Technical evaluation and standardization of the human thyroid microtissue assay.

Area of Science:

• Toxicology and Environmental Health
• In Vitro Toxicology
• New Approach Methodologies (NAMs)

Background:

• Reducing reliance on animal testing necessitates reliable in vitro and in silico methods for human health risk assessment.
• Organotypic culture models using primary human cells offer human relevance but face challenges in standardization and validation.
• Establishing performance standards is crucial for validating new approach methods (NAMs) without animal data.

Purpose of the Study:

• To define technical parameters for donor procurement, thyrocyte qualification, and performance in the human thyroid microtissue (hTMT) assay.
• To establish benchmark ranges for reference chemical responses within the hTMT assay.
• To ensure human-relevant predictions from in vitro NAMs by addressing inter-individual variability.

Main Methods:

• Characterized thyrocytes from 32 diverse donors for morphological and functional variability.
• Utilized the human thyroid microtissue (hTMT) assay with primary human thyrocytes.
• Profiled reference chemical responses to assess donor-dependent variability.

Main Results:

• Defined data-informed minimum acceptance criteria for donor qualification.
• Established benchmark parameters for method transfer, proficiency testing, and validation.
• Demonstrated the variability of donor-dependent effects in response to reference chemicals.

Conclusions:

• The study provides a framework for standardizing and validating the hTMT assay for thyroid-disrupting chemical assessment.
• Setting performance benchmarks is essential for the reliable application of human cell-based NAMs.
• This work supports the U.S. EPA's goals for reducing, refining, and replacing animal testing.