Tox21 Resources

Tox21’s scientific tools include robotic screening, assay development and informatics resources, all of which enable the program’s large-scale screening and data analysis efforts.


  • Phenotypic readouts
    • Cytotoxicity assays (i.e., cell viability [measures ATP], kinetic measurement of cytotoxicity and viability in cells)
    • Apoptosis assays (i.e., caspase assays [measures activity of caspase 3/7, 8, 9])
    • Membrane integrity assays (i.e., LDH and protease release)
    • Mitochondrial toxicity assays (i.e., mitochondrial membrane potential)
    • Gene toxicity assays
      • Differential cytotoxicity (DNA damage repair gene–deficient cell lines, DT40 and mouse cell lines)
      • ATADS (ELG1)
      • Micronucleus assay
    • Phospholipidosis assays
    • Genetic variation (more than 1,000 HapMap lines)
  • Pathway-specific toxicological mechanisms
    • Pathway assays using reporters (e.g., luciferase, β-lactamase) measuring hypoxia, ER stress, NF-κB*, p53*, ARE/Nrf2*, HSE, CREB, AP-1, STAT, NFAT, HIF-1α*, ESRE*, real-time cytotoxicity and viability*
    • Developmental pathway assays (retinol signaling*, Hedgehog/Gli* and SBE/SMAD*)
  • Target-specific toxicological mechanisms
    • Nuclear receptor assays, including AR*, AhR*, ERα/β*, FXR*, GR*, LXR, PPARδ*, PPARγ*, PR*, PXR*, RXR*, TRβ*, VDR*, RORγ, CAR* and ERR*
    • G-protein-coupled receptor signaling assays, including TRHR* and TSHR*
    • Enzyme activity assays, including AChE*
    • hERG channel assay
    • Cytokine assays including IL-8 and TNFα

​*Assays screened against the Tox21 10K library

Genomic Methods

  • Genomic engineering using CRISPR
  • Induced pluripotent stem cell culture and differentiation, including endothelial, cardiomyocyte and neuronal models
  • 3-D cell culture
  • High-throughput gene expression profiling (e.g., RNAseq)

Informatics Tools

  • BioPlanet
    • Integrated pathway database that annotates molecular pathways by source, species, biological function/process, disease/toxicity relevance and availability of probing assays
    • Continued development of this platform includes compound activity data, sequence data, gene/protein expression data, pathways from non-human species and organization of assays according to pathways/diseases/toxicity end points
    • Allows easy browsing, visualization and analysis of the universe of pathways to facilitate systematic analysis and modeling of toxicity responses
  • Repository hosting all Tox21 qHTS data
    • Includes a graphic user interface for easy browsing and retrieval
  • Online system for tracking the status of all assays, protocols and experimental conditions
    • Includes a mechanism for generating standard laboratory protocols for each assay
  • Chemical browser hosting the Tox21 10K library compound structures and annotations, including analytical quality control results
  • Point-of-departure modeling of gene expression profiling data

Robotic Platform

The Tox21 robot tests hundreds of thousands of chemicals against multiple in vitro assays to establish a signature of chemical compounds that can be used to predict in vivo human and rodent toxicity. This robust, automated platform uses plate readers, liquid handlers, incubators and a centrifuge to run a wide variety of biochemical and cell-based assays yielding reproducible, high-quality data.

The screening system consists of a series of peripherals and workstations arranged around a central anthropomorphic robotic arm. The key components include the Kalypsys Director software, a pin tool device for nanoliter compound transfer, plate storage and environmentally controlled assay incubation units, nanoliter reagent dispensers (BioRAPTR, Multidrop, acoustic/noncontact), centrifuge (V-spin), and plate readers (ViewLux, EnVision and FDSS).

This robotic system provides unparalleled speed, reliability and high-quality reproducible data. The millions of data points generated from robotic screening will be transformed into in vitro chemical signatures that may be used to study the mechanism of action of compounds, predict toxicity and minimize traditional animal toxicity testing