A collection of 1,408 substances (except where noted, the term “substance” is used interchangeably with “compound” here) was constructed for characterization in qHTS assays (Smith et al. 2007 ; Tice et al. 2007 ); 1,408 is the number of substances that can fit in a single 1,536-well plate exclusive of controls. To allow evaluation of assay reproducibility, 55 of the compounds were represented twice in the collection, giving a total of 1,353 unique compounds. Of these, 1,206 had been tested by the NTP in one or more in vitro and/or in vivo assays, including those for Salmonella typhimurium mutagenicity (68%), chronic toxicity/carcinogenicity (23%), reproductive toxicity (3%), developmental toxicity (3%), and immunotoxicity (1%). Also included were 147 reference compounds identified by the ICCVAM for the development and/or validation of alternative in vitro test methods for dermal corrosivity, acute toxicity, and endocrine activity. Molecular weights of all compounds ranged from approximately 32 (methanol) to 1,300 (actinomycin D), with 95% of the compounds having a molecular weight that was < 400. Functionally, the NTP library of 1,408 compounds includes solvents, fire retardants, preservatives, flavoring agents, plasticizers, therapeutic agents, inorganic and organic pollutants, drinking-water disinfection by-products, pesticides, and natural products. Compounds excluded from this NTP collection were those considered excessively volatile and those not soluble in dimethylsulfoxide (DMSO), the solvent used for compound transfer. A complete list of the NTP 1,408 compounds and full chemical descriptions are publicly available (PubChem 2007a ).
All compounds were received from suppliers via the NTP chemistry support contract in 1-mL aliquots at 10 mM dissolved in DMSO and stored at −80°C in Matrix TrakMates 2D bar-coded storage tubes (Thermo Fisher Scientific, Hudson, NH). Purity and identity information for the compounds was obtained from the suppliers and, in the case of compounds used in NTP studies, from the characterizations performed in support of those studies. With the exception of natural compounds and other known mixtures, most compounds were > 90% pure.
Sets of compounds prepared as 10-mM stock solutions and stored in 96-well plates were compressed into 384-well plates. From these plates, fifteen 384-well plates containing the 1,408 compounds at 2.236-fold dilutions were prepared using an Evolution P3 system (PerkinElmer, Inc., Wellesley, MA). The sets of 384-well plates composing the dilution series were then compressed into multiple 1,536-well plates by interleaved quadrant transfer. During screening, working copies of the 1,536-well compound plates were stored at room temperature for up to 6 months; back-up copies were heat sealed and stored at −80°C.
All compounds were received from suppliers via the NTP chemistry support contract in 1-mL aliquots at 10 mM dissolved in DMSO and stored at −80°C in Matrix TrakMates 2D bar-coded storage tubes (Thermo Fisher Scientific, Hudson, NH). Purity and identity information for the compounds was obtained from the suppliers and, in the case of compounds used in NTP studies, from the characterizations performed in support of those studies. With the exception of natural compounds and other known mixtures, most compounds were > 90% pure.
Sets of compounds prepared as 10-mM stock solutions and stored in 96-well plates were compressed into 384-well plates. From these plates, fifteen 384-well plates containing the 1,408 compounds at 2.236-fold dilutions were prepared using an Evolution P3 system (PerkinElmer, Inc., Wellesley, MA). The sets of 384-well plates composing the dilution series were then compressed into multiple 1,536-well plates by interleaved quadrant transfer. During screening, working copies of the 1,536-well compound plates were stored at room temperature for up to 6 months; back-up copies were heat sealed and stored at −80°C.
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