ToxCast Data Generation: Chemical Workflow
This page describes the process EPA follows to select chemicals, procure chemicals, register chemicals, conduct a quality review of the chemicals, and prepare the chemicals for high-throughput screening. There is also information about each phase of testing.
ToxCast Phase I screened 310 unique chemicals, mostly pesticides. The pesticides in Phase I included 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.
Thirty non-pesticidal environmental chemicals of research or regulatory interest were also evaluated in Phase I, including:
- Perfluorinated compounds - 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).
The original 310 chemicals tested in ToxCast Phase I are referred to as “ph1_v1” and are available on the CompTox Chemicals Dashboard. A listing of the ToxCast ph1_v1 chemicals, including modified or discontinued compounds labeled and those with solubility and degradation issues.
An extensive amount of toxicity data was already available for most Phase I chemicals and included data for subchronic, chronic, multigenerational reproductive, and developmental endpoints across several species (rat, mouse, dog and rabbit). The data was available in data evaluation records from the US EPA's Office of Pesticides Programs' review of registrant study data. EPA researchers extracted the data from EPA’s Data Evaluation Records and systematically captured these data in a database of animal toxicity data called the Toxicity Reference Database (ToxRefDB).
Data from the screening of Phase I chemicals is available with other previously published ToxCast Data.
Phase II included a majority of the Phase I chemicals and expanded the chemicals screened to include data‐poor chemicals of research and regulatory interest. 293 unique chemicals from Phase I (ph1_v2) were chosen, excluding chemicals which we learned from Phase I were 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. The complete list of Phase II chemicals are available on the CompTox Chemicals Dashboard.
768 unique new ToxCast chemicals identified through a selection process were added in Phase II (ph2). ToxCast Phase II also includes a list of 799 chemicals of interest to EPA’s Endocrine Disruption Screening Program (e1k). Specifically, the e1k library contains many known estrogen receptor (ER) and androgen receptor (AR) active reference chemicals. The e1k list is specifically identified because the chemicals were not screened in the entire set of Phase II high-throughput assays, but a subset of assays to evaluate potential endocrine‐related activity.
The majority of 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). A complete list of the donated pharmaceuticals (“donated pharma” chemical list) is available on the ToxCast chemical libary file (DSSTox_TOXCST, date‐stamped 20160129), available from the EPA DSSTox Data download page.
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 added to EPA’s Toxicity Reference database.
ToxCast Phase II screened the newly added ph2 inventory in the majority of the original 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 ToxCast data resulting from the screening of 1,863 unique ToxCast chemicals is available with other previously published ToxCast Data.
ToxCast Phase III launched in late 2014. Phase III added new assays, endpoints, and chemicals. Five hundred chemicals and a small set of donated mixtures and water samples were added to ToxCast. 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 chemical list for ToxCast Phase III 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). Each of these chemical lists is available on the CompTox Chemicals Dashboard including the complete Phase III chemical list.
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 chemicals comprising ToxCast is over 4,200 unique chemicals. (Note: this number of chemicals does not include chemicals only tested in Tox21). The most recent ToxCast data release was completed in October 2018 and includes the release of Phase III data which is available on the EPA ToxCast data download website.
EPA follows a standard process for procuring and registering chemicals which includes purchasing chemicals from a supplier and working closely with a contractor to register and catalogue the chemicals. The majority of ToxCast chemicals are purchased from commercial suppliers.
- First, EPA provides the supplier a list of generic CAS and chemical names that need to be purchased. EPA may also provide chemical structure information in the form of SMILES to the contractor to help with structure searching of larger aggregated commercial chemical services, such as eMoleculesor ChemNavigator .
- EPA purchases two bottles of analytical grade samples (> 98% purity) from a supplier. A majority of samples are then shipped directly from the supplier to the 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).
- When the contractor receives the chemicals, bottles are scanned, weighed, registered into the 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 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 small percentage of the chemicals deviate from the standard process (less than 10% of Phase II and Tox21 chemicals). Most common deviations from this process are the use of specialty suppliers and procurement of larger or smaller quantities for hard‐to‐locate chemicals.
EPA requires, wherever possible, a Certificate of Analysis (COA) and Material Safety Data Sheet (MSDS) from the chemical supplier. The chemical supplier provides the COA and MSDS to the contractor and the contractor provides it to EPA. Occasionally samples are accepted by EPA without documentation when the supplier has problems obtaining the documentation (COAs are missing for approximately 15% of unique lot‐batch chemical samples).
The goal of chemical quality check (QC) is to have accurate and reliable information on every chemical undergoing ToxCast screening. There are practical limits as well as a balance that must be struck between cost/time/efficiency and the larger objectives of ToxCast. 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, but also in handling and storage procedures. At the same time, the ToxCast analytical QC processes attempt to detect, understand, document, and communicate actual errors and problems impacting particular chemicals under testing conditions.
In the QC process, EPA may request its chemical management services contractor to analyze chemical samples to confirm the supplier's identification of the chemical 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 is required to confirm 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
The QC analysis requires the availability of analytical methods suitable to analyzing 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 documentation QC review include:
- Certificate of Analysis (COA) Chemical Validation – To establish chemical identity (chemical name, CAS, MW) from the supplier‐provided COA.
- DSSTox Chemical Information 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 Preparation Process and Timeline
EPA has developed a step-by-step process to prepare chemicals for sending to EPA contract laboratories and to those requesting the chemical library for research purposes. This process ensures that the neat chemical or DMSO solution stocks 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 ToxCast mission.
The process is as follows:
- Requestors submit an EPA ToxCast Chemical Request Form 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.
- The Center for Computational Toxicology and Exposure (CCTE) Chemical Management reviews the request, prepares the order, and provides it to the NCCT chemical services contractor.
- Based on the order, the contractor aliquots, packages, and ships chemical samples in various formulations (neat, dried down, or plated solutions), with sufficient quantities of dry ice (to ensure no thawing), to the designated laboratories or investigators within or outside the U.S.
It takes an average of 2-6 weeks (depending on whether chemical stocks are available for plating) to process a chemical order request from receipt of a request to shipment of the samples.
The chemical library is typically requested by contractors hired by EPA to screen the chemicals, EPA research collaborators with a signed agreement and EPA staff. External organizations requesting the chemical library must agree to NCCT’s policy of only providing blinded plate maps during assay testing. These organizations should provide EPA NCCT 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, NCCT will provide either partially or fully unblinded plate maps.