br />
Active aerobic metabolism<br />
Biochemical parameters (MTT, PGE 2 )<br />
Repetitive application<br />
Similar to human skin<br />
(mammalian)<br />
Long application period(5 h)<br />
Repetetive application<br />
similar to human skin (mammalian)<br />
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Dose-effect relationship<br />
PIG SKIN<br />
Triton-X 100<br />
260<br />
240<br />
220<br />
200<br />
FR, %<br />
180<br />
160<br />
140<br />
120<br />
100<br />
80<br />
0,36788 1 2,71828 7,38906<br />
ED 50<br />
= 1,19%<br />
Triton X, %<br />
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PIG SKIN<br />
Nr.<br />
Code<br />
FREE<br />
RADICALS, %<br />
Effective<br />
concentration<br />
of Triton X<br />
1<br />
Untreated skin<br />
100 ± 41<br />
0<br />
2<br />
Treated with 10 % Triton X<br />
205 ± 45<br />
10 %<br />
3<br />
Placebo (O/W)<br />
100 ± 31<br />
0<br />
4<br />
10% Triton X and Placebo<br />
165 ± 36<br />
1,19 %<br />
5<br />
10% Triton X and Product A<br />
181 ± 54<br />
1,24 %<br />
6<br />
10% Triton X and Product B<br />
89 ± 6 *<br />
0<br />
Tab. 2<br />
* Different from the treated skin (10% Triton, negative control) at a significance (p
PIG SKIN<br />
Excess in FR<br />
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250<br />
18<br />
200<br />
150<br />
100<br />
50<br />
0<br />
10% Triton X<br />
no Placebo Product A Product B<br />
treatment<br />
free radicals, %<br />
Inhibition<br />
of FR
700<br />
PIG SKIN<br />
600<br />
0,5 % NaOH<br />
500<br />
free radicals, %<br />
400<br />
300<br />
200<br />
100<br />
0<br />
without P 1 P 2 P 3 P 4 P 5 P 6<br />
protectors<br />
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Correlation between inducible FR and cell vitality:<br />
BUS model<br />
Cytotoxic and<br />
inflammatory effects<br />
measured by MTT / PGE 2<br />
-<br />
test<br />
Free radicals<br />
measured by ESR<br />
spectroscopy<br />
MTT score<br />
PGE 2 score<br />
Radical Status of the skin<br />
Protection factor<br />
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BUS model<br />
Noxa: coolant<br />
Protector: HERWESAN AiO,<br />
2mg/cm 2 , 20 min prior to the application of the coolant<br />
260<br />
240<br />
220<br />
Graph<br />
correlation:<br />
R = 0,997<br />
2,5<br />
2,0<br />
Free Radicals (%)<br />
200<br />
180<br />
160<br />
140<br />
120<br />
100<br />
80<br />
1,5<br />
1,0<br />
0,5<br />
0,0<br />
-0,5<br />
MTT score<br />
untreated<br />
Protector<br />
Coolant<br />
Protector + Coolant<br />
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nOctan 5h<br />
Protector + nOctan 5h<br />
SDS 10% 5h<br />
Protector + SDS 5h<br />
Triton X 10% 5h<br />
Protector+ Triton X 5h<br />
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Protector + Toluol 5h<br />
Toluol 5 h<br />
Protector 5 h<br />
control 5h<br />
350<br />
300<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
Toluol<br />
controls<br />
noxe treatments<br />
protection + noxe BUS model<br />
n Octan<br />
SDS<br />
Triton X<br />
free radicals, %
Protection factor: FR (unprotected skin)<br />
FR (protected skin)<br />
BUS model<br />
Irritant<br />
Toluol<br />
n-Octan<br />
SDS 10%<br />
Triton-X 10%<br />
Protection Factor of the Skin<br />
Protection Product<br />
After 1 h<br />
5,7<br />
1,4<br />
2,8<br />
1,7<br />
After 5 h<br />
1,5<br />
5,1<br />
1,1<br />
1,5<br />
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4<br />
3<br />
MTT score<br />
24<br />
P + Triton X<br />
Triton-X<br />
P + Toluol<br />
Toluol<br />
protector<br />
control<br />
BUS model<br />
220<br />
200<br />
180<br />
160<br />
140<br />
120<br />
Free radicals, %<br />
100<br />
80<br />
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1<br />
0
5<br />
4<br />
5h<br />
BUS model<br />
total score MTT+PGE 2<br />
3<br />
2<br />
1<br />
0<br />
R = 0,98<br />
80 100 120 140 160 180 200 220<br />
FR<br />
Good correlation between the inducible FR and the cell vitality<br />
and irritation parameters.<br />
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time, t<br />
26<br />
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Skin Antioxidative Retension (SAR)<br />
Penetration time<br />
produkt<br />
ESR measurement<br />
PIG SKIN<br />
Penetration time<br />
marker<br />
before UV<br />
0,3 MED UV<br />
0,5 MED UV<br />
0,7 MED UV<br />
1 MED UV<br />
1 MED UV + sunscreen<br />
1 0 0<br />
8 0<br />
6 0<br />
4 0<br />
2 0<br />
0<br />
rel. SAP, %
BUS model<br />
<strong>SEPAWA</strong> <strong>2011</strong><br />
120<br />
100<br />
27<br />
80<br />
60<br />
40<br />
20<br />
0<br />
**<br />
**<br />
control Triton X Toluol<br />
treatment<br />
Antioxidative Power of the skin, SAP (%)
Conclusion<br />
• The Radical Staus Factor is a valuable tool for the<br />
determination of the skin‘s defence system.<br />
• The inducibility of free radicals is directly related to<br />
the cell vitality, the inflammation process and the<br />
antioxidative systems of the skin.<br />
• The Radical Status is able to detect both<br />
toxic/negative and the protective/positive effect of<br />
topically applied substances, raw materials, or final<br />
product.<br />
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Thank you for your kind attention!<br />
<strong>SEPAWA</strong> <strong>2011</strong><br />
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Pig ear Application of sunscreen 20 min time<br />
ESR tissue cell<br />
Biopsy 4 mm<br />
UV irradiation<br />
ESR measurement<br />
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Collaborations<br />
• BASF SE<br />
• Botanica GmbH, Schweiz<br />
• Crodarom, France<br />
• Forschungsinstitut für molekulare Pharmakologie FMP, Berlin, Deutschland<br />
• GABA International, Schweiz<br />
• GfN-Selco GmbH, Deutschland<br />
• Hallstar, USA<br />
• Henkel KGaA, Deutschland<br />
• Kuhs GmbH, Deutschland<br />
• LEO Pharma, Denmark<br />
• Lipotec S.A., Spain<br />
• Mann&Schröder GmbH, Germany<br />
• Marico s.l., India<br />
• MAXIM Markenprodukte GmbH, Deutschland<br />
• Merck KGaA, Deutschland<br />
• Mibelle Cosmetics AG, Schweiz<br />
• Qenax AG, Schweiz<br />
• ROVI Cosmetics International GmbH, Deutschland<br />
• Schering-Plough, USA<br />
• Semper Idem GmbH, Deutschland<br />
• Spirig Pharma AG, Schweiz<br />
• SLI Chemicals GmbH<br />
• The Dial Corp., USA<br />
• Unilever Thai Trading Lim., Thailand<br />
• Weleda AG<br />
• Universitätsklinik Charité, Dermatologie, Berlin, Deutschland<br />
• Technische Hochschule Berlin, Deutschland<br />
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