Analytical Quality Evaluation of the Tox21 Compound Library.

The analytical quality of compounds subjected to high-throughput screening (HTS) impacts accurate interpretation of assay results, with poor quality samples potentially leading to false negatives or positives. The Tox21 "10K" library consists of over 8900 unique compounds, spanning a diverse landscape of environmental and pharmaceutical chemicals, posing opportunities and challenges for analytical quality control (QC) determinations. Tox21 sample plates stored in DMSO at ambient conditions for 0 (T0) and/or 4 months (T4), totaling more than 13K unique sample identifiers (Tox21 IDs), were subjected to various analyses, including liquid and gas chromatography mass spectrometry (LC-MS, GC-MS) and nuclear magnetic resonance (NMR). Results for each sample at T0 or T4 underwent expert review and, where possible, a QC grade conveying purity, identity, and concentration was assigned. Herein, we relate details of the methods applied and report on the original (v0) Tox21 ID level results. Thirteen QC grades were condensed to 5 quality scores to aid global analysis, resulting in reinterpretation and improvement of >700 sample grades. Of the 92% T0 samples successfully graded, 76% exceeded 90% purity. For 76% of samples that were also tested at T4, 89% showed no evidence of sample loss or degradation. Prioritized quality bins were used to summarize thousands of replicate sample-level QC results to a compound-level QC score to support structure-based analyses. ToxPrint chemotype analysis identified structural features enriched in unstable compounds, as well as in high and low quality T0 subsets. Predicted vapor pressure was weakly correlated with low-concentration QC indicators, reflecting likely entanglement with method amenability and quality issues. Finally, an ongoing EPA effort to re-evaluate the original QC spectra is generating insights that will further modify QC grades. Tox21 QC spectra and results will be made available in a new public QC browser, facilitating further evaluation to support HTS interpretation and modeling applications.