Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

Optimizing androgen receptor prioritization using high-throughput assay-based activity models.

Introduction: Computational models using data from high-throughput screening assays have promise for prioritizing and screening chemicals for testing under the U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). The purpose of this work was to demonstrate a data processing method for the determination of optimal minimal assay batteries from a larger comprehensive model, to provide a uniform method of evaluating the performance of future minimal assay batteries compared with the androgen receptor (AR) pathway model, and to incorporate chemical cluster analysis into this evaluation. Although several of the assays in the AR pathway model are no longer available through the original vendor, this approach could be used for future evaluations of minimal assay models for prioritization and screening. Methods: We compared two previously published models and found that an expanded 14-assay model had higher sensitivity for antagonists, whereas the original 11-assay model had slightly higher sensitivity for agonists. We then investigated subsets of assays in the original AR pathway model to optimize overall testing strategies that minimize cost while maintaining sensitivity across a broad chemical space. Results and Discussion: Evaluation of the critical assays across subset models derived from the 14-assay model identified three critical assays for predicting antagonism and two critical assays for predicting agonism. A minimum of nine assays is required for predicting agonism and antagonism with high sensitivity (95%). However, testing workflows guided by chemical structure-based clusters can reduce the average number of assays needed per chemical by basing the assays selected for testing on the likelihood of a chemical being an AR agonist, according to its structure. Our results show that a multi-stage testing workflow can provide 95% sensitivity while requiring only 48% of the resources required for running all assays from the original full models. The resources can be reduced further by incorporating in silico activity predictions. Conclusion: This work illustrates a data-driven approach that incorporates chemical clustering and simultaneous consideration of antagonism and agonism mechanisms to more efficiently screen chemicals. This case study provides a proof of concept for prioritization and screening strategies that can be utilized in future analyses to minimize the overall number of assays needed for predicting AR activity, which will maximize the number of chemicals that can be tested and allow data-driven prioritization of chemicals for further screening under the EDSP.

Chemistry domain of applicability evaluation against existing estrogen receptor high-throughput assay-based activity models.

Introduction: The U. S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP) Tier 1 assays are used to screen for potential endocrine system-disrupting chemicals. A model integrating data from 16 high-throughput screening assays to predict estrogen receptor (ER) agonism has been proposed as an alternative to some low-throughput Tier 1 assays. Later work demonstrated that as few as four assays could replicate the ER agonism predictions from the full model with 98% sensitivity and 92% specificity. The current study utilized chemical clustering to illustrate the coverage of the EDSP Universe of Chemicals (UoC) tested in the existing ER pathway models and to investigate the utility of chemical clustering to evaluate the screening approach using an existing 4-assay model as a test case. Although the full original assay battery is no longer available, the demonstrated contribution of chemical clustering is broadly applicable to assay sets, chemical inventories, and models, and the data analysis used can also be applied to future evaluation of minimal assay models for consideration in screening. Methods: Chemical structures were collected for 6,947 substances via the CompTox Chemicals Dashboard from the over 10,000 UoC and grouped based on structural similarity, generating 826 chemical clusters. Of the 1,812 substances run in the original ER model, 1,730 substances had a single, clearly defined structure. The ER model chemicals with a clearly defined structure that were not present in the EDSP UoC were assigned to chemical clusters using a k-nearest neighbors approach, resulting in 557 EDSP UoC clusters containing at least one ER model chemical. Results and Discussion: Performance of an existing 4-assay model in comparison with the existing full ER agonist model was analyzed as related to chemical clustering. This was a case study, and a similar analysis can be performed with any subset model in which the same chemicals (or subset of chemicals) are screened. Of the 365 clusters containing >1 ER model chemical, 321 did not have any chemicals predicted to be agonists by the full ER agonist model. The best 4-assay subset ER agonist model disagreed with the full ER agonist model by predicting agonist activity for 122 chemicals from 91 of the 321 clusters. There were 44 clusters with at least two chemicals and at least one agonist based upon the full ER agonist model, which allowed accuracy predictions on a per-cluster basis. The accuracy of the best 4-assay subset ER agonist model ranged from 50% to 100% across these 44 clusters, with 32 clusters having accuracy ≥90%. Overall, the best 4-assay subset ER agonist model resulted in 122 false-positive and only 2 false-negative predictions compared with the full ER agonist model. Most false positives (89) were active in only two of the four assays, whereas all but 11 true positive chemicals were active in at least three assays. False positive chemicals also tended to have lower area under the curve (AUC) values, with 110 out of 122 false positives having an AUC value below 0.214, which is lower than 75% of the positives as predicted by the full ER agonist model. Many false positives demonstrated borderline activity. The median AUC value for the 122 false positives from the best 4-assay subset ER agonist model was 0.138, whereas the threshold for an active prediction is 0.1. Conclusion: Our results show that the existing 4-assay model performs well across a range of structurally diverse chemicals. Although this is a descriptive analysis of previous results, several concepts can be applied to any screening model used in the future. First, the clustering of the chemicals provides a means of ensuring that future screening evaluations consider the broad chemical space represented by the EDSP UoC. The clusters can also assist in prioritizing future chemicals for screening in specific clusters based on the activity of known chemicals in those clusters. The clustering approach can be useful in providing a framework to evaluate which portions of the EDSP UoC chemical space are reliably covered by in silico and in vitro approaches and where predictions from either method alone or both methods combined are most reliable. The lessons learned from this case study can be easily applied to future evaluations of model applicability and screening to evaluate future datasets.