IN-VITRO Skin sensitization test

Haptenation Resorption Irritation index Sensitization index Keratinocytes NCTC2544

Activation / Migration 1 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

Inflammation/ Cell damage Activation / Migration 1 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

Inflammation/ Cell damage Activation / Migration 1 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

Haptenation Resorption Irritation index Sensitization index Keratinocytes NCTC2544

Resorption: During flux Test molecule

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

Haptenation Resorption Irritation index Sensitization index Keratinocytes NCTC2544

Filtrate was quantified Haptenation: Filtrate was quantified Centrifuge for 2H at 10.000g Centrifuge for 2H at 10.000g 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

Resorption Haptenation Irritation index

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

Sensitization: Antigens are added

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

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

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

Haptenation Resorption Irritation index Sensitization index Keratinocytes NCTC2544

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

Haptenation Resorption Irritation index Sensitization index Keratinocytes NCTC2544