Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway

Reinke, Emily N.; Petersen, Elijah J.; Gordon, John D.; Uhl, Rick; Adams, Valerie H.; Rua, Diego; Johnson, Victor J.; Burleson, Gary R.; Strickland, Judy; Gutierrez, Robert; Sprankle, Catherine S.; LaPratt, Tripp; Lehmann, David M.; Germolec, Dori R.; Kleinstreuer, Nicole C.

doi:10.3390/toxics14060511

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Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway

by

Emily N. Reinke

^1,2,*

,

Elijah J. Petersen

^3,*

,

John D. Gordon

⁴,

Rick Uhl

⁴,

Valerie H. Adams

⁵

,

Diego Rua

⁶,

Victor J. Johnson

⁷

,

Gary R. Burleson

⁷,

Judy Strickland

¹

,

Robert Gutierrez

³

,

Catherine S. Sprankle

^1,2,

Tripp LaPratt

^1,2

,

David M. Lehmann

⁸,

Dori R. Germolec

⁹ and

Nicole C. Kleinstreuer

¹⁰

¹

Inotiv, Inc., Morrisville, NC 27560, USA

²

GDIT, Inc., Falls Church, VA 22042, USA

³

National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

⁴

U.S. Consumer Product Safety Commission, Rockville, MD 20850, USA

⁵

Defense Centers for Public Health—Aberdeen, Aberdeen, MD 21005, USA

⁶

DRX2 Consulting LLC, Washington, DC 20001, USA

⁷

Burleson Research Technologies, Inc. Morrisville, NC 27506, USA

⁸

Intertox, LLC, Seattle, WA 98101, USA

⁹

National Institute of Environmental Health Sciences, Durham, NC 27709, USA

¹⁰

Division of Program Coordination, Planning, and Strategic Initiatives, National Institutes of Health, Bethesda, MD 20892, USA

^*

Authors to whom correspondence should be addressed.

Toxics 2026, 14(6), 511; https://doi.org/10.3390/toxics14060511

Submission received: 7 April 2026 / Revised: 4 June 2026 / Accepted: 7 June 2026 / Published: 11 June 2026

(This article belongs to the Special Issue Applicability of New Approach Methodologies (NAMs) to Skin Sensitization: Insights from Industry and Regulatory Perspectives)

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Abstract

The electrophilic allergen screening assay (EASA) uses small-molecule probes as surrogates for skin proteins to measure hapten protein carrier complex formation, Key Event (KE) 1 in the adverse outcome pathway for skin sensitization. Although multiple assays are accepted for this purpose, the EASA has higher throughput and needs less specialized equipment than other commonly used KE1 assays. This validation study assessed transferability of the EASA to naïve laboratories and within- and between-laboratory reproducibility. The predictive capacity of the assay in relation to reference data both from the murine local lymph node assay (LLNA) and, where available, human predictive patch tests, was estimated. The validation study was conducted using performance standards developed for methods that are under evaluation for inclusion in relevant test guidelines. The within- and between-laboratory reproducibility were cumulatively 96% and 85%, respectively. These scores exceeded the performance standard thresholds of 80%. Compared to reference LLNA data, the cumulative EASA results from the four laboratories had an overall sensitivity of 87%, specificity of 76%, and accuracy of 83%. The consensus results among the four laboratories had an overall sensitivity of 85%, specificity of 80%, and accuracy of 83%. For human reference data, the cumulative EASA results had an overall sensitivity of 81%, specificity of 76%, and accuracy of 70%. The EASA predicted sensitizers very well, although it had a slightly higher rate of misclassifying some negative test chemicals as positive with a specificity below the performance standards criterion of 80%. Our findings support further exploring use of the EASA in defined approaches to identify potential skin sensitizers.

Keywords: hapten-carrier formation; haptenation; skin sensitization; Key Event 1; KE1; adverse outcome pathway; molecular initiating event; validation; transferability; reproducibility; performance standards

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MDPI and ACS Style

Reinke, E.N.; Petersen, E.J.; Gordon, J.D.; Uhl, R.; Adams, V.H.; Rua, D.; Johnson, V.J.; Burleson, G.R.; Strickland, J.; Gutierrez, R.; et al. Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway. Toxics 2026, 14, 511. https://doi.org/10.3390/toxics14060511

AMA Style

Reinke EN, Petersen EJ, Gordon JD, Uhl R, Adams VH, Rua D, Johnson VJ, Burleson GR, Strickland J, Gutierrez R, et al. Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway. Toxics. 2026; 14(6):511. https://doi.org/10.3390/toxics14060511

Chicago/Turabian Style

Reinke, Emily N., Elijah J. Petersen, John D. Gordon, Rick Uhl, Valerie H. Adams, Diego Rua, Victor J. Johnson, Gary R. Burleson, Judy Strickland, Robert Gutierrez, and et al. 2026. "Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway" Toxics 14, no. 6: 511. https://doi.org/10.3390/toxics14060511

APA Style

Reinke, E. N., Petersen, E. J., Gordon, J. D., Uhl, R., Adams, V. H., Rua, D., Johnson, V. J., Burleson, G. R., Strickland, J., Gutierrez, R., Sprankle, C. S., LaPratt, T., Lehmann, D. M., Germolec, D. R., & Kleinstreuer, N. C. (2026). Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway. Toxics, 14(6), 511. https://doi.org/10.3390/toxics14060511

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Validation of the Electrophilic Allergen Screening Assay for Detection of Key Event 1 of the Skin Sensitization Adverse Outcome Pathway

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