Defined Approaches to Identify Potential Skin Sensitizers

Skin sensitizers are substances with the potential to cause allergic contact dermatitis. Allergic contact dermatitis is an allergic reaction that is often initiated through covalent binding of substances to skin proteins.

Potential skin sensitizers can be identified without animal testing using defined approaches to testing and assessment. A defined approach consists of:

• Input data generated from identified methods.
• A data interpretation procedure that is used to translate data produced from a defined set of information sources into a prediction.

An adverse outcome pathway can provide a framework for the development of a defined approach. An adverse outcome pathway is a model that identifies the sequence of biological events required to produce a toxic effect when an organism is exposed to a substance. In vitro methods can be used to observe or measure the biological events and predict whether a substance will produce a toxic effect.

The events that result in allergic contact dermatitis have been described in an adverse outcome pathway for skin sensitization. NICEATM, working with NTP, ICCVAM, and industry scientists, has created and evaluated defined approaches to identify potential skin sensitizers based on this adverse outcome pathway.

Regulatory Applications of Defined Approaches

In June 2021, OECD issued Guideline 497, Defined Approaches on Skin Sensitisation. Drafted and sponsored by NICEATM and international partners, Guideline 497 is the first internationally harmonized guideline to describe a non-animal approach that can be used to replace an animal test to identify skin sensitizers. Guideline 497 was updated in 2023, and a proposal to broaden the number and type of test methods it includes has been accepted by OECD for evaluation in 2024. A poster presented at the 2023 annual meeting of the Society of Toxicology (SOT) (Reinke et al. Alternatives to Mammalian Models II session) summarizes validation of the EpiSensA assay, which may improve upon existing assays that measure keratinocyte activation, Key Event 2 in the skin sensitization adverse outcome pathway, potentially in turn improving defined approaches for this endpoint.

NICEATM has developed the DASS App, a user-friendly web app that uses the defined approaches from OECD Guideline 497 to predict skin sensitization hazard and potency. The most recent updates to the DASS App were presented in a poster (To et al.) at the 2024 annual meeting of the American Society for Cellular and Computational Toxicology.

The defined approaches described in Guideline 497 were evaluated using primarily chemicals relevant to the cosmetics industry. NICEATM evaluated performance of the Guideline 497 defined approaches using 185 substances nominated by NTP, EPA, and CPSC. Results of the study were presented in an oral presentation (Strickland et al., Session S459, "What's New in Skin Sensitization?") at the 12th World Congress on Alternatives and Animal Use in the Life Sciences, and will be described in a technical report to be published in 2025.

Guidance finalized by FDA in June 2023 stated that they will consider skin sensitization data generated using a battery of in silico, in chemico, and in vitro studies that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods.

In April 2018, EPA released a draft Science Policy to reduce animal use by using defined approaches to identify potential skin sensitizers. The draft policy is the result of national and international collaboration among ICCVAM, NICEATM, Cosmetics Europe, the European Union Reference Laboratory for Alternatives to Animal Testing, and Health Canada's Pest Management Regulatory Agency.

Defined Approaches Developed by ICCVAM

Defined approaches developed by NICEATM and ICCVAM use non-animal data to predict skin sensitization hazard and potency. These defined approaches combine data from the following sources:

• Direct peptide reactivity assay (DPRA).
• KeratinoSens assay.
• Human cell line activation test (h-CLAT).
• Read-across predictions generated by the QSAR Toolbox software package.
• Physical properties (e.g., partition coefficients).

The defined approaches were described in papers published in 2016 and 2017:

• Strickland et al. 2016 described prediction of murine local lymph node assay outcomes.
• Strickland et al. 2017 used data from human exposures to predict human skin sensitization hazard.
• Zang et al. 2017 described prediction of human or animal skin sensitization potency, enabling the classification of skin sensitizers as "weak" or "strong" without animal tests.

To expand the potential applicability of these defined approaches to a broader range of chemical types, ICCVAM agencies nominated over 200 chemicals for additional testing in the DPRA, KeratinoSens, and h-CLAT tests. Chemicals tested included pesticide ingredients and formulations, industrial chemicals, and personal care product ingredients. NTP conducted this testing, which began in 2017 and was completed in 2020; reports are being prepared for publication in 2023. Strickland et al. (2022) described the application of defined approaches to potency classification of isothiozolinones, antimicrobial compounds that are widely used in consumer products.

Collaboration With Cosmetics Europe to Evaluate Defined Approaches

NICEATM and Cosmetics Europe collaborated to evaluate multiple defined approaches for skin sensitization safety assessment that had been submitted to OECD as case studies. The collaboration produced two publications:

• Hoffman et al. 2018 describes a database including data from human, animal, and five non-animal tests for 128 substances. The substances in the database have a variety of chemical structures and use categories. The database is a proposed point of reference for the evaluation and development of new non-animal approaches to skin sensitization safety assessment.
• Kleinstreuer et al. 2018 describes an analysis of multiple defined approaches for skin sensitization safety assessment of cosmetic ingredients using the database described above. Many of these approaches were found to perform as well or better than animal methods to predict human skin sensitization hazards.

Collaboration With P&G to Develop an Open-Source Integrated Testing Strategy

An integrated testing strategy is a type of defined approach that relies on:

• Input data generated from identified methods.
• A data interpretation procedure such as a machine-learning model, flowchart, or decision tree, through which the data are evaluated.
• A simultaneous assessment of the input data to arrive at either a hazard prediction or a decision that more testing is needed.

NICEATM and other NTP scientists collaborated with scientists at Procter & Gamble (P&G) to develop an integrated testing strategy to identify potential skin sensitizers. Using data from non-animal tests and other information (e.g., substance solubility), the strategy produces a numerical probability that a chemical should be placed in one of the following skin sensitization hazard classes:

• Strong
• Moderate
• Weak
• Nonsensitizer

This probability could potentially be used to determine if a substance requires hazard labeling. P&G and NTP scientists collaborated to develop this integrated testing strategy using free, publicly available software (Pirone et al. 2014).

P&G updated the integrated testing strategy in 2015. The updated strategy (Jaworska et al. 2015) uses only validated non-animal tests, simplifies the bioavailability inputs, and nearly doubles the size of the database used to derive the previous network. The updated P&G integrated strategy is included in a web tool produced by Edelweiss Connect that can be used to predict a substance's skin sensitization potency.