IN-VITRO Skin sensitization test

Sensitization index

Keratinocytes NCTC2544

Irritation index

HaptenationResorption

Non Sensitizer(No Effect)

Non Sensitizer(Irritant)

Sensitizer0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cyto-toxicity

1

2Fixing

Detection

3

Cytokine production Activation /

Migration

PresentationResorption

Haptenation

Sensitization index

Irritation index

1

2

3

4

Xenobiotic

Endogenous protein

Keratinocyte

Dendritic cell (Langerhans cell)T Lymphocyte

Interleukin 18

1

2Fixing

Detection

3

Cytokine production Activation /

Migration

4

Xenobiotic

Endogenous protein

Keratinocyte

Dendritic cell (Langerhans cell)T Lymphocyte

Interleukin 18

Resorption

Haptenation

Sensitization index

Irritation index

1

2

3

4

Presentation

Inflammation/ Cell damage

1

2Fixing

Detection

3

Cytokine production Activation /

Migration

Xenobiotic

Endogenous protein

Keratinocyte

Dendritic cell (Langerhans cell)T Lymphocyte

Interleukin 18

Resorption

Haptenation

Sensitization index

Irritation index

1

2

3

4

Presentation

4

Inflammation/ Cell damage

Sensitization index

Keratinocytes NCTC2544

Irritation index

Haptenation

Non Sensitizer(No Effect)

Non Sensitizer(Irritant)

Sensitizer0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cyto-toxicity

Resorption

Resorption:During flux

Test molecule

Resorption:During flux

The resorption kinetics was determined by measuring the appearance of the molecules

in the donor compartment

Resorption

Sensitization index

Non Sensitizer(No Effect)

Non Sensitizer(Irritant)

Sensitizer0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cyto-toxicity

Keratinocytes NCTC2544

Irritation index

Haptenation

Haptenation:

Solution of the test molecule was mixed with Human serum

albumin

Centrifuge for 2H at 10.000g

Addition of urea

powder

Filtrate was quantified

Test molecule

Endogenous protein

Centrifuge for 2H at 10.000g

Incubated 4H at 20°C

Incubated 1H at 20°C

Irritation index

Haptenation

Non Sensitizer(No Effect)

Non Sensitizer(Irritant)

Sensitizer0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cyto-toxicity

Resorption

Rinsing

Antibodies are deposited in well

Sensitization:

Sensitization:

Antigens are added

Sensitization:

An enzyme-linked antibodies that

can react with the antigen is added

Sensitization:

The colorless substrate for the enzyme is then

added

Sensitization:

Development of color indicates the

presence of the antigen

This coloration was measured by

spectrophotometer

Sensitization index

Keratinocytes NCTC2544

Non Sensitizer(No Effect)

Non Sensitizer(Irritant)

Sensitizer0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cyto-toxicity

HaptenationResorption

Irritation index

Irritation:

Culture cells in micro plate

Remove medium and add MTT solution

Incubate 3HRemove MTT

and add isopropanol

Read with micro plate Reader after incubation

Add to the cell culture the xenobiotic

Sensitization index

Keratinocytes NCTC2544

HaptenationResorption

Irritation index Non Sensiti

zer

(No Eff

ect)

Non Sensitize

r

(Irrit

ant)

Sensiti

zer

0

0.5

1

1.5

2

Results interpretation

Factor A Resorption

Factor BHaptenation

Factor C↑IL18

Factor DAcute Cytotox-icity