Table of Contents

In Vitro Skin Sensitization [Human Cell Line (U937-U-SENS) Activation Test (h-CLAT)] (OECD 442E)

The Human Cell Line Activation Test (h-CLAT) is another promising in vitro method for predicting skin sensitization potential, addressing the third key event in the Adverse Outcome Pathway (AOP): dendritic cell activation. As with the other assays you mentioned, it offers an alternative to animal testing. Here’s a breakdown of h-CLAT:

Test materials:

Human monocytic leukemia cell line (U937): These cells differentiate into dendritic cells in the presence of specific cytokines.
Test chemicals: Chemicals being tested for skin sensitization potential are dissolved in an appropriate solvent.
Positive and negative controls: Known skin sensitizers and non-sensitizers are included as controls.

Test Procedure:

Cell differentiation: U937 cells are treated with cytokines to differentiate into dendritic cells.
Treatment: Differentiated dendritic cells are then treated with the test chemicals for 24-48 hours.
Flow cytometry: Cells are stained with antibodies against specific cell surface markers associated with dendritic cell activation (e.g., CD86 and CD54).
Analysis: The expression of activation markers is quantified using flow cytometry and compared between treated and control cells.

Data analysis:

The percentage of cells expressing activation markers is calculated.
The mean fluorescence intensity (MFI) of activation markers is also measured.
The fold induction in activation markers is calculated by dividing the percentage of positive cells or MFI of the treated cells by the corresponding values of the untreated control cells.
The fold induction is compared to a pre-defined cut-off value to categorize the test chemical as a sensitizer or non-sensitizer.

Interpretation of results:

Chemicals with a fold induction above the cut-off value are considered potential skin sensitizers.
Chemicals with a fold induction below the cut-off value are considered non-sensitizers.

Advantages of h-CLAT:

Mechanistic: h-CLAT targets a key event in the AOP for skin sensitization, providing mechanistic insights into the sensitizing potential of chemicals.
Animal-free: h-CLAT is an in vitro assay, which means it does not require the use of animals.
Fast and cost-effective: h-CLAT is a relatively quick and inexpensive test compared to animal-based skin sensitization assays.
High throughput: h-CLAT can be used to test a large number of chemicals in a short period of time.

Limitations of h-CLAT:

Does not address all key events in skin sensitization: h-CLAT only assesses the third key event in skin sensitization. It does not address other key events, such as T cell activation and sensitization.
False positives and negatives: h-CLAT may give false positive or negative results for some chemicals.
Limited to certain types of chemicals: h-CLAT may not be suitable for testing all types of chemicals, such as metals and metal salts.

h-CLAT is a valuable tool for screening chemicals for skin sensitization potential. It is a relatively simple and inexpensive test that can be used to quickly identify potential sensitizers. However, like other in vitro methods, it has limitations and its results should be interpreted in conjunction with other information, such as the chemical structure and known skin sensitization hazards of similar chemicals.