Computational Toxicology Research Program
SDF Download Page
KIERBL: EPA Estrogen Receptor Ki Binding Study (Laws et al.)
** Launch Version 1a, 17 February 2009
Quick & Easy File Downloads: FTP Download Instructions
New Users: For general information, see DSSTox Project Goals and About DSSTox. For additional information on DSSTox SDF (Structure Data Format) files and their use in Chemical Relational Databases, see More on SDF and More on CRDs.
Description: This study was conducted by EPA researchers to evaluate the validity of the rat uterine cytosolic (RUC) estrogen receptor (ER) competitive binding assay for use in the Endocrine Disruption Screening Program (EDSP). The assay measures the ability of radiolabeled 17-beta-estradiol (3H-E2) to bind with RUC ER in the presence of increasing concentrations of a test chemical. The goal is to employ this in vitro assay as a 1st tier sceening tool for assessing the potential of structurally diverse environmental chemicals to bind to the ER, as well as to support research efforts to develop computational models to predict ER binding. A distinguising feature of the present study from other ER competitive binding studies (see, e.g., NCTRER) was the use of secondary experimental analysis to confirm that measured IC50 values corresponded to true positive results. This was accomplished by secondary analysis using Lineweaver-Burk plots and slope replots to determine true competitive inhibition and an experimental Ki value (inhibitor binding constant) -- see also: Relating IC50 values to Ki values . The study concluded that a number of experimental conditions can confound interpretation of IC50 values obtained from the RUC ER-binding assay data and lead to false positive results. In particular, some chemical treatments likely alter the stability of the assay by changing the buffer pH, denaturing ER, or disrupting the ER binding kinetics. In addition, in some cases, neither an IC50 or Ki value could be determined due to solubility constraints (i.e., sufficient concentration of the chemical to produce greater than 40% inhibition could not be experimentally achieved).
The chemicals used in this study were selected as part of a validation test for two Quantitative Structure-Activity Relationship (QSAR) models designed to predict ER binding [Fed. Reg. 67(250), 79618, Dec. 30, 2002]. Several hundred chemicals were initially screened, including approximately 100 chemicals selected from among the High Production Volume (HPV) chemicals included in EPA's Toxic Substances Control Act (TSCA) inventory that were predicted to be positive for ER binding, with the rest selected at random from the TSCA inventory. All chemicals were initially tested in ER competitive binding assays by Battelle Pacific Northwest Laboratories, Richland, Washington [EPA Contract Number 68-W-99-033, Task 6]. Approximately 50 chemicals produced binding curves that: (1) strongly implied competitive inhibition of ER binding, or (2) produced partial curves or irregular (non-sigmoid) binding curves. These chemicals were further evaluated in an ER competitive binding assay with secondary analysis to confirm true competitive inhibition and to determine an experimental Ki [U.S. EPA Contract 3D-5967-NTEX]. The results of RUC ER screening for these 50 structurally diverse chemicals, for which secondary confirmatory analyses were performed, were published in the Main Citation (Laws et al., 2006). The remaining 228 chemicals were designated as non-binders (i.e., inactives) since these chemicals at concentrations up to 100 micromolar failed to inhibit the maximum binding of radiolabeled estradiol more than 20%.
The DSSTox KIERBL data files include all published IC50 and Ki experimental results for the 50 chemicals included in the Main Citation (Laws et al., 2006) (denoted Group 1), as well as previously unpublished results for an additional 228 structurally diverse TSCA chemicals for which no ER binding was observed (denoted Group 2). IC50 values were determined for 32 compounds in Group 1. For 19 of these having nearly complete or partial competitive binding curves, i.e. displacing >75% and >50% 3H-E2, respectively, secondary analysis was able to determine Ki values for a total of 17 compounds (designated "active"), whereas two of the 19 compounds were determined by secondary analysis to be non-binders. Of the remaining 31 Group 1 chemicals, no Ki could be determined for 8 compounds due to solubility limitations (designated "inconclusive"), and 23 chemicals were confirmed as non-binders (designated "inactive"). Hence, of the original 32 compounds in Group 1 with IC50 values indicating some level of competitive ER binding, Ki values could be determined for only 17 of these (53%), whereas 7 compounds (22%) were confirmed to be non-binders, and 8 (25%) produced inconclusive results due to solubility constraints. These results indicate that the incidence of true ER binders detected by the RUC ER assay was substantially less than implied by IC50 values alone. The 228 compounds in Group 2 were tested in the RUC ER competitive binding assay and designated as non-binders (i.e., inactives) since these chemicals at concentrations up to 100 micromolar failed to inhibit the maximum binding of radiolabeled estradiol more than 20%.
Source Contacts: Susan C. Laws, U.S. EPA, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Endocrinology Branch, MD-72, Alexander Drive, Research Triangle Park, NC 27711; Email: firstname.lastname@example.org;
Jim Kariya, EPA, OPPTS, Office of Science Coordination and Policy, EACPD, EPA East Building, 1200 Pennsylvania Ave, N.W., Mail Code 7203M, Washington, DC. 20460; Email: email@example.com
Laws SC, Yavanhxay S, Copper RL, Eldridge JC. 2006. Nature of the binding interaction for 50 structurally diverse chemicals with rat estrogen receptors. Toxicological Sciences. 94(1), 46-56; doi:10.1093/toxsci/kfl092.
U.S.EPA. 2002. Endocrine Disruptor Screening Program: Chemical Selection Approach for Initial Round of Screening. Federal Register, 67(250): 79618.
USEPA. 2002. Development of estrogen binding data for approximately 300 chemicals: final data package submittal. EPA Contract No. 68-W-99-033, Work Assignment 3-04.
Guidance for Use: A user of the DSSTox KIERBL SDF files is encouraged to consult the Main Citation (Laws et al., 2006) for further information and experimental details pertaining to this study (for further information on assay protocol, see also ICCVAM/NTP document). Although IC50 values are provided in the DSSTox KIERBL data file for completeness sake (IC50_microM), their use as a quantitative activity endpoint for QSAR modeling of ER binding is not recommended based on the overall conclusions of this study. Rather, reported Ki values are believed to provide the more definitive measure of true ER binding and the more accurate quantitative measure of binding inhibition. Hence, DSTox PubChem activity assignments, ActivityOutcome_KIERBL and ActivityScore_KIERBL, are based on Ki values of confirmed binders (Ki_microM_mean), with 17compounds classified as "active", 8 compounds labeled "inconclusive" due to solubility constraints preventing determination of Ki values, and the remaining 253 compounds classified as non-binders and labeled as "inactive". ActivityScore_KIERBL values project log10(Ki_microM_mean) values onto a 10-100 activity range, with the strongest binder (E2) assigned a score of "100", the weakest confirmed binder assigned a score of "10", inconclusives assigned a score of "5", and inactives assigned a score of "0".
The KIERBL Field Definition File (see Download Table below) contains essential documentation and should be downloaded with, and accompany any use of the DSSTox KIERBL SDF file. The KIERBL Log File provides database summary information (field, chemical counts, etc.) and a description of procedures and quality assurance checks used in SDF file creation. In addition, the Log File documents modifications incorporated into version/revision updates of the DSSTox KIERBL SDF file. For additional information on DSSTox SDF files and their use in Chemical Relational Databases, see More on SDF and More on CRDs. To report errors in any KIERBL documentation or SDF data file, click on File Error Report here or below.
Source_ChemicalName new field added Jan2009
* Note: For detailed information on SDF content, see KIERBL_FieldDefinitionFile in Download Table below.
Log File (PDF) provides SDF data file version history and summary information (field, chemical counts, etc.), and a description of procedures and quality assurance checks used in SDF file creation;
Field Definition File (PDF or MS Word doc file) provides field definitions and essential documentation, and should be downloaded with and accompany any use of the DSSTox SDF file;
Structure Data File (SDF) is the main DSSTox product, providing the complete inventory of chemical structures, DSSTox Standard Chemical Fields, and all Source-specific data fields [Note: the structure field is blank for all records containing mixtures or undefined substances];
Data Table MS Excel (MS Office 2003) file contains the full SDF data contents in spreadsheet table form, minus the chemical structure field [file created with CambridgeSoft ChemDraw Ultra 2004 plug-in to MS Excel 2003];
Structures Table (PDF) file contains a tiled format graphical view of all chemical structures contained in the SDF file, annotated with TestSubstance_CASRN and truncated TestSubstance_ChemicalName field entries for the tested form of the chemical [file created with ACD ChemFolder, ver. 11.02, ACD Labs].
|You will need Adobe Acrobat Reader, available as a free download, to view the Adobe PDF files on this page. See EPA's PDF page to learn more about PDF, and for a link to the free Acrobat Reader.|
These files constitute the main DSSTox products. DSSTox Documentation Files use standard templates, and DSSTox Structure Data Files and DSSTox File Names adhere to strict formatting standards and conventions. For additional information, see More on DSSTox Standard Chemical Fields, Known Problems & Fixes, Chemical Information Quality Review Procedures, and How to Use DSSTox Files.
Quick & Easy File Downloads: FTP Download
Return to Top
Acknowledgements: The DSSTTox SDF file for KIERBL includes the 50 chemicals in the original Laws et al. publication and an additional 228 chemicals tested by Battelle Pacific Northwest Laboratories, Richland, WA (USEPA Contract 68-W-99-033, Task 6). The list of additional chemicals was kindly provided by Jim Kariya, EPA. The file was converted to DSSTOX format by Maritja Wolf (Lockheed Martin, Contractor for EPA) and Ann Richard (EPA). We additionally thank Michelle Hotchkiss, Janet Ferrell (EPA) and Rocky Goldsmith (EPA) for assistance with the review of the final files and documentation.
DSSTox Citation: S. Laws, J. Kariya, M. Wolf, and A.M. Richard (2009) DSSTox EPA Estrogen Receptor Ki Binding Study (Laws et al.) Database - (KIERBL): SDF file and documentation, Launch version: KIERBL_v1a_278_17Feb2009, www.epa.gov/ncct/dsstox/sdf_kierbl.html
Disclaimer: Every effort is made to ensure that DSSTox SDF files and associated documentation are error-free, but neither the DSSTox Source collaborators nor the EPA DSSTox project team make guarantees of accuracy, nor are any of these persons to be held liable for any subsequent use of these public data. The contents of this webpage and supporting documents have been subjected to review by the National Center for Computational Toxicology and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. See additional disclaimers.