Impact of Chemical Quality on High-Throughput in Vitro Assays: A Tox21 Study

The
Tox21 10K chemical library, an in vitro toxicology
toolbox consisting of more than 8900 unique chemical entities including
environmental chemicals and drugs, has undergone analytical quality
control (QC) testing after storage at room temperature for 0 and 4
months (T0 and T4). Each chemical was previously assigned a QC grade
based on purity, identity, and concentration. In parallel, the Tox21
10K library has been tested across approximately 90 in vitro assays in a quantitative high-throughput screening (qHTS) format,
generating >120 M data points to date. These data were used to
analyze
the correlation between chemical quality and bioassay activity, as
well as chemical structure. The chemical characteristics of poor-quality
and unstable compounds were explored to identify structural features
that should be avoided. In addition, one of the high-throughput assays
measuring the induction of p53 activity by small molecules was used
to test the Tox21 10K compound library at T0 and T4 due to its robust
performance and reproducibility. Approximately 2% of compounds in
the library showed a significant change in activity in the p53 assay
between T0 and T4 (active to inactive or vice versa), which also correlated
with chemical stability. Here, machine learning models were constructed
using bioassay data or chemical structures to predict poor-quality
(low QC grades at T0) and unstable (grade drop from T0 to T4) chemicals.
Chemical structure was found to be highly predictive (0.75) of chemical
quality and stability, whereas bioassay data was less predictive (0.66)
but still showed better than random performance. Taken together, these
findings provide valuable guidance for interpreting the Tox21 assay
results and informing best practices for future chemical selection
and handling.