1 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 additi onal 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 select ed 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.

Includ ed 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 chron ology 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

i ngredients 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, b isphenol 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 toxicit y 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/w ater 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 compound2 sample s 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 Re search 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 Chemis try, 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 compoun ds, 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, t he 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 majo rity 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 pre

tared, 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 s ubsequently 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 (SD F) 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 m olecular 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 s upplier

- provided vials (requiring splitting, weighing and transfer to CFI bar

coded 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 concen tration 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

- 2 0 °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 comp ounds 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 we ll), solution volume, shipping address and contact. In most cases, a particular sample

-3 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 a utomated 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

purpose s) 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 plat e shipment, EPA processes the CFI

generated 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 dat abase, 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 p rovided 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 i nformation 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 caut ions 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 h ydrate, 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 to4 confirm chemical identities can s ometimes 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 CF I 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, fur ther review takes place within the EPA DSSTox project to ensure consistency and accuracy of assigned DSSTox

substance and structure annotations 1 . Once this final review is completed, primary DSSTox identifiers (GSID, TestSubstance_ChemicalName, TestSubsta nce_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 a nd NCGC), EPA performed a substantial amount of chemical information QC on the NCGC supplier

provided samples, and both the NCGC and NTP chemical inventories were incorporated into DSSTox and were subject to the standard DSSTox chemical annotation review p rocedures. 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, conce ntration 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 wel l 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 o f 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

G rade 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.html5 C The expected molecular weigh t 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 A nalysis (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 stabil ity 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,6 accompanied by the final QC grade for each Tox21 ID solution

- level sample, will be made publicly available to inform th e 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 mixt ure 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 (chemic al 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 rep licates, 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 strengthe n independent assay result verification. The non

overlapping 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 h ave considerable overlaps and structural analogs across the complete Tox21 inventory, which in turn creates expanded coverage and context for the EDSP21 chemical

- assay results.