Tox21 Scientific Capabilities

This system is capable of weekly triplicate screening of the 10K library across 15 concentrations (1.2 nM to 92 µM). Other areas of expertise include development of human cell models using stem cells and 3-D cell culture, as well as genomics approaches, such as genome editing and high-throughput gene expression profiling. A dedicated informatics group performs chemi-informatics and bioinformatics analyses, including concentration-response and toxicological modeling. 

Within this format, the Tox21 program offers a variety of scientific capabilities available to NIH investigators and to other scientists whose assays have been submitted to and accepted by the program. Learn about the assay nomination and evaluation process. 

Tox21 Expertise

Tox21 experts have expertise in the following areas:

Large-Scale Screening 

• Assay development 
• Assay design 
• Assay validation (assay screened in triplicate against pharmacologically active compounds and 88 additional compounds selected by the Tox21 Chemical Selection Working Group to evaluate reproducibility and positive control consistency) 
• Assay optimization 
• High-content screening 
• Quantitative high-throughput screening (qHTS) 
• Assay miniaturization (adapted for the qHTS platform) 

Genomic Analyses 

• Gene expression profiling (e.g., RNAseq and scRNAseq) 
• Epigenetic analysis 

Human Cell Model Development 

• Stem cell–derived models of neurons, endothelium and cardiomyocytes 
• Immortalized cell line models of neurons, keratinocytes and renal proximal tubule epithelial cells 
• Genome engineering using CRISPR 

Data Analysis 

• Normalization and correction 
• Concentration-response curve-fitting to generate potency and efficacy 
• Classification of curves based on significance of fit (by p-values), completeness of fit and efficacy
• Reproducibility evaluation 
• Assignment of activity outcomes 
• Concentration-response point-of-departure determination from gene expression data 

Data Dissemination 

Data available in these public databases encourage independent evaluations of Tox21 findings: 

• PubChem (National Library of Medicine) 
• Tox21 Data Browser (NCATS) 
• Chemical Effects in Biological Systems (National Toxicology Program) 
• Aggregated Computational Toxicology Resource (Environmental Protection Agency)

Selected Publications

View a list of selected Tox21 publications: PubMed; NCATS.

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.

Assays

• 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 assay*)
  • 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, PPARα*, PPARδ*, PPARγ*, PR*, PXR*, RXR*, TRβ*, VDR*, RORγ, CAR* and ERR*
  • G-protein-coupled receptor/Calcium signaling assays, including TRHR*, TSHR*, GnRHR*, CHRM1*, HTR2A*, and KISS1R
  • G-protein-coupled receptor/cAMP signaling assays, including DRD2*, ADRB2*, and ADRB1*
  • Enzyme activity assays, including AChE*
  • hERG potassium channel assay*
  • Cytokine assays including IL-8 and TNFα
  • Cytochrome P450 assay including CYP 1A2*, CYP 2C9*, CYP 2C19*, CYP 2D6*, CYP 3A4*, and CYP26A1*

* 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.