NAMs: Defined Approaches for Skin Sensitization Studies

Skin sensitization is defined as an allergic response triggered by a substance that induces an overreaction of the immune system, and it is a crucial concern for pharmaceutical and medical device developers. Rigorous testing for skin sensitization is an essential part of preclinical drug development, ensuring both patient safety and regulatory compliance. 

Skin sensitization testing has evolved in recent years, as regulators and drug developers have moved away from in vivo assays towards New Approach Methodologies (NAMs), spurred by a renewed focus on the 3Rs (replacement, reduction, refinement) and concerns over poorly translatable data. 

Developers adapting to an NAM-enabled future must understand the drawbacks while appreciating the benefits of these new technologies and be prepared with a robust plan of action.  

The Adverse Outcome Pathway for Skin Sensitization 

The covalent binding to proteins, —i.e., the cause of skin sensitization— is described in an Adverse Outcome Pathway (AOP) and follows four key events: 

1. Molecular initiating event (KE1) 
2. Keratinocyte activation (KE2) 
3. Dendritic cell activation (KE3) 
4. T-cell activation and a resulting allergic response 

Although no individual NAM assay or technology can completely replace in vivo testing, a Defined Approach (DA) combining multiple assays with in silico and in chemico predictions offers a more comprehensive approach. This rule-based approach can provide predictive performance equivalent or superior to traditional preclinical tests when assessing human skin sensitization. 

How NAMs Measure the Pathway 

Different NAMs can be used to study specific events in the AOP, and the results are then combined to determine whether a chemical will cause skin sensitization. Three of the most common approaches include:  

• Direct Peptide Reactive Assay (DPRA) is one of the in chemico methods used to assess the first stage of the AOP. During the test, the chemical is incubated with synthetic peptides containing cysteine or lysine, amino acids commonly found in skin proteins. After incubation, the amount of peptide remaining is measured, indicating the strength of the chemical reaction. The more the peptide has been depleted, the greater the potential for skin sensitization. 

• KeratinoSens^TM is used to study the second stage of the AOP and to explore the response of keratinocytes, the primary cells in the outer layer of the skin. KeratinoSens^TM measures activation of the Nrf2-ARE signaling pathway, which is triggered when cells respond to chemical or oxidative stress. If there’s an increase in reporter activity, the substance has likely activated a response associated with skin sensitization. Other methods, such as LuSens and EpiSensA, test the same key event through related cellular responses. 

• Human Cell Line Activation Test (h-CLAT) exposes human THP-1 cells to the chemical and measures changes in the surface markers CD86 and CD54. This helps assess the third stage, in which the activation of dendritic cells alerts the immune system to potential threats. Increased expression of surface markers suggests that cells are in an activated state, consistent with a sensitization response. Other assays, such as GARD®skin, U-SENS, and IL-8 Luc, also assess this stage using different biological markers. 

Each of these methods has limitations when used alone, so test results are usually interpreted together. Data from these assays is then combined with in silico prediction tools such as Derek Nexus and the OECD QSAR Toolbox to help predict whether a chemical is likely to be a skin sensitizer.

Defined Approaches Under OECD Test Guideline 497 

The OECD Test Guideline 497 describes two principal approaches for skin sensitization assessment: the 2-out-of-3 (2o3) approach and the integrated testing strategy (ITS). 

The 2o3 approach is primarily used for hazard identification and combines results from assays covering at least two of the first three AOP events. If the first two assays produce the same result, the substance is classified either as a skin sensitizer or not. If the first two assays disagree, a third assay covering the remaining key event is conducted, and the final prediction is determined by the two results that align. The 2o3 battery usually comprises the DPRA, KeratinoSens^TM, and h-CLAT explained above.

The ITS combines quantitative results from an in chemico KE1 assay, an in vitro KE3 assay, and an in silico prediction. A classic ITS uses a combination of DPRA and h-CLAT results, along with a prediction from either Derek Nexus or the OECD QSAR Toolbox. The KE1 and KE3 results are converted into scores ranging from zero to three, and the in silico prediction receives a score of one for a positive result and zero for a negative result. The combined score determines the classification of the chemical: 

• 6-7: Strong sensitizer 
• 2-5: Moderate or weak sensitizer 
• 0-1: Not classified

The Benefits and Limitations of the Defined Approach

Defined Approaches reduce the reliance on in vivo testing by combining results from in chemico, in silico, and in vitro methods. They also provide a broader assessment than any single test, as they are based on several key stages in the skin sensitization AOP. 

Performance data cited in OECD Guideline 497 demonstrates that these approaches outperform the traditionally used Local Lymph Node Assay (LLNA). The 2o3 approach reported accuracy of 83-95%, while the ITS achieved 83-91%, compared with 82% for the LLNA. 

However, each assay has  limitations. For example, DPRA, KeratinoSens^TM, and h-CLAT may not be suitable for substances that require metabolic activation. In addition, the assays each have specific restrictions: 

• DPRA may not be appropriate for substances that react with other amino acids besides cysteine and lysine, complex mixtures, or metals;
• KeratinoSens^TM may not properly address substances that interact with amino acids other than cystine;
• H-CLAT may be unsuitable for complex mixtures and highly lipophilic or insoluble substances.

A Final Word 

NAMs are revolutionizing the way skin sensitization is assessed by replacing reliance on a single in vivo test with evidence from in chemico, in vitro, and in silico methods. When these methods are combined using the Defined Approaches of OECD Test Guideline 497, they can support hazard identification and potency categorization within a transparent, mechanistically based framework. 

Developers seeking to leverage new technology and comply with guidelines on NAM use must choose a platform based on the classic Defined Approaches. This supports a more humane and efficient skin sensitization assessment while upholding a scientifically robust approach to safety evaluation.