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Appendix D: Section 4 Supplemental, Details of Chemical Library

The ToxCast chemical library consists of a total of 1,860 unique chemical substances. 

By design, this library includes the full ToxCast Phase I (v2, 293 unique compounds) and Phase 

II (767 unique compounds) active plating inventories, as well as an additional set of 800 

compounds selected to serve EDSP21 program objectives. ToxCast Phase I & II chemicals 

(1,060 total) are analyzed in the full suite of ToxCast HTP assays (>500), whereas the additional 

set of 800 unique EDSP21 compounds are run in a selected subset of these assays (approx 60) 

that probe various measures of endocrine activity; hence, all 1,860 ToxCast compounds generate 

results for this subset of 60 endocrine-related assays. Additionally, all 1,860 unique compounds 

in the ToxCast inventory are included in the final Tox21 EPA chemical inventory (3,726 

compounds). When combined with other Tox21 partner chemical inventories (from the National 

Toxicology Program – NTP, and NIH’s Chemical Genomics Center - NCGC), the Tox21 

inventory totals 8,307 unique substances, spanning considerable chemical structural and 

functional diversity. This inventory is undergoing HTP screening at the NCGC Tox21 robotics 

facility in Bethesda, MD, with initial Tox21 assays focused on endocrine related assays. 

Included within the HTP libraries are various sets of plate replicate samples, samples 

procured from 2 or 3 independent sources, and a set of reference chemicals. For the Tox21 

library, we have also run a set of 88 chemicals on every 1536-well plate. 

The chronology of the formation of the various chemical libraries described above 

strongly influenced the composition of EPA’s final ToxCast inventory. ToxCast chemicals were 

selected in a multi-phased process. Phase I compounds were primarily pesticidal active 

ingredients selected for testing due to the availability of extensive in vivo data through EPA’s 

pesticide regulatory program activities (in addition, a handful of compounds of high interest to 

EPA programs were included, such as perfluorinated compounds, bisphenol A, and a few known 

pesticide metabolites). Beyond Phase I, significant input was solicited and received from EPA 

scientists and regulators, other federal Agencies, non-profits, collaborators, and outside 

stakeholders in constructing the full EPA inventory, from which ToxCast Phase II compounds 

were selected. In addition, with the concurrent development of the EPA ACToR database, 

hundreds of EPA and non-EPA chemical inventories pertaining to commercial use, 

environmental occurrence and/or toxicity were captured from public sources and standardized 

for cross-searching by CAS Registry Number. 

The final nomination list of nearly 17K substances (with unique CASRN) were assigned 

chemical structures from public sources (DSSTox, PubChem, ECOTOX, etc.) and roughly 

filtered by calculated phys-chem properties (using EPA’s EpiSuite) to eliminate compounds that 

would be considered highly volatile (low molecular weight - MW, high vapor pressure), unlikely 

to transmit through cell membranes (high log octanol/water partition coefficient - logP), or less 

suitable for testing and modeling (mixtures, inorganics, reactives, etc.). Note that this filtering 

process is independent of that used to develop the EDSP21 Universe. The resulting 9K 

substances were submitted to EPA’s chemical contractor (Compound Focus, Inc.) for 

procurement, subject to the contract specifications pertaining to cost, availability, etc. 

Of the final set of more than 4,400 procured chemicals, approximately 8% were 

determined to be insoluble in DMSO or otherwise unsuitable for inclusion in the final Tox21 

EPA plating inventory (e.g., volatile, reactive, etc.). In addition to procured chemicals, EPA 

entered into agreements with several outside partners to directly supply physical compound 
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samples for inclusion in ToxCast Phase II (and, by default, Tox21), including 6 major 

pharmaceutical companies (135 failed drugs), chemical manufacturers (green plasticizer 

alternatives and specialty chemicals), and the FDA’s National Center for Toxicological Research 

laboratory (drugs with known liver toxicity). Further ToxCast Phase II compounds were selected 

from the remainder of the Tox21 EPA inventory on the basis of their importance to EPA 

program office objectives (e.g., CCL, EDSP, TSCA, HPV, Green Chemistry, etc.), availability of 

in vivo toxicity data (EPA pesticidal programs, NTP bioassay data, FDA food additives), and 

known bioactivity or target interactions (drugs, reference compounds). Prior to finalizing the 

Tox21 EPA inventory, another 800 compounds, including 60 reference compounds with known 

endocrine activity (most of which were separately procured for this purpose), were selected for 

inclusion in EPSP21 testing. The majority of these compounds were already part of the Tox21 

inventory. Hence, the final Tox21 EPA library of 3,726 unique substances fully incorporates the 

ToxCast 1,860 compound library. 

D.1 Ordering, Processing and Plating

The standard process of chemical procurement for the Tox21 and ToxCast projects, 

which applied to the majority of samples (>95%), involves the purchase of 2 identical bottles of 

100 mg neat (dry) or 10 ml pure liquid, analytical grade (>98% purity as reported in the 

Certificate of Analysis -COA) samples, obtained from preferred suppliers where possible, 

(Sigma-Aldrich and Light Biologicals, primarily). Samples are shipped directly to CFI in pretared, barcoded vials from the supplier (empty vials provided by CFI to the supplier). Upon 

receipt, bottles are scanned, registered into the CFI COMIT database, and subsequently stored in 

a -20 °C freezer. The majority of samples are accompanied by a COA and MSDS (Material 

Safety Data Sheet) from the supplier, which CFI provides to EPA in electronic form. The 

supplier routinely also supplies a structure-data file (SDF) of chemical structures (required for 

pure compounds), and chemical name and CAS (in the majority of cases) for each sample. 

Additionally, CFI's COMIT database records the Supplier, Catalog number, Lot/Batch, CFI 

shipment batch code, supplier-provided molecular weight, and physical form of the chemical 

received (Solid or Liquid). Most common deviations from this standard process for a small 

percentage of chemicals involves use of specialty suppliers for hard-to-locate chemicals, and 

samples shipped in supplier-provided vials (requiring splitting, weighing and transfer to CFI barcoded vials upon receipt). 

Prior to plating, one of the pair of procured neat sample bottles (i.e., 100 mg) is 

solubilized in DMSO to create a stock solution at a target concentration of 20 mM (or the highest 

concentration achievable). Solubilizations were carried out at room temperature, by a single 

Chemical Project Leader at CFI for the entire Tox21 library, with final solubility determined by 

visual inspection. For large MW compounds, polymers, or ill-defined substances with unknown 

or ill-determined MW, a weight-based stock solution concentration of 10 mg/ml is targeted. 

Stock solutions are stored in one or more bar-coded vials (depending on total volume of solution) 

at -20 °C. Solubilization results are reported to EPA, with each bar-coded solution vial assigned 

the designation "Soluble" or "Insoluble", and accompanied by an approximate stock solution 

concentration and total quantity of solution available. Insoluble compounds remaining in DMSO 

are returned to the freezer for storage. 

Plating orders shipped to ToxCast and Tox21 assay vendors and partners generally 

involved EPA providing CFI with a list of solution vial barcodes, and specifying a plate type (96 

or 384 well), solution volume, shipping address and contact. In most cases, a particular sample-
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well plating order and inclusion of randomly placed sample replicate solutions are specified by 

EPA. All CFI plates are bar-coded and the process of plating is fully automated and 

accompanied by final dilutions and precise determination of plated solution concentrations and 

volumes. Plates are then sealed, frozen and shipped overnight, well-packed on dry ice. At the 

time of shipment (usually within 1-2 days of plating), CFI provides the EPA Contract Officer 

Representative (COR) with an electronic plate map file, generated from CFI's COMIT database, 

listing all CFI compound identifiers, the plate barcode and well address of each sample, along 

with the supplier-provided sample identifiers (name, CAS, MW, etc.) and CFI-determined final 

concentration and volume of each sample. 

D.2 QC for ID and chemical structure

EPA maintains a record of every CFI shipment, vendor, plate barcode, inventory, and 

volume of sample plated. EPA also created and maintains an independent EPA ChemInventory 

MS ACCESS database that stores information on all EPA-procured bottles, solutions, and 

sample IDs used in plating. This database fully incorporates, (for additional consistency and QC 

purposes) the CFI COMIT database records for EPA's entire Tox21 inventory, which are 

accessed securely on-line to perform regular updates. EPA’s database also incorporates 

information resulting from EPA’s review of the supplier-provided chemical annotations and 

COAs, as well as further information pertaining to solutions, plating status (active, retired), 

corrected concentrations (resulting from corrected molecular weights), test inventory (Phase I, II, 

etc.), bottle barcode lot-matches, etc. At the time of plate shipment, EPA processes the CFIgenerated plate map file, strips the file of chemical identifiers, and replaces these identifiers with 

a blinded EPA_Sample_ID. This ID is identical to the CFI solution barcode in almost all cases, 

but new EPA_Sample_IDs are assigned by EPA to the randomly placed sample replicates to 

mask their identities. The EPA ChemInventory database maintains a record of every assigned 

EPA_Sample_ID provided to assay vendors and collaborators, mapped to the original solution 

barcode. A blinded plate map file that includes this EPA_Sample_ID, along with the plate 

barcode, plate well address, and solution concentration for each plated sample is provided by 

EPA, usually within a day of the plate shipment, to the assay vendor or partner. All assay results 

subsequently generated by assay vendors and partners, in turn, are reported back to EPA by 

EPA_Sample_ID, which is decoded using tables from the ChemInventory database that have 

been fully incorporated into EPA’s assay-data-handling database, ToxMiner (described 

elsewhere). 

A significant step in the process of chemical information quality review associated with 

new chemical procurements is the EPA verification of the chemical identity (chemical name, 

CAS, MW) from the COA information provided by the supplier, and the accurate substance and 

structure annotation of the generic chemical (independent of supplier, lot, batch variation) within 

the DSSTox project. Once EPA is provided with the supplier-provided documents (COA and 

MSDS), COA information pertaining to chemical identity (name, CAS, hydration, salt form, 

molecular weight) and analysis (analytical method, purity determination, expiration date) is 

manually extracted and entered into the ChemInventory database, along with safety cautions 

extracted from the MSDS (reacts violently with water, stench, reacts with air, avoid light, etc). 

For a substantial fraction of procurements, supplier-provided information in COMIT is either 

missing entirely (name or CAS), incomplete (e.g., missing hydrate, salt or complex form) or, in a 

small number of cases, entirely incorrect (wrong CAS, name assigned to substance based on 

supplier error) on the basis of this review. In the absence of COA's, further information to 
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confirm chemical identities can sometimes be located on manufacturer's or supplier's websites. 

Most often, when a chemical annotation is corrected, the MW also requires correction; the 

revised MW is then used by EPA to adjust the solution concentration based on the originally 

reported CFI MW and concentration. Corrected solution concentrations are replaced in the plate 

map files prior to being provided to assay vendors and partners. Once the chemical identity of a 

sample has been sufficiently established from supplied documentation, further review takes place 

within the EPA DSSTox project to ensure consistency and accuracy of assigned DSSTox 

substance and structure annotations1. Once this final review is completed, primary DSSTox 

identifiers (GSID, TestSubstance_ChemicalName, TestSubstance_CASRN) are entered into the 

ChemInventory Bottle and Sample ID tables to establish linkage between the DSSTox Master 

and the ChemInventory databases. DSSTox structures (used to generate a final SDF file) and 

chemical identifiers (corrected names, CAS, MW) override the CFI supplier-provided identifiers 

in all cases where they disagree. 

Lastly, with regards to the Tox21 inventory, although EPA did not procure or manage the 

plating of chemical samples from the two other contributing Tox21 partners (NTP and NCGC), 

EPA performed a substantial amount of chemical information QC on the NCGC supplierprovided samples, and both the NCGC and NTP chemical inventories were incorporated into 

DSSTox and were subject to the standard DSSTox chemical annotation review procedures. 

Hence, final chemical substance and structure annotations for the entire Tox21 inventory are 

assigned within the DSSTox project, and the master mapping of unique Tox21 solution IDs, used 

for reporting of Tox21 assay results, to DSSTox substance identifiers and IDs centrally resides in 

EPA’s ChemInventory database.

D.3 Analytical QC

In addition to the chemical information quality review and curation of COA information 

for each sample, analytical QC procedures to establish purity, identity, concentration and 

stability of all plated Tox21 samples, including Tox21 EPA samples, are being carried out in 

association with the Tox21 program under an NCGC-administered contract with OpAns 

Analytical Laboratory, located in Durham, NC. A full set of 384 well parent plates, identical to 

those subjected to the start of Tox21 assay testing, were submitted at time zero (t=0) for LC/MS 

(liquid chromatography mass spectrometry) analysis. The concentration chosen for the 

analytical analysis was 3 mM and a volume of 20 μL. Those passing identity (MW) checks with 

purity greater than 50% (Grade A, >90%, Grade B, 75-90%, Grade C, 50-75%, etc. Full set of 

QC grades are given in Table D-1) are not subject to further t=0 analysis. 

Table D-1 Grades for analytical QC 

Grade Explanation

A The expected molecular weight has been confirmed and the purity is >90% based on 

the analytical technique employed. There is a high degree of confidence in the purity 

and identity of the compound being tested. No additional analytical testing is 

planned.

B The expected molecular weight has been confirmed, however, the purity is estimated 

to be between 75% and 90% based on the analytical technique employed. No 

additional analytical testing is planned.

 1 http://www.epa.gov/ncct/dsstox/ChemicalInfQAProcedures.html
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C The expected molecular weight has been confirmed, however, the purity is estimated 

to be between 50% and 75% based on the analytical technique employed. No 

additional analytical testing is planned.

D The expected molecular weight has been confirmed, however, the purity is estimated

to be <50% based on the analytical technique employed. The major component in this 

sample is not the sample of interest. Additional analytical testing is planned to 

confirm these results.

X Concentration < 5% - FAILED

E Analysis by the analytical technique employed gave no information. Additional 

testing is planned using an alternate analytical technique to confirm purity and 

identity. 

F The sample fails identity and purity. The molecular weight determined is not the

expected molecular weight for this compound. There is no confidence in any results 

derived from this sample. Additional analytical testing is planned to confirm these 

results.

G The expected molecular weight has been confirmed, however, no information on the 

purity of the compound could be obtained based on the analytical technique 

employed. Additional testing is planned using an alternate analytical technique to 

determine the purity of the compound. 

H The purity of the compound is >90%, however, no information on the identity of the 

compound could be obtained based on the analytical technique employed. Additional 

testing is planned using an alternate analytical technique to confirm the identity of the 

compound. 

I This sample contains two or more isomers. The expected molecular weight has been 

confirmed and the composite purity is >90% based on the analytical technique 

employed. No additional analytical testing is planned.

M This sample is a known mixture. Two or more components which are part of this 

solution have been identified.

W This compound has been withdrawn. Data subsequent to plating indicates that the 

sample is volatile or insoluble. Compound is not present in the well.

Those failing the identity or purity check, or for which no usable results are generated and the 

LC/MS method is deemed unsuitable (such as for low MW compounds and suspected volatiles), 

undergo follow-up GC/MS (gas chromatography mass spectrometry) at the National Institutes of 

Standards & Technology (NIST). Other failed compounds are potentially subject to follow-up 

LC/MS testing to increase the effective MW range, improve detection of polar compounds, and 

confirm insoluble samples using Flow Injection Analysis (FIA). An initial review of the LC/MS 

chromatograms is carried out by OpAns, with follow-up review, ordering of additional analysis, 

and final analytical QC Grade assignments provided by an NCGC analytical chemist experienced 

with HTP operations. In addition to the initial set of analytical QC plates analyzed at t=0, a 

second identical set of Tox21 plates, stored at room temperature under the same conditions as 

those undergoing assay analysis, will be analyzed at t=4 months to assess sample stability over 

time across the entire Tox21 compound library. For the subset of samples passing at t=0 but 

failing at t=4 months, testing will additionally be carried out for t=3 months. This information 

will be used to inform subsequent assay analysis, and to set an overall “expiration” date on the 

Tox21 plates undergoing assay testing. Finally, a summary report of the QC analytical results, 
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accompanied by the final QC grade for each Tox21 ID solution-level sample, will be made 

publicly available to inform the use and interpretation of Tox21 assay data. 

D.4 EDSP21 subset of chemicals

Finally, the Tox21 project and complete chemical library, i.e. including the NTP and NCGC 

contributions, has additional importance and relevance to the larger EDSP21 program objectives. 

Firstly, the complete Tox21 chemical library is being tested in all NCGC endocrine-related 

assays, which comprise a subset of the EDSP21 ToxCast endocrine assay subset. Secondly, the 

Tox21 NTP library includes a pilot study set of 118 defined mixtures, with each mixture 

comprised of 2 to 64 chemical components. Each mixture component is a known ER or AR 

active chemical and also is present individually in the larger Tox21 library. Thirdly, the Tox21 

chemical library partner contributions from NTP and NCGC add substantial chemical structure 

diversity, functionality (chemical and biological), and overlap (overlapping chemicals, as well as 

structurally related chemicals) to the chemical-property space covered in relation to the NCGC 

endocrine-related assays. To elaborate this last point, the Tox21 chemical library consists of 

8,307 unique substances, but when the 3 partner plate contributions to the common library, 

separately procured and prepared, are considered, there are a combined total of 10,444 unique 

plated substances. Hence, there are 2,225 independently sourced replicates, in addition to the 

official Tox21 replicate set of 88 compounds included in duplicate on all Tox21 1,536 well 

plates. In particular, the Tox21 EPA and NTP compound libraries have considerable substance 

overlap (1,328 compounds) that can strengthen independent assay result verification. The nonoverlapping portion of NTP’s library contains many more toxicity reference chemicals and 

chemicals pertaining to the NTP toxicity research and the bioassay testing program. NCGC’s 

Tox21 inventory consists of over 3,500 unique substances, all marketed, commercially available 

drugs that could be procured for testing and solubilized in DMSO, with approximately 600 

compounds that overlap the EPA Tox21 inventory. Hence, the EDSP21 1,860 library is 

expected to have considerable overlaps and structural analogs across the complete Tox21 

inventory, which in turn creates expanded coverage and context for the EDSP21 chemical-assay 

results.