Analytical\nQuality Evaluation of the Tox21 Compound\nLibrary

The analytical quality of compounds subjected to high-throughput screening (HTS) impacts\naccurate interpretation of assay results, with poor quality samples\npotentially leading to false negatives or positives. The Tox21 “10K”\nlibrary consists of over 8900 unique compounds, spanning a diverse\nlandscape of environmental and pharmaceutical chemicals, posing opportunities\nand challenges for analytical quality control (QC) determinations.\nTox21 sample plates stored in DMSO at ambient conditions for 0 (T0)\nand/or 4 months (T4), totaling more than 13K unique sample identifiers\n(Tox21 IDs), were subjected to various analyses, including liquid\nand gas chromatography mass spectrometry (LC-MS, GC-MS) and nuclear\nmagnetic resonance (NMR). Results for each sample at T0 or T4 underwent\nexpert review and, where possible, a QC grade conveying purity, identity,\nand concentration was assigned. Herein, we relate details of the methods\napplied and report on the original (v0) Tox21 ID level results. Thirteen\nQC grades were condensed to 5 quality scores to aid global analysis,\nresulting in reinterpretation and improvement of >700 sample grades.\nOf the 92% T0 samples successfully graded, 76% exceeded 90% purity.\nFor 76% of samples that were also tested at T4, 89% showed no evidence\nof sample loss or degradation. Prioritized quality bins were used\nto summarize thousands of replicate sample-level QC results to a compound-level\nQC score to support structure-based analyses. ToxPrint chemotype analysis\nidentified structural features enriched in unstable compounds, as\nwell as in high and low quality T0 subsets. Predicted vapor pressure\nwas weakly correlated with low-concentration QC indicators, reflecting\nlikely entanglement with method amenability and quality issues. Finally,\nan ongoing EPA effort to re-evaluate the original QC spectra is generating\ninsights that will further modify QC grades. Tox21 QC spectra and\nresults will be made available in a new public QC browser, facilitating\nfurther evaluation to support HTS interpretation and modeling applications.