Technical evaluation and standardization of the human thyroid microtissue assay
Data files
Dec 12, 2024 version files 35.87 KB
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CSS.405.1.1.4_Supplementary_Tables_v6.xlsx
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README.md
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Abstract
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 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 human thyroid microtissue 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.
README: Technical Evaluation and Standardization of the Human Thyroid Microtissue Assay
SUPPLEMENTARY TABLES LEGENDS
Supplementary Table 1. Human Thyroid Microtissue (hTMT) Medium. Formulation of hTMT medium components, vendor, catalog number, and final working concentrations.
Supplementary Table 2. Assay Performance Parameters. Performance criteria were established from the T4 median, median absolute deviation (MAD), and range values derived from the qualified donor cohort under assay-specific conditions with required priority levels for all metrics.
Supplementary Table 3. Donor Procurement Parameters. Donor procurement specifications extrapolated from linear regression modeling using the acceptance criteria defined for thyroglobulin, thyroxine (T4), and triiodothyronine (T3). Not applicable (NA).
Supplementary Table 4. Multiple Linear Regression Statistics. Multiple linear regression model statistics for microtissue biomass, thyroglobulin, T4, T3, and T4/T3 ratio. Analysis of Variance (ANOVA) parameters include the residual sum of squares (SS), degrees of freedom (DF), mean square (MS), F statistic, and p-value. The parameter estimates include the dependent variable, estimate, standard error, 95% confidence interval (CI), t statistic, p-value, and p-value summary. The goodness-of-fit reports the degrees of freedom (DF), multiple R value, R2 value, adjusted R2 value, sum of squares, Sy.x, root mean squared error (RMSE), and corrected Akaike Information Criterion (AICc). The multicollinearity reports the independent variables, the variance inflation factor (VIF), and R2 with other variables. The normality of the residuals statistics and data summary are also reported.
Supplementary Table 5. Simple Linear Regression Statistics. Simple linear regression model statistics for microtissue thyroglobulin, T4, and T3 relative to age and BMI. Best fit values, 95% confidence intervals, model goodness-of-fit, significance of slope, model equation, and data summary are reported. Dfn and DFd are the degrees of freedom for the numerator and denominator of the F ratio, respectively.
Supplementary Table 6. Literature-derived Reference Chemical Potency Values. Specifications for chemical name, target MIE, species, assay method, assay source material, assay type, potency, potency 95% confidence interval (CI), potency unit, potency category, and references are noted. n/a: not applicable
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Donor Micrographs at Day 14. Representative micrographs across three treatment groups (Tx-1, Tx-2, Tx-3) for each donor evaluated in the study (N=32). Scale bar is 1,000 µM.
Supplementary Figure 2. Endpoint Correlations. Pearson correlation of characterization endpoints at day 14 in the (A) Tx-1 and (B) Tx-3 treatment groups. Values greater than 0 indicate a positive correlation, less than 0 a negative correlation, and 0 as no correlation.
Supplementary Figure 3. Evaluation of Thyroid Hormone Synthesis Inhibition by Reference Chemical – Methomyl. (A) Population-level concentration-response modeling of the total (N=32) versus qualified (N=24) donor cohort. The values were normalized by donor and plotted in aggregate for data modeling. The red band represents the 95% confidence interval of the model fit. The gray band indicates the 95% prediction interval where 95% of future donors would be expected to exhibit bioactivity.
Supplementary Figure 4. Reference Chemical Cytotoxicity Plots. Population-level concentration-response modeling of ATP measurements at day 14 for the total (N=32) versus qualified (N=24) donor cohort in response to (A) methomyl, (B) methimazole, (C) 6-propyl-2-thiouracil, and (D) sodium perchlorate. The values were normalized by donor and plotted in aggregate for data modeling. The red band represents the 95% confidence interval of the model fit. The gray band indicates the 95% prediction interval where 95% of future donors would be expected to exhibit bioactivity. The horizontal dashed line represents the baseline.
Supplementary Figure 5. Assay Plate Maps. The 96-well plate map defined by the assay workflow on designated days for seeding (Day 0), medium exchanges (Day 2-8), and chemical treatments (Day 8-14). Control wells (Tx-1, Tx-2, Tx-3) and the chemical concentrations (µM) are noted. The table specifies TSH supplementation, compound treatments, concentration range and units, technical replicates, the molecular initiating event (MIE) target of the treatment, and chemical classification as defined by the assay context. Not applicable (NA).