New OECD TG 497: Interpretation and application of defined approaches for skin sensitization assessment

The advent of requiring multiple non-animal in vitro alternatives for skin sensitization assessments has prompted the Organisation for Economic Co-operation and Development (OECD) to publish new guidance to outline the approaches for data interpretation from multiple non-animal test methods. When used together in specific combinations, these methods will aid with skin sensitization hazard identification and, potentially, potency characterization. A detailed scientific understanding of how best to apply these methods and/or test strategies is needed to address the requirements of regulators for different test chemicals (molecules, chemicals, substances, etc.). This blog will provide an overview of these defined approaches (DAs).

Assessing skin sensitization: The need for DAs

Historically, the assessment for a test chemical’s potential to induce skin sensitization was performed using animal tests, such as the murine local lymph node assay (LLNA). While the mapping out of the adverse outcome pathway (AOP) has paved the way for a whole animal test system to be replaced with a battery of in chemico and in vitro test methods (e.g., direct peptide reactivity assay [DPRA], KeratinoSens^TM and human cell line activation test [h-CLAT] among others), none of these alternatives are considered sufficient standalone replacements of animal data to make conclusions on skin sensitization potential of test chemicals or to provide information on their potency. Data from multiple non-animal test methods need to be used in combination; however, a standardized approach has only recently been adopted at the OECD level.

OECD Test Guideline (TG) 497 (published in June 2021) describes three DAs:

1. The 2 out of 3 (2o3) DA to skin sensitization hazard identification based on in chemico (key event [KE] 1) and in vitro (KE2/KE3) data
    
2. The integrated testing strategy (ITSv1) for United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) potency categorization based on in chemico(KE1), in vitro (KE3) data and in silico (Derek Nexus) predictions
    
3. A modification of the integrated testing strategy, ITSv2, for UN GHS potency categorization based on in chemico (KE1), in vitro (KE3) and in silico (OECD QSAR Toolbox) predictions

Each DA uses data derived from a different combination of validated non-animal test methods (in chemico, in vitro, and sometimes in silico) and interprets these using a fixed data interpretation procedure to provide a prediction on skin sensitization potential (and potency).

Summary of DAs in OECD TG 497

Table adapted from OECD Guideline No. 497: Defined approaches on skin sensitization¹
 
1A, strong sensitizer; 1B, other sensitizer; BA, balanced accuracy (average of sensitivity and specificity); DA, Defined approach; ITS, integrated testing strategy; NA, not applicable; NC, no categorization; Sens, sensitivity (true positive rate); Spec, specificity (true negative rate)

OECD TG 497: DAs for use in skin sensitization hazard determination

OECD TG 497 outlines the 2o3 approach to distinguish whether a test chemical is potentially skin sensitizing. Testing is performed in an undefined order and in up to three of the non-animal test methods mapping to KEs 1-3 of the AOP:

• KE1: protein binding (e.g., DPRA [in chemico])
• KE2: keratinocyte activation (e.g., KeratinoSens^TM [in vitro])
• KE3: dendritic cell activation (e.g., h-CLAT [in vitro])

The data interpretation procedure specifies that two results addressing at least two of the first three KEs of the AOP need to be in agreement to determine the final classification. There will be cases where two assays will be sufficient to determine whether a test chemical is a sensitizer or a non-sensitizer (only if both results are consistent); however, a third assay may have to be conducted to provide a definitive result if the first two assays provide discordant results. There may be instances where results are borderline, and care should be taken when evaluating these individual cases.

OECD TG 497: DAs for skin sensitization potency classification and the rise of k-DPRA

For some industries, if a test chemical is known to cause skin sensitization, it is a regulatory requirement that the degree of potency is assessed. Like skin sensitization hazard identification, potency of a test chemical was traditionally assessed using animals in tests such as the murine LLNA.

The new OECD TG 497 describes two DAs that include the assessment of test chemical potency, which, in addition to in chemico and in vitro, also make use of in silico information sources(Derek Nexus® or OECD QSAR Toolbox). An ITS is applied to separate test chemicals into one of three UN GHS potency categories (Category 1A = strong sensitizers, Category 1B = other sensitizer, and NC = no categorization).

There may be instances where an additional or alternative testing method for potency is required. Kinetic DPRA (k-DPRA), a modification of in vitro DPRA (OECD TG 442C), has been accepted by the OECD and is now included in OECD TG 442C. k-DPRA uses kinetic rates of cysteine peptide depletion to determine and distinguish between two levels of skin sensitization potency (classification, labeling and packaging/GHS subcategories 1A and 1B/non-sensitizer) and can be used (i) as a follow-up test method for subcategorization of chemicals identified as UN GHS Category 1 skin sensitizers or (ii) on its own by using positive results for direct classification of a chemical into UN GHS subcategory 1A, depending on the regulatory framework (OECD TG 442C, 2021). Utilization of k-DPRA would help further decrease the use of animal test methods for assessing skin sensitization.

Reaching a conclusion on skin sensitization potential: Two IATA testing strategies to consider

All data generated from the non-animal test methods utilized by the OECD TG 497 should be considered within the context of an integrated approach to testing and assessment (IATA), that is, in combination with other complementary information.* By combining readouts from a variety of information sources, the predictive capacity of skin sensitization by the assessed test chemical can be enhanced.

A DA to testing and assessment can take the form of a sequential testing strategy (STS) or an ITS.

STS involves a fixed stepwise approach for obtaining and assessing test data. It includes decision steps after application of each test method to inform whether the prediction result provides sufficient evidence to meet the specific regulatory need on its own or whether it can be used with other sources of information within an IATA.

In contrast, ITS involves an approach where information or data from multiple sources are assessed at the same time. Here, a variety of specific methodologies (statistical and mathematical models) are applied to convert inputs from the different information sources into a prediction.

The decision to proceed with either STS or ITS is driven by a client’s priorities. For example, STS may not be suitable if predictions are required within a short time frame. ITS, although higher in cost, may be a preferred approach if speed is an important consideration.

Sometimes, however, there is a third scenario that falls between STS and ITS. In these cases, some information on the test chemical already exists either from previous experience/tests or because of its similarity to other molecules. In this situation, we, at Labcorp, use our extensive knowledge and experience of conducting skin sensitization assessments to provide an approach that is specifically tailored to address your regulatory needs and aligned with your test chemical’s specific properties.

*Information can include chemical structure and physicochemical properties, extrapolations from grouping approaches, QSAR predictions, results from in vitro tests, relevant human or animal data

Skin sensitization assessment: What’s next in innovation?

The past few years have seen significant advances in the development of comparable non-animal test substitutes. Progress in testing strategies has also allowed for greater predictive capacity of a test chemical to cause skin sensitization.

Innovation is essential to match the ever-changing needs and demands of industry and regulators. At Labcorp, we are helping to lead innovation in this area. Labcorp has played an active role in the validation and support of industry adoption of various in vitro technologies. This places us at the forefront of in vitro methodologies and innovations, generating insights that benefit you when partnering with us.

Utilization of k-DPRA would help further decrease the use of animal test methods for assessing skin sensitization, enabling greater contribution toward the three Rs—reduce, replace, refine—a framework used by many regulatory institutions globally for more targeted, humane and ethical animal research. Our team of scientists is currently validating k-DPRA and looking to make this available in late 2022. Adding k-DPRA to the current battery of non-animal skin sensitization assessments we offer would give rise to a fully in vitro testing strategy. Contact us today to find out more.

Conclusion

The OECD has recently published new guidance (OECD TG 497) to describe three DAs to aid with skin sensitization hazard identification and potency characterization. The employment of DAs may be straightforward for “simpler” test chemicals, but its application may be more challenging when faced with those that are more “complex.” A deep understanding in the use of non-animal test methods and the application of the testing strategies is needed to reliably characterize the safety profile of a test chemical to meet regulatory requirements.

Labcorp has a team of experts with significant regulatory and scientific insight that can help navigate the complexity of the various test methods and approaches to assess the skin sensitization potential of test chemicals. We can provide the in vitro, in chemico and in silico testing to support regulatory requirements.

Please contact us for more information on individual assays or approaches to in vitro, in chemico and in silico skin sensitization testing.