ToxCast Data Generation: Chemical Procurement Workflow

EPA follows a standard process to select, procure, register, conduct quality reviews, and prepare chemicals for high-throughput screening through its ToxCast program. The chemicals evaluated by EPA’s ToxCast program are organized into Phases.

On this page:

ToxCast Chemical Library
Procuring and Registering Chemicals
Chemical Quality Control
Chemical Order Process

ToxCast Chemical Library

Additional Resources

The initial ToxCast chemical library was organized into three phases. Screening results for the ToxCast Chemical library are available in ToxCast.

Phase I

Chemical Types: Phase I includes 310 unique chemicals, mostly pesticides. The pesticides have diverse chemical structures and a broad range of pesticidal mechanisms. The chemicals already had significant toxicity data (approximately 275 compounds had near complete guideline data coverage) and a variety of chemical reactivity features and mechanistic diversity.

Pesticides were selected to be screened first because of EPA’s extensive animal toxicity data from testing pesticides. The ToxCast screening results from Phase I were compared to these animal toxicity study results as proof-of-concept for using ToxCast to evaluate chemicals for potential health effects. 30 non-pesticidal environmental chemicals of research or regulatory interest were also evaluated in Phase I, including:

Perfluorinated compounds, such as Perfluorooctanoic acid (PFOA), a surfactant used in fluoropolymers such as Teflon; and Perfluorooctanesulfonic acid (PFOS), used in the semiconductor industry, ScotchGuard™ formulations, and flame-retardant foams.
Bisphenol A (BPA) and a set of phthalate alternatives used as plasticizers.
10 toxicologically active metabolites of included phthalates and pesticides such as monobutyl phthalate (a metabolite of dibutyl phthalate) and diazoxon (a metabolite of diazinon).

ToxCast Screening: The original 310 chemicals tested in Phase I are referred to as “ph1_v1”, and this list includes modified or discontinued compounds as well as compounds with solubility and degradation issues that are removed in “ph1_v2”.

Toxicity Data: An extensive amount of toxicity data is available for most Phase I chemicals, including data for subchronic, chronic, multigenerational reproductive, and developmental endpoints across several species (rat, mouse, dog and rabbit). These data were extracted into the Toxicity Reference Database (ToxRefDB) from data evaluation records (DERs) obtained from the US EPA's Office of Pesticides Programs' review of registrant-submitted study data.

Phase II

Chemical Types: Phase II includes a majority of the Phase I chemicals with the addition of 768 data‐poor chemicals of research and regulatory interest. From Phase I, 293 unique chemicals were selected (ph1_v2), excluding chemicals determined to be not suitable for high-throughput screening. These excluded chemicals were 14 sulfurons determined to undergo rapid hydrolysis in DMSO, 3 chemicals deemed insufficiently soluble in DMSO, and 1 chemical originally tested in parent form that was instead tested in a salt form.

Phase II also includes a list of 799 chemicals of interest to EPA’s Endocrine Disruption Screening Program (e1k). The e1k library contains many known estrogen receptor (ER) and androgen receptor (AR) active reference chemicals, which were identified specifically for testing in a subset of assays to evaluate potential endocrine‐related activity, as opposed to across all ToxCast assays.

Many Phase II chemicals were purchased from commercial suppliers and approximately 150 chemicals were donated by research collaborators external to EPA. Some chemicals were donated by the chemical industry and the U.S. Food and Drug Administration’s National Center for Toxicological Research, (“green” plasticizer alternatives and reference liver toxicants, respectively). Pharmaceutical companies donated the remaining 136 “failed pharma” compounds (i.e., drug candidates discontinued due to toxicity in preclinical or clinical trials).

Toxicity Data: The donation of failed drug compounds, along with some preclinical and clinical data, introduced to ToxCast a set of chemicals designed to be bioactive at specific human (or veterinary) targets. All compatible donated data on these failed pharmaceuticals were extracted into the Toxicity Reference Database (ToxRefDB).

ToxCast Screening: Phase II screened the newly added ph2 inventory in most of the Phase I ToxCast assays, as well as testing of the ph2 and reprocured ph1_v2 inventory in newly acquired ToxCast and Tox21 assays. In addition, the e1k chemicals were screened in a limited subset of Phase II endocrine‐related assays, including many assays run using the Tox21 robotics technology. Phase II concluded in 2015 and the data resulting from the screening of 1,863 unique Phase II chemicals is available in ToxCast.

Phase III

Chemical Types: Phase III launched in late 2014 and encompassed screening in new ToxCast assays and endpoints for over 500 chemicals and a small set of donated mixtures and water samples were added. Phase III also added selected chemicals from Tox21 and e1K to test in the new assays. Unique Phase III chemicals include flame retardants and chemicals of interest to EPA’s Endocrine Disruption Screening Program (EDSP).

The Phase III chemical list does not overlap with earlier chemical inventories having full or partial testing coverage in Phases I and II (ph1_v1, ph1_v2, ph2, and e1k).

ToxCast Screening: Phase III chemicals were not run through all ToxCast assays. Using the results from Phase I and Phase II, EPA identified the assays most appropriate for the Phase III chemicals selected.

With the addition of Phase III chemicals, the total combined set of unique chemicals comprising the ToxCast chemical library is over 4,200. (Note: This number of chemicals does not include chemicals only tested in Tox21).

Procuring and Registering Chemicals

EPA follows a standard process for procuring and registering chemicals. The majority of ToxCast chemicals are purchased from commercial suppliers.

EPA identifies a list of generic CAS and chemical names to purchase from a Chemical Supplier as well as chemical structure information (in the form of SMILES) to help with structure searching of larger aggregated commercial chemical services, such as eMolecules or ChemNavigator .
EPA purchases two bottles of analytical grade samples (> 98% purity) from the Chemical Supplier. Most samples are then shipped directly from the Chemical Supplier to the Chemical Contractor in pre‐tared, barcoded vials. One of two bottles is designated for solubilization and the other stored in neat‐powder form. The Chemical Supplier also provides a chemical structure file and molecular weight for defined pure compounds (SMILES or Structure Data Format [SDF] file).
- EPA requires, wherever possible, a Certificate of Analysis (COA) and Safety Data Sheet (SDS) from the Chemical Supplier. The Chemical Supplier provides the COA and SDS to the Chemical Contractor, which the Chemical Contractor provides to EPA.
When the Chemical Contractor receives the chemicals, bottles are scanned, weighed, registered into the chemical contractor’s chemical management tracking system, and either immediately solubilized, or stored in a ‐20°C freezer under inert conditions until a solubilization order is placed. The contractor's chemical management tracking system records: the Supplier, Catalog number, Lot number, the Contractor shipment sample code, SMILES, molecular weight (usually derived from the structure file), physical form of the chemical received (solid or liquid), quantity (ul or mg), and the date the sample was registered in the system.
- In cases where a chemical name, CAS or structure are not provided, the Chemical Contractor attempts to fill in this information through reference to the original orders or supplier website catalogs, or through internal database matches to the compound structures provided.

A percentage of the chemicals may deviate from this standard process. Most common deviations from this process are the use of specialty suppliers, procurement of larger or smaller quantities for hard‐to‐locate chemicals, and other “external” reference chemicals that may be screened.

Chemical Quality Control

The goal of chemical quality control (QC) is to have accurate and reliable information on every chemical undergoing screening. In the absence of perfect knowledge and certainty, the ToxCast chemical QC process minimizes controllable sources of errors, particularly in the chemical information QC review and registration process, and also in handling and storage procedures. Simultaneous, analytical QC efforts attempt to detect, understand, document, and communicate actual errors and problems impacting chemicals under testing conditions.

EPA may request its Chemical Contractor to analyze chemical samples to confirm the Chemical Supplier’s identification of the sample and to estimate sample purity (ideally >90% for commercial grade and >99% for pharmaceutical or pestinal analytical grade samples) using appropriate analytical methods. Analytical QC provides an experimental standard of verification and confirms the chemical identity and purity in the plated DMSO solutions undergoing testing at the time of plating, as well as at later time points (to assess sample stability over time).

The QC analysis requires the availability of analytical methods suitable for the various types of chemical compounds derived from the chemical domains identified. The standard analysis would consist of high-throughput, low-resolution liquid chromatography mass spectrometry (LCMS), using UV and ELS detection methods run in positive and negative ion mode, of solutions in microtiter 96 well plates.

The types of QC review include:

Certificate of Analysis (COA) Validation: To establish chemical identity (chemical name, CAS, MW) from the chemical supplier‐provided COA.
DSSTox Review: To ensure accurate and consistent substance (CAS, name, description) and structure annotation of the generic chemical (independent of supplier, lot, batch) as part of the DSSTox chemical registration process.

The results of the analytical QC review include:

An analytical determination and report supplied to EPA in electronic format.
All spectra and supporting information, along with summary purity and identity confidence score.

Chemical Order Process

The EPA chemical library was set up to provide the ToxCast phased chemical library to screening partners for ToxCast and other research efforts. The chemical library was expanded to include the Tox21 library and has continued to expand to add PFAS, EDSP, and DNT-relevant chemical libraries. Additional “Phases” of chemical lists may be released in the future as research progresses. Outside entities can request chemical libraries for research purposes and the transfer of these libraries are facilitated through Material Transfer Agreements.

The chemical order process ensures chemical samples are provided at quantities and concentrations primarily designed for high-throughput testing applications, to be run in assays deemed of sufficient value to EPA’s mission.

The process is as follows:

Screening Partners submit a chemical order outlining the number of chemicals, quantities, and concentrations needed; background information on the proposed assay testing; pertinent plating details; and the recipient of the shipment and desired shipment date. For external requestors, an approved Material Transfer Agreement must be in place prior to submission of the chemical order form.
EPA reviews the chemical order request and prepares the order for Chemical Contractor.
The Chemical Contractor aliquots, packages, and ships chemical samples in various formulations (neat, dried down, or plated solutions), with dry ice (to ensure no thawing), to the designated screening partner.
Screening Partners receive blinded plate maps for screening testing and agree to provide EPA with raw or processed screening results prior to the unblinding of the plates. However, when collaborators have a legitimate need for unblinding earlier in testing (e.g., for method development only) or additional details, such as actual concentration, EPA may provide either partially or fully unblinded plate maps.

The time it takes to process a chemical order varies and may depend on whether chemical stocks are available for plating.