In the not-too-distant past, the determination of whether or not a material was a skin sensitizing agent was achieved through testing conducted on either lab animals (most commonly guinea pigs) or human subjects. Advances were made along the way, and eventually, mice replaced guinea pigs in conducting the Local Lymph Node Assay. Today, several in vitro and in chemico assays are used in skin sensitization testing, thus eliminating the need to conduct testing on lab animal or human subjects.
The REACH Regulation (Annex VII, section 8.3.1) includes a requirement for in chemico and in vitro assays as an initial step for determining skin sensitization. Manufacturers and formulators are now able to utilize such tests to determine the sensitization potential of their compounds. Many are now using this integrated skin sensitization testing strategy for skin sensitization hazard identification and classification as per the Globally Harmonized System of Classification and Labelling of Chemicals. Within the United States, however, in vitro and in chemico assays alone may not be sufficient for safety substantiations. This may change in the future, but for now, FDA still views animal, or human testing as viable and necessary elements of safety testing.
The following assays are frequently used for skin sensitization testing.
Direct Peptide Reactivity Assay (DPRA) (OECD TG 442C)
A skin sensitizer, as defined by the United Nations Globally Harmonized System, is a substance that, after skin contact, will elicit an allergic response. In humans, multiple exposures are required for the phenomenon to occur. As a result of initial exposures, a sensitizing substance may bind to proteins, increasing the possibility of a sensitization reaction. This potential binding is the first key event within skin sensitization, adverse outcome pathway (AOP). The greater the binding, the greater the sensitization potential of the substance.
The DPRA is an in chemico test which duplicates this in vivo binding and quantifies the reactivity of substances towards synthetic peptides containing either cysteine or lysine. The percent peptide depletion values of the cytsteine and lysine are then evaluated to place the substance in question into 1 of 4 reactivity classes (no or minimal reactivity, low reactivity, moderate reactivity and high reactivity). Subsequently, predictions can be made as to the relative sensitization potential of various substances.
High-Performance Liquid Chromatography (HPLC), and more recently Ultra-High-Performance Liquid Chromatography (UPLC) is the technique used to make these determinations.
ARE-Nrf2 Luciferase Test Method (OECD TG 442D)
In humans, multiple exposures to a source are required for skin sensitization to occur. As a result of initial exposures, a sensitizing substance may initiate keratinocyte induction of a cytoprotective gene pathway linked to skin sensitization. This is the second key event within the skin sensitization, adverse outcome pathway (AOP). The greater the keratinocyte induction, the greater the sensitization potential of the substance.
In this in vitro test method, the gene expression of antioxidant/electrophile response element (ARE)-dependent pathways are measured using luminescence detection. To this end, an immortalized, adherent cell line, derived from human keratinocytes, containing a luciferase reporter gene under the control of the antioxidant response element of human AKR1C2 genes are utilized. The AKR1C2 gene has been well documented as being up-regulated by skin sensitizers.
The luciferase gene is contained in this cell line and it is under the transcriptional control of a constitutive promoter fused with the ARE element. Sensitizers activate endogenous Nrf2 dependent genes and the luciferase signal reflects that. The dependence of the luciferase signal in the recombinant cell line on Nrf2 has been well documented. The luminescence elicited by gene induction, as measured utilizing a luminometer, is then quantified.
Substances are considered positive (skin sensitizing agent) in this test method if they induce a statistically significant induction of the luciferase activity above a given, defined threshold.
Human Cell Line Activation Test (h-CLAT) (OECD TG 442E)
In humans, multiple exposures to a source are required for skin sensitization to occur. As a result of initial exposures, a sensitizing substance may initiate the activation of dendritic cells. This is the third key event within the skin sensitization, adverse outcome pathway (AOP). Using flow cytometry, the h-CLAT assay measures the expression of specific cell surface markers linked to dendritic cell maturation (CD86 and CD54).
This in vitro assay utilizes a human monocytic leukemia cell line (THP-1 cells). Following a 24 hour exposure to the test substance, changes of cell surface marker expression are quantified. Dendritic cell activation may be mimicked by these surface molecules, which are typical markers of monocytic THP-1 activation. This phenomenon plays a critical role in T-cell priming.
Following cell staining with fluorochrome-tagged antibodies, flow cytometry is used to quantify the changes in cell surface marker expression. As upregulation of surface marker expression can occur at sub-cytotoxic concentrations, cytotoxicity measurements are concurrently conducted.
The relative fluorescence intensity of the surface markers of cells exposed to a test substance compared to those of cells exposed to solvent/vehicle controls are determined and used to differentiate between sensitizers and non-sensitizers.