In vitro Cutaneous Hypersensitivity test. h-CLAT method (human Cell-Line Activation Test).

(OECD 442E: 2017 Standard)

 

Test accredited by ENAC (National Accreditation Entity).

Test with the Certificate of Good Laboratory Practices (GLPs).

 

 

Contact allergic dermatitis (CAD: Contact Allergic Dermatitis) is an altered immune response to certain chemical substances that are capable of inducing a delayed hypersensitivity response (type IV) in the immune system, with the participation of TDTH cells.

Some chemical substances, due to their molecular structure, are more prone than others to induce a type of delayed hypersensitivity response (type IV). For this reason, when chemical substances that come into contact with the skin are used, it must be ruled out that they can induce a phenomenon of delayed hypersensitivity.

To detect the ability to induce delayed hypersensitivity responses tests with experimental animals have been used, such as: 1) Guinea Pig Maximization Test -GPMT; 2) Büehler occlusive patch method; 3) Local lymph node test in mouse -LLNA: Local Lymph Node Assay. This last test (LLNA) is currently considered the reference method, but encounters the drawback of using H3-thymidine whose incorporation is detected in the local lymph nodes.

The progressive social concern for animal welfare and its use in laboratory tests has been the reason why alternative methods to the use of experimental animals in laboratories have been investigated over the years. In this regard, since March 2013, its use with cosmetic products has been banned in the European Community, so alternative methods must be used.

Among the alternative methods to detect skin sensitization of chemical substances, several methods have been developed:

  1. Activation of dendritic cells. The problem with the use of dendritic cells lies in the fact that the response is conditioned by the origin of the peripheral blood used to obtain the dendritic cells, and in the requirement of a considerable volume of blood.
  2. Activation of surrogate cells of dendritic cells, such as TPH-1 cells, to obviate the afore mentioned problems with dendritic cells. TPH-1 cells are a monocytic human leukemic cell line, which can activate a series of transduction signals with expression of genes and costimulatory molecules after in vitro exposure to skin sensitizing substances.

The h-CLAT test (human Cell-Line Activation Test) detects phenotypic changes, such as the expression of cell surface markers CD86 and CD54, expressed in both dendritic cells and TPH-1 cells. The advantage of these latter cells is that they have an enhanced expression of the CD86 and CD54 markers, when exposed in vitro to sensitizing substances. The correlation between the results obtained with the h-CLAT and LLNA test is around 84%. Some false positive and false negative results may occur because some chemicals induce one of the two markers and other substances another.