Generation of Historical Control Database, Acceptance and Interpretation Criteria in a Comparative T4-Glucuronidation Assay Using Primary Human and Rat Hepatocyte (TruVivo®) for Regulatory Acceptance

Ahtasham Raza¹, Stephanie Kellum¹, Justin Brown¹, Suhair Al Janabi¹, Lauren Kent², Marco Corvaro³, Jinlan Dong¹, Jessica LaRocca⁴, Edward LeCluyse⁵, Raja Settivari¹ and Shadia M. I. Catalano¹. 

¹Corteva Agriscience (DE, USA), ²Corteva Agriscience (Oxford, U.K), ³Corteva Agriscience (Cremona, Italy), ⁴Corteva Agriscience (IN, USA), ⁵LifeNet Health LifeSciences (NC, USA).

Introduction:

Evaluating the human relevance of liver-mediated thyroid hormone disruption remains a significant challenge within the European Union (EU) regulatory framework. Currently, no in vitro methodologies exist with established guidelines, acceptance criteria, or interpretation frameworks that can quantitatively assess such disruption across species. This gap hinders both applicants and regulators in determining the human relevance of chemically induced thyroid toxicity. New Approach Methodologies (NAMs) offer a promising solution by enabling direct comparisons between human and rat hepatic responses. Among these, the comparative in vitro hepatic enzyme assay is specifically highlighted in the ECHA-EFSA guidance on endocrine disruptors. However, its development and implementation have proven technically complex and difficult to standardize. This study contributes to the advancement of NAMs by utilizing TruVivo®, a tri-culture hepatic model composed of hepatocytes and feeder cells (growth-inactivated fibroblast and epithelial cells), to evaluate thyroxine glucuronide (T4G) formation, a key metabolite in thyroid hormone metabolism and a critical endpoint for assessing liver-mediated thyroid disruption. The work focuses on quantifying interspecies differences in T4G formation following exposure to nuclear receptor reference agonists (NR-RFs), while also establishing historical control data (HCD), assay acceptability criteria, a suitable positive control, and interpretation criteria.