Roadmap Implementation: Skin Sensitization

The SSWG and representatives from other U.S. federal agencies published a scoping document to identify U.S. agency information requirements, needs, and decision contexts for skin sensitization testing (Strickland et al. 2018). A white paper characterizing international regulatory requirements for skin sensitization testing (Daniel et al. 2018) was developed through interactions between ICCVAM and NICEATM and their international counterparts.

ICCVAM and NICEATM facilitate public-private partnerships to share data and information on the development and evaluation of alternative methods for skin sensitization assessment. For example, EPA established a stakeholder group to discuss the development, evaluation, and implementation of alternatives for the “six-pack” of acute toxicity tests required for pesticide registration. Activities within this group have informed EPA efforts toward adoption of alternative methods.

To provide high-quality data to support the development of new testing approaches, NICEATM is working with stakeholders to expand its database of in vivo and in vitro skin sensitization testing data. Contact NICEATM if your organization has skin sensitization data that it would like to share.

• NICEATM compiled high-quality reference data for the local lymph node assay (LLNA) from the published literature and data submissions. NICEATM has also compiled data for in chemico and in vitro methods from the public literature. These data sets are available through the NICEATM Integrated Chemical Environment (ICE).
• EPA provided NICEATM with data submitted to support Federal Insecticide, Fungicide, and Rodenticide Act registrations of pesticide formulations and active ingredients. These data, which include LLNA data, are also available via ICE.
• NTP tested approximately 200 chemicals of interest to ICCVAM agencies in three in chemico/in vitro assays: the direct peptide reactivity assay, the KeratinoSens assay, and the human cell line activation test. Testing was completed in 2020 and data will be made available via ICE.
• NICEATM worked with the German Federal Institute for Risk Assessment (BfR) to compile and curate a database of human predictive patch test data (Strickland et al. 2023). These data, available via ICE, will serve as reference data for assessing performance of defined approaches and facilitate analysis of reliability and variability of human data. Herzler et al. (2024) describes how these data might be applied to improve potency classification of skin sensitizers.
• NICEATM and ICCVAM are currently identifying additional potential sources of skin sensitization data.

ICCVAM activities to support the regulatory acceptance and facilitate the use of non-animal approaches include:

• Collaboration with Cosmetics Europe to evaluate the performance of defined approaches (described in "Developing IATA and Defined Approaches" below).
• Validation of the electrophilic allergen screening assay. A poster presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences (Reinke et al., Human-Centered Biomedical Research session) describes outcomes of the validation study, which involved four ICCVAM agencies.
• Serving on a peer review panel for an OECD-coordinated study evaluating the use of the kinetic direct peptide reactivity assay for classifying substances for skin sensitization potency.
• Submission of comments on OECD test guidelines for the direct peptide reactivity assay, the KeratinoSens assay, and the human cell line activation test.
• Serving on the OECD expert group for skin sensitization since 2015.
• Leading the OECD project to establish a guideline for defined approaches to skin sensitization (described in “Developing IATA and Defined Approaches” below).

NICEATM and ICCVAM agencies are considering a number of different approaches to reduce animal use for required testing. These include the use of integrated approaches to testing and assessment (IATA) and defined approaches to enable prediction of skin sensitization hazard through high-throughput screening assays and in silico models.

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 effort produced a set of human, animal, and non-animal test data for 128 chemicals (Hoffman et al. 2018) and an analysis of multiple defined approaches for skin sensitization assessment of cosmetics ingredients (Kleinstreuer et al. 2018). More recently, NICEATM and the consumer products company Unilever collaborated to develop the Skin Allergy Risk Assessment-Integrated Chemical Environment (SARA-ICE) Model. This defined approach uses a combination of human, mouse, and new approach methodology (NAM) data to generate a numerical prediction of skin sensitization potency. The SARA-ICE Model was described in a presentation (Maxwell et al., Session S459, "What's New in Skin Sensitization") at the 12th World Congress on Alternatives and Animal Use in the Life Sciences.

In April 2018, EPA released a draft Science Policy to reduce animal use through defined approaches to identify potential skin sensitizers. Under this policy, EPA's Office of Pesticide Products and Office of Pollution Prevention and Toxics will accept submissions for single chemicals tested using non-animal methods used in two specific defined approaches. The draft policy is the result of the Cosmetics Europe collaboration described above as well as other collaborations among ICCVAM, NICEATM, the European Union Reference Laboratory for Alternatives to Animal Testing, and Health Canada’s Pest Management Regulatory Agency. Similarly, guidance finalized by the U.S. Food and Drug Administration 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.

ICCVAM coordinates evaluation of skin sensitization test methods in part through its participation in ICATM. SSWG members participated in an October 2016 ICATM workshop on skin sensitization. The work products from the workshop included:

• A white paper (Daniel et al. 2018) characterizing international regulatory requirements for skin sensitization testing.
• A position paper (Casati et al. 2017) summarizing workshop outcomes and ICATM recommendations.
• An OECD guideline for defined approaches to testing and assessment for skin sensitization (described in detail below).

NICEATM also participated in the peer review of a validation study of the EpiSensA assay conducted by the Japanese Center for the Validation of Alternative Methods. The validation study is described in a poster (Reinke et al. Alternatives to Mammalian Models II session) presented at the 2023 SOT annual meeting. The EpiSensA assay may improve upon other assays that measure the skin sensitization key event of keratinocyte response. A proposal for a test guideline for the EpiSensA assay is under consideration by OECD.

NICEATM and ICCVAM actively participate in the OECD Test Guidelines Programme, which supports the development of IATA and defined approaches. Recent activities include:

• Serving on the drafting group for Guidance Document 255, Guidance Document on the Reporting of Defined Approaches to be Used within Integrated Approaches to Testing and Assessment, and Guidance Document 256, Guidance Document on the Reporting of Defined Approaches and Individual Information Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitisation.
• Drafting and sponsoring OECD Guideline 497, Defined Approaches on Skin Sensitisation, adopted in June 2021. This 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. NICEATM has developed a web application to facilitate using the Guideline 497 defined approaches; the application is described in a poster (To et al., Regulatory Acceptance and Global Harmonization session) presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences.
• Leading proposals submitted to OECD for acceptance of additional defined approaches and test methods to be used within defined approaches.

NICEATM and ICCVAM are collaborating with stakeholder groups to convene events to raise awareness about the guideline and promote best practices for implementation of defined approaches to skin sensitization testing and assessment.

ICCVAM evaluations (Kleinstreuer et al. 2018; Strickland et al. 2016; Strickland et al. 2017; Zang et al. 2017) indicate that most defined approaches evaluated so far perform better than standard animal methods in predicting human skin sensitization hazard and potency.

The Division of Translational Toxicology within NIEHS has completed testing 185 substances of regulatory interest to ICCVAM agencies (including mixtures) using the in vitro direct peptide reactivity assay, KeratinoSens assay, and human cell line activation test. Reports on this testing are being prepared for publication in 2023. Defined approaches that use these test methods will be evaluated for predictive performance across this chemical set and for applicability to the different regulatory programs. The results of this project were presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences (Strickland et al., Session S459, "What's New in Skin Sensitization"), and a report is in preparation that will describe the data set in detail. Further efforts in this area were described in Strickland et al. (2022a), which summarizes a public-private collaboration that evaluated performance of these test methods individually and in the defined approach for testing of 27 agrochemical formulations.

NICEATM and ICCVAM played key roles in the development of draft human health and ecological risk assessments issued by EPA in 2020. These risk assessments evaluate dermal sensitization potential for a group of antimicrobial chemicals known as isothiazolinones. In a May 2020 announcement, EPA requested comment on the use of an in vitro and artificial neural network-based defined approach to determine points of departure used in the isothiazolinone draft risk assessments instead of using laboratory animal data. NICEATM analyzed the in vitro data and ran the artificial neural network-based defined approach to provide quantitative potency predictions used to determine points of departure (Strickland et al. 2022b). The Skin Sensitization Expert Group reviewed the NTP testing report and the NICEATM analyses before data were provided to EPA for development of the risk assessments. The use of in vitro and in chemico assays and neural network-based defined approaches is the first use of such information in regulatory risk assessment.

A common theme that has emerged from past NICEATM workshops is the need for training opportunities to inform industry and regulatory staff about the use and interpretation of in vitro and in silico data and defined approaches. Thus, efforts to facilitate regulatory acceptance and use of defined approaches and other alternative methods will include the development of training materials and technical support to assist agencies in issuing guidance.