1. IN-VITRO Skin
sensitization test

2. Haptenation Resorption Irritation index Sensitization index
Keratinocytes NCTC2544

3. Activation / Migration1 2
Fixing
Detection 3
Cytokine production
Activation / Migration
Presentation
Xenobiotic
Endogenous protein
Keratinocyte
Dendritic cell
(Langerhans cell)
T Lymphocyte
Interleukin 18
Resorption
Haptenation
Sensitization index
Irritation index 1 2 3 4

4. Inflammation/ Cell damage Activation / Migration1 2
Fixing
Detection 4
Inflammation/ Cell damage 3
Cytokine production
Activation / Migration
Presentation
Xenobiotic
Endogenous protein
Keratinocyte
Dendritic cell
(Langerhans cell)
T Lymphocyte
Interleukin 18
Resorption
Haptenation
Sensitization index
Irritation index 1 2 3 4

5. Inflammation/ Cell damage Activation / Migration1 2
Fixing
Detection 4
Inflammation/ Cell damage 3
Cytokine production
Activation / Migration
Presentation
Xenobiotic
Endogenous protein
Keratinocyte
Dendritic cell
(Langerhans cell)
T Lymphocyte
Interleukin 18
Resorption
Haptenation
Sensitization index
Irritation index 1 2 3 4

7. Haptenation Resorption Irritation index Sensitization index
Keratinocytes NCTC2544

8. Resorption:
During flux
Test molecule

9. Resorption:
During flux
The resorption kinetics was determined by measuring the appearance of the molecules in the donor compartment

10. Haptenation Resorption Irritation index Sensitization index
Keratinocytes NCTC2544

11. Filtrate was quantified
Haptenation:
Filtrate was quantified
Centrifuge for 2H at g
Centrifuge for 2H at g
Addition of urea
powder
Incubated 1H at 20°C
Incubated 4H at 20°C
Solution of the test molecule was mixed with Human serum albumin
Test molecule
Endogenous protein

12. Resorption
Haptenation
Irritation index

13. Antibodies are deposited in well
Sensitization:
Rinsing
Antibodies are deposited in well

14. Sensitization:
Antigens are added

15. An enzyme-linked antibodies that can react with the antigen is added
Sensitization:
An enzyme-linked antibodies that can react with the antigen is added

16. The colorless substrate for the enzyme is then added
Sensitization:
The colorless substrate for the enzyme is then added

17. Sensitization: This coloration was measured by spectrophotometer
Development of color indicates the presence of the antigen

18. Haptenation Resorption Irritation index Sensitization index
Keratinocytes NCTC2544

19. Irritation: Incubate 3H Read with micro plate Reader after incubation
Add to the cell culture the xenobiotic
Remove medium and add MTT solution
Culture cells
in micro plate
Read with
micro plate
Reader after incubation
Remove MTT
and add
isopropanol
Incubate 3H

20. Haptenation Resorption Irritation index Sensitization index
Keratinocytes NCTC2544