1

Appendix F: Section 6 Supplemental, Descriptions of HTP Assays for the Detection of 

Estrogen Receptor Agonists

F.1 Estrogen Receptor Transactivation Assay (Attagene ERα) 

• Short names: ATG ERα TRANS and ATG ERE CIS. 

• Purpose of assay: Detection of chemicals that activate ERα in the context of a 

multiplexed panel of other transcription factors.

• Source of assay: Developed for medium throughput by Attagene, Inc. The company has 

published methods for conducting the assay. Components of assay are covered by 

patents and patent applications filed in the USA and worldwide by Attagene, Inc. 

(http://www.attagene.com/technology.php)

• Primary journal reference for the assay: Romanov et al., Nature Methods 5(3):253-

60(2008); Martin et al., Chem Res Toxicol. 23(3):578-90 (2010).

• General description: 

o Multiplexed reporter gene assays utilizing a library of uniformly constructed 

Reporter Transcription Units (RTUs). Each FACTORIALTM RTU is made in a 

common plasmid backbone and contains a unique TF-inducible promoter that is 

fused to a transcribed reporter sequence. When co-introduced into cell of interest, 

the RTUs produce reporter RNAs in amounts commensurable with the activities 

of the corresponding TFs present in a cell. To provide equal detection 

opportunities for different transcription factors, all FACTORIALTM RTUs are 

essentially identical reporter sequences. To distinguish reporter sequences 

produced by different RTUs, each sequence is provided with a short processing 

tag (HpaI restriction cleavage site), position of which varies among the RTUs. 

Thus, reporter sequences can be discriminated upon cleavage with the 

corresponding processing enzyme. The cleaved reporter species are separated by 

high resolution capillary electrophoresis (sequencing) and quantified. 
2

o A key aspect of the FACTORIALTM technology is that all steps of the detection 

protocol are performed using a homogeneous set of reagents in a single reaction 

tube format, providing highly uniform conditions for detecting multiple 

transcription factors.

o ATG_ERα_cis assay:

 RTU’s for all multiplexed reporter genes are transiently co-transfected in 

to the human HepG2 cell line variant HG19.

 Full length, wild type, human ERαNM_000125) is transiently cotransfected into same cells for the ATG_ERα_cis assay.

 RTU for ATG_ERα_cis contains a 5X ERE synthetic response element.

o ATG_ERα_TRANS assay

 GAL4-ERa-Ligand Binding Domain fusion protein construct is transiently 

co-transfected in to HepG2 cell line variant HG19 along with the RTU 

controlled by a 5X-UAS-TATA promoter.

 Cells from individual co-transfections for each of the nuclear receptors 

that are multiplexed in the TRANS assays are mixed and plated for 

testing.

• Notes relating to other ER assays: 

o See accompanying data for replication and accuracy using blinded chemical 

replicates and reference chemicals

o Assay has not been performed between laboratories

o Assay has advantage of much higher information content for multiple 

transcription factor activities

Assay protocol

• Assay components and general procedure:

o Source/characteristics of ER cell line: 

 Human liver hepatoma HepG2 cell line (clonally selected HG19 variant)

 Cis assay uses transiently transfected full-length, human ERα

 Trans assay uses transfected hERα-LBD/GAL4 DNA-binding domain 

fusion protein in mammalian two-hybrid format

o Cell culture and plating conditions: 

 DMEM supplemented with 1% charcoal-stripped FBS.
3

 Cells transfected in suspension and transfected cells plated at 3x105

cells/well or 0.8x105 cells/cm2.

 Cells are treated with chemicals for 24 hr following a 24 hr attachment 

incubation.

 Cell passage number limited to 36 for transfections or replaced with fresh 

thaws from a single batch every 3 months, whichever is less.

 High quality tissue culture reagents including large batch of HyCLone 

FBS are used. All reagents are tested by FACTORIAL assay before using 

for EPA ToxCast screenings.

 Basal TF activities assessed weekly for phenotypic drift and replaced with 

newly thawed, early passage cells if drift detected.

• a) Specifically, cells replaced if the correlation coefficient of tested 

basal profile vs. standard profile is less than 0.85. Cells replaced if 

basal activity of TGF endpoint is higher than 0.8. The increase of 

TGFβ endpoint correlates well with reduced responses to multiple 

inducers.

• b) Cells replaced if significantly reduced response (less than 2 

fold) to the following control inducers is observed: estradiol (ER, 

ERE), Dex (GRE-cis, GR-trans), CDCA (FXR), Rifampicin (PXRcis, PXR-trans), Rosiglitazone (PPRE-cis, PPARg-trans).

o Media pH: 7.2-7.4.

o Time, temperature, incubation period: 24 hr exposure, 37°C

o Solvent: All ToxCast testing has used DMSO as a universal solvent. Attagene has 

also used water and water/2% serum for testing other samples (e.g. nanomaterials)

o Test chemical: 

 Chemicals are almost all 20 mM stocks in DMSO

 Concentration response series used with 8 concentrations, 3-fold dilutions, 

high concentration = 200 micromolar (0.091, 0.274, 0.823, 2.469, 7.407, 

22.22, 66.67, 200 micromolar)

 Serial dilutions prepared in DMSO 

 No solubility testing performed

o Cytotoxicity test: tested for cytotoxicity against HepG2 cells in XTT (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay 

following 24 h chemical exposure to five concentrations with an upper 

concentration of 200 μM and 10-fold dilutions. All concentrations were run in 

triplicate. AC50 values were determined on the basis of 50% cell death, as

measured by decreasing XTT conversion to formazan.

o Detection system: Total RNA using TriZol reagent (Invitrogen), treated it with 

DNAse I (Ambion) for 3 min, and reverse transcribed the RNA using oligo(dT) 

primer and Mo-MLV reverse transcriptase (Invitrogen). PCR-amplified one-tenth 

of the reverse-transcribed RNA using Taq DNA polymerase (Invitrogen) and two 

reporter sequence–specific primers. Labeled PCR products by primer extension 

with 6-carboxyfluorescein (6-FAM) 5’-labeled reporter sequence–specific primer 

(2 min at 95 1C, 20 s at 68o

C and 10 min at 72oC) and then digested products with 

5U of HpaI (New England Biolabs) for 2 h at 37o

C. Using Qiaquick PCR columns 

(Qiagen), purified the fragments and analyzed on an ABI 3130xL genetic analyzer 
4

(Applied Biosystems). Peaks’ positions were identified by using a set of Xrhodamine (ROX)-labeledMapMarker1000 molecular weight standards 

(BioVentures).

o Endpoint reported: quantity of specific cDNA transcripts (i.e. the 5X-ERE-RTU 

or the ERα-5X-UAS-TATA RTU for the CIS and TRANS assays, respectively) of 

each RTU in chemical-treated cells relative to quantity of that transcript in 

DMSO-treated cells. Units are Fold-Change. This was then normalized to 

maximum Fold-Change of the 17β-estradiol positive control compound run on the 

concurrent control plate and expressed as % of Control.

• Reference estrogens and positive/negative /solvent controls

o Negative control: 0.33% DMSO (or 1.0% for the 200 μM sample treatment)

o Positive control: 0.2 μM 17β-estradiol

o Use of controls: 

 Concurrent 17β-estradiol positive control run in triplicate for each assay 

run at 2.00E-07 M. This is used to normalize the data on a screening run 

basis. Controls available for all ToxCast assay runs.

• Plate format

o Forty eight (48) chemicals are serially diluted and generate four 96-well plates 

(two concentrations per plate). Four samples are replaced with DMSO and 

processed separately to allow inclusion of DMSO controls on assay plate.

o Each 96-well plate is applied to eight 12-well plates with cells. Thirty two 12-well 

plates per each FACTORIAL assay (CIS or TRANS).12-well plate provides 

sufficient cell number for robust mRNA isolation. Each plate contains 5 

chemicals at two concentrations plus two DMSO controls

o Following cell lysis, eight 12-well plates are re-assembled in to 96-well plates (4 

total) for sample processing (RNA isolation, DNase treatment, RT, PCR, 

Capillary electrophoresis)

o Replication

 Chemicals tested in blinded format

 Internal chemical replicates included in chemical libraries

 Concentration-response format for all chemicals with one well per 

concentration

 Reference chemicals are run in triplicate on a separate plate, one set of 

reference chemicals per run.

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid 

o Cells: 

 Morphology and growth efficiency of cells is assessed before transfection 

for normal appearance. 

 Basal profile of TF activities using multiplexed reporter gene endpoints is 

assessed every week by comparing reporter gene results with historic 

activity for possible phenotype drift. If qualitative or quantitative drift is 

detected, cells are replaced with fresh ones.

o Compound treatment:

 Visual assessment of cell viability in each treated well before cells 

collected. Presence of precipitated chemicals or crystals is also recorded.
5

 Correlation between visual and XTT viability data is assessed.

o RNA

 Presence of RNA in the sample is assessed if PCR problems are detected.

o PCR:

 All PCR samples are assessed by agarose electrophoresis.

 Presence of amplified plasmid DNA is assessed. It is different in size from 

cDNA. 

 The strength and position of amplified cDNA PCR fragment is assessed. 

 Presence of non-specific amplification (wrong fragment size) is assessed.

 Correlation of PCR data with XTT viability data is assessed.

o Capillary electrophoresis QC criteria:

 Software fails to extract meaningful data from readout. 

 Uniformity and size of red markers (see figure below).

 M-19, M-32 marker values out of normal range

 M-19/M-32 marker ratio out of normal range

 0 value of any endpoint

 Negative value of any endpoint

 Presence of non-specific peaks on capillary electrophoresis (PCR 

specificity problem). 

Known issues with assay performance 

• False negatives due to compound solubility issues

• Cytotoxicity can result in increased transcription of ER reporter together with a group of 

other transcription factors which may be false positive with respect to binding to the ER 

ligand-binding domain. The mechanism of this is unknown and was empirically observed 

during chemical testing.

• Limits on concentration of test chemicals: Limited by DMSO tolerance of assay (0.5%)

• Metabolic capability of cell line:

o Consensus is that HepG2 cells have metabolic capabilities for xenobiotics 

although not at the level of liver or primary hepatocytes (Wilkening S, Stahl F, 

Bader A. Comparison of primary human hepatocytes and hepatoma cell line 

Hepg2 with regard to their biotransformation properties. 2003 Drug Metab 

Dispos. 31:1035-42.)

o The assay uses clone HG19 variant derived at Attagene that was selected for 

higher level of CYP expression and transfected with fl hPXR.
6

• Cytotoxicity

o Can cause a group of transcription factors to become activated at similar 

concentrations. See previous description of cytotoxicity filter.

o Can cause biphasic concentration-response curve and limit detection of true Emax 

potential

• Stability of reagents: No known issues

• Solubility, volatility, physical properties, and stability of test chemicals

o Chemical library filtered by predicted solubility and volatility parameters. On 

average, there should be minimal problems. 

o Chemical structures are confirmed by GC/MS or other appropriate technique in 

the DMSO stock within one year of preparation. The available analytical 

chemistry will be included in the results.

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 

F.2 Estrogen Receptor Transactivation Assays (Tox21_ERα_BLA) 

• Short name: Tox21 BLA

• Purpose of Assay: Transactivation assays to identify ERα agonists and antagonists

• Source of assay: Tox21_ ERα_BLA: commercial cell line available from InVitrogen, 

Inc. developed to support the drug discovery community in HTS format. β-lactamase 

reporter originally developed for 1536- and 3456-well plates.

• Primary Journal Reference for the Assay: Tox21_ ERα_BLA: Wilkinson et al., J 

Biomol Screen. 13(8):755-65 (2008).

• General description: 

o Mammalian two-hybrid system consisting of HEK293T cells stably transfected 

with 5X_UAS_β-lactamase and a human ERα ligand-binding domain fused to a 

GAL4 DNA binding domain. 

o Should only report ERα activity unless there is heterodimerization with 

endogenous ERβ (in which case it could theoretically report 

ERα:ERβ heterodimer activity if ERβ is expressed in these cells)

o Expression of other steroid receptors not very relevant to two-hybrid system
7

o Reporter gene product is β-lactamase (bla). Cells are loaded with an engineered 

fluorescent substrate containing two fluoroprobes, coumarin and fluorescein. In 

the absence of bla expression, the substrate molecule remains intact. In this state, 

excitation of the coumarin results in fluorescence resonance energy transfer to the 

fluorescein moiety and emission of green light. In the presence of bla expression, 

the substrate is cleaved, separating the fluorophores, and disrupting energy 

transfer. Excitation of the coumarin in the presence of enzyme bla activity results 

in a blue fluorescence signal. The resulting blue:green ratio provides a normalized 

reporter response.

o This assay is mechanistically and functionally similar to a fully validated ER TA 

assay described in Draft OECD TG 457 which is a medium through put version of 

the BG1 Luc ER TA assay; NICEATM-ICCVAM version:

http://iccvam.niehs.nih.gov/methods/endocrine/end_eval.htm.

The major difference is use of the mammalian one-hybrid (GAL4) system 

incorporating the ligand-binding domain of the human ERα. 

• How was the assay developed?

o Developed for commercial purposes by Invitrogen using standard HTS guidelines

o See accompanying data for replication and accuracy using blinded chemical 

replicates and reference chemicals

o Reference compound behavior available from Invitrogen for comparison

o Assay has advantage of internal control for toxicity (green fluorescence channel) 

and can easily be miniaturized to 1536-well format

o Assay not subject to luciferase inhibitors and stabilizers

Assay protocol

• Assay components and general procedure

o Source/characteristics of ER cell line: Tox21_ ERα_BLA: Recombinant human 

ERα-LBD_GAL4-DBD

o Cell culture and plating conditions: 

 Used between passages 5 and 7 from Invitrogen

 5000 cells/well in 1536-well plate

 Cells plated for 5-6 hrs before treatment

 Chemical exposure for 18 hrs

 Medium:

• 98% Phenol red-free DMEM

• 2% Charcoal stripped FBS

• 0.1mM NEAA

• 1mM sodium pyruvate

• 100U/ml Penicillin and 100μg/ml Streptomycin

• pH 7.0

 Antagonist mode: 0.5 nM 17β-estradiol

 Run as three independent replicate experiments

o Solvents: All Tox21 testing has used DMSO as a universal solvent with final 

concentration of 0.5%. 

o Test chemical: 

 Chemicals are almost all 20 mM stocks in DMSO
8

 Concentration response series used with 15 concentrations, 2-fold 

dilutions, concentration range = 92 μM – 6 nM 

 Serial dilutions prepared in DMSO

 Compounds added from DMSO stocks by pin tool addition

 No solubility testing performed

o Cytotoxicity test: Cell lines were tested concurrent with the antagonist version of 

the assay using Tox21_ ERα_BLA: Promega CellTiter Glo (luminescence), an 

ATP-dependent luciferase assay

o Detection system: 

 Cells are loaded with an engineered fluorescent substrate (CCF4-AM from 

Invitrogen) containing two fluorophores, coumarin and fluorescein. In the 

absence of bla expression, the substrate molecule remains intact. In this 

state, excitation of the coumarin results in fluorescence resonance energy 

transfer to the fluorescein moiety and emission of green light. In the 

presence of bla expression, the substrate is cleaved, separating the 

fluorophores, and disrupting energy transfer. Excitation of the coumarin in 

the presence of bla activity results in a blue fluorescence signal. The 

resulting coumarin:fluorescein ratio provides a normalized reporter 

response which can minimize experimental noise that can mask the 

underlying biological response of interest.

 Endpoint reported: coumarin:fluorescein ratio normalized to negative 

control (DMSO) and positive control (17β-estradiol).

• Reference estrogens and positive/negative /solvent controls

o Negative control: 0.5% DMSO 

o Positive control: 17β-estradiol

 Tox21_ ERα_BLA concentration-response curve (3.8-06, 1.3E-06, 4.3E07, 1.4E-07, 4.7E-08, 1.6E-08, 5.3E-09, 1.8E-09, 5.8E-10, 1.95E-10, 

6.5E-11, 2.2E-11, 7.2E-12, 2.4E-12, 8.0E-13, 2.7E-13 M)

 Tox21_ERα_Luc_BG1: (4.6E-6, 1.5E-08, 5.1E-09, 1.7E-09, 5.7E-10, 

1.9E-10, 6.3E-11, 2.1E-11, 6.99E-12, 2.3E-12, 7.8E-13, 2.6E-13, 8.6E14, 2.9E-14, 9.6E-15, 3.2E-15 M)

o Use of controls: 

 Concurrent 17β-estradiol positive control tested in triplicate for each assay 

run.

 Results for the positive controls, including, 17β-estradiol, are available for 

all assays run for EPA chemicals.

• Plate format

o Assays have been miniaturized to 1536-well format and have been conducted in 

lower density format as well. 

o Each 1536-well plate contains 1408 test chemical wells along with columns of 

reference chemicals. The 15-point concentration-series is run across 15 plates. 

The 3 replicates are run with the 1536-well master plate with different 

arrangements. See figures below.

o Replication: 

 Chemicals tested in blinded format

 All chemicals tested in triplicate (or sometimes more replicates)
9

 Replicates present on different plates in different plate positions

 Many internal chemical replicates included in screening set

 Concentration-response format for all chemicals

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid 

o Plate Failure Criteria

 no signal

 failed controls (Z’<0)

 large areas of contamination

 strong tip patterns

 extremely large variations (CV>15%)

 low signal to background ( S/B <2)

Known issues with assay performance 

• False negatives due to compound solubility issues

• Cytotoxicity can result in false negatives

• Potential for cross-contamination from incomplete washing of pin tools used for 

compound delivery

• Blue fluorescent compounds can create false positives in ERα-bla

• Compounds that quench blue or green fluorescence can create false negatives. Data from 

this assay is not sufficient to detect these false negatives, and one would need to look at 

other assays. 

• Limits on concentration of test chemicals: limited by DMSO tolerance of assay (0.5%)

• Metabolic capability of cell line: Considered low for HEK-293 cells

• Cytotoxicity

o Can result in false negatives.

o Can cause biphasic concentration-response curve and limit detection of true Emax 

potential

• Stability of reagents: No known issues

• Solubility, volatility, physical properties, and stability of test chemicals

o Chemical library filtered by predicted solubility and volatility parameters. On 

average, there should be minimal problems. 

o Chemical structures are confirmed by GC/MS or other appropriate technique in 

the DMSO stock within one year of preparation. The available analytical 

chemistry will be included in the results.

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 

The two Tox21 / NCGC assays are run in a format that is significantly different than most HTS 

assays, and understanding plate preparation and layout issues are necessary to understand some 

of the QC criteria discussed elsewhere.
10

Figure F-1: Mapping of source 384-well plates to the 1536-well master plate. The technical 

replicates are ordered slightly differently so in each replicate, the neighbors of a sample are 

slightly different.

Figure F-2: Plate layout with controls and test sample wells described.
11

Figure F-3: Showing how the concentration-response is carried out by using a 15-point serial 

dilution across plates.

15-point

concentrationresponse series
12

F.3 Estrogen Receptor Transactivation Assays (Tox21_ERα_Luc_BG1) 

• Short name: Tox21 BG1 LUC.

• Purpose of Assay: Transactivation assays to identify ERα agonists and antagonists

• Source of assay: Tox21_ERα_Luc_BG1: developed in an academic laboratory (Michael 

Dennison, UC Davis) for use in detection of xenobiotic compounds with estrogen 

receptor activity in HTS format. Modified to 1536-well format by NIH/NCTT.

• Primary journal reference for the assay: Tox21_ERα_Luc_BG1: Gorden et al., 

Organohalogen Compounds, Dioxin 2003, Volume 65, p.78-81. 

• General description: 

o The BG1Luc ER TA utilizes an ER responsive human ovarian adenocarcinoma 

cell line, BG-1Luc4E2, which has been stably transfected with a firefly luciferase 

reporter construct under control of four estrogen response elements placed 

upstream of the mouse mammary tumor virus promoter.

o Cell line obtained under an MTA from Mike Dennison (UC Davis) who 

developed the cell line. 

o BG-1 cells endogenously express both ERα and ERβ; ratio not known.

o Reporter gene product is luciferase enzyme with signal read by a one-step 

luciferase assay. Cells are lysed and luciferin substrate added along with signal 

stabilizers and bioluminescence read using a CCD camera-based reader.

o This is a high-throughput version of Draft OECD TG 457 describes the medium 

through put version of the BG1 Luc ER TA assay; NICEATM-ICCVAM 

version: http://iccvam.niehs.nih.gov/methods/endocrine/end_eval.htm

• How was the assay developed: 

o Developed for high-throughput screening of environmental estrogens in the 

laboratory of Michael Dennison, UC Davis, using standard HTS guidelines.

o See accompanying data for replication and accuracy using blinded chemical 

replicates and reference chemicals

o Reference compound behavior available from published ICCVAM validation 

work

o Assay has higher throughput than OECD validated bench top version

Assay protocol

• Assay components and general procedure

o Source/characteristics of ER cell line: endogenous ERα and ERβ

o Cell culture and plating conditions: 

 Used between passages 8 and 10 from UC Davis.

 4000 cells/well in 1536-well plate

 Cells plated for 22-24 hrs before treatment

 Chemical exposure for 24 hrs

 Medium:

• 90% DMEM phenol red free - low glucose

• 10% Charcoal/dextran treated FBS

• 2mM L-Glutamine

• 100U/ml Penicillin and 100μg/mL Streptomycin

• Media (pH) 7.0

 Antagonist mode: 0.5 nM 17β-estradiol

 Run as three independent replicate experiments
13

o Solvent: All Tox21 testing has used DMSO as a universal solvent with final 

concentration of 0.5%. 

o Test chemical: 

 Chemicals are almost all 20 mM stocks in DMSO

 Concentration response series used with 15 concentrations, 2-fold 

dilutions, concentration range = 92 μM – 6 nM 

 Serial dilutions prepared in DMSO

 Compounds added from DMSO stocks by pin tool addition

 No solubility testing performed

o Cytotoxicity test: Cell lines were tested concurrent with the antagonist version of 

the assay using Promega CellTiter Fluo (fluorescence), measures a constitutive 

protease activity within live cells 

o Detection system: 

 ONE-Glo luciferase reagent from Promega

 Endpoint reported: Luminescence normalized to negative control (DMSO) 

and positive control (17β-estradiol).

• Reference estrogens and positive/negative /solvent controls

o Negative control: 0.5% DMSO

o Positive control: 17β-estradiol 

 Concentrations tested on each plate: (4.6E-6, 1.5E-08, 5.1E-09, 1.7E-09, 

5.7E-10, 1.9E-10, 6.3E-11, 2.1E-11, 6.99E-12, 2.3E-12, 7.8E-13, 2.6E-13, 

8.6E-14, 2.9E-14, 9.6E-15, 3.2E-15 M)

o Use of Controls: 

 Concurrent 17β-estradiol positive control is tested in triplicate for each 

assay run.

 Results for the positive controls, including, 17β-estradiol, are available for 

all assays run for EPA chemicals.

• Plate format: identical to previous assay

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid 

o Plate Failure Criteria

 no signal

 failed controls (Z’<0)

 large areas of contamination

 strong tip patterns

 Extremely large variations (CV >15%)

 low signal to background ( S/B <2)

Known issues with assay performance 

• False negatives due to compound solubility issues

• Cytotoxicity can result in false negatives

• Potential for cross-contamination from incomplete washing of pin tools used for 

compound delivery

• False positives from carryover of luminescent signal from very bright wells to adjacent 

wells 
14

• Compounds that inhibit luciferase activity could be false negatives or false positives in 

ERα_Luc_BG1. Inhibitors of luciferase paradoxically show up as luciferase activators as 

they can stabilize the enzyme from proteolytic degradation (Thorne, N., J. Inglese, et al. 

(2010). "Illuminating Insights into Firefly Luciferase and Other Bioluminescent 

Reporters Used in Chemical Biology." Chemistry and Biology 17(6): 646-657.)

• Limits on concentration of test chemicals: Limited by DMSO tolerance of assay (0.5%)

• Metabolic capability of cell line: BG-1 parental cells express some level of aromatase; 

complete characterization of xenobiotic metabolism capacity not known

• Cytotoxicity

o Can result in false negatives.

o Can cause biphasic concentration-response curve and limit detection of true Emax 

potential

• Stability of reagents: No known issues

• Solubility, volatility, physical properties, and stability of test chemicals

o Chemical library filtered by predicted solubility and volatility parameters. On 

average, there should be minimal problems. 

o Chemical structures are confirmed by GC/MS or other appropriate technique in 

the DMSO stock within one year of preparation. The available analytical 

chemistry will be included in the results.

o Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library.
15

F.4 Estrogen Receptor Transactivation Assays (Odyssey Thera ERα)

• Short name: OT LUC 

• Purpose of Assay: Transactivation assay to identify ERα and ERβ agonists 

• Source of assay: Developed by Odyssey Thera to compliment Protein Complementation 

Assays (PCA) that measure receptor dimerization and translocation; Developed for HTS 

in 384-well plates; Cell lines developed are proprietary to Odyssey Thera although there 

is nothing particularly unique about this system

• Primary Journal Reference for the Assay: Vanden Heuvel, J.P., Thompson, J.T., 

Frame, S.R., and Gillies, P.J. (2006). Differential activation of nuclear receptors by 

perfluorinated fatty acid analogs and natural fatty acids: a comparison of human, mouse, 

and rat peroxisome proliferator-activated receptor-alpha, -beta, and -gamma, liver X 

receptor-beta, and retinoid X receptor-alpha. Toxicol Sci. 92(2):476-89. 

• General description: 

o The ERα and ERβ TA assays are stable cell lines expressing full length human 

ERα or ERβ along with a luciferase reporter gene controlled by 5 copies of the 

estrogen response element placed upstream of the mouse mammary tumor virus 

promoter. Parental cell line is CHO-K1. 

o Express endogenous hamster ERα and ERβ. Other receptors present in cell line 

not characterized. The response with mock-transfected cells to 17β-estradiol is 

more than 10-fold less in maximal response compared to either ERα- or ERβtransfected cells.

o Reporter gene product is luciferase enzyme with signal read by a one-step 

luciferase assay. Cells are lysed and luciferin substrate added along with signal 

stabilizers and bioluminescence read using a CCD camera-based reader.

o This assay is similar to OECD TG 457 but different parental cell line.

• How was the assay developed: 

o Developed using industry standard guidelines for HTS 

(see http://www.ncbi.nlm.nih.gov/books/NBK53196/)

o Developed for compound profiling purposes by Odyssey Thera using standard 

HTS guidelines

o See accompanying data for replication and accuracy using blinded chemical 

replicates and reference chemicals

o Assay has higher throughput than OECD validated version

Assay protocol

• Assay components and general procedure

o Source/characteristics of ER cell line:

 Recombinant full length human ERα or ERβ transiently transfected in 

bulk followed by freezing of aliquots of cells for subsequent assay. All 

chemicals screened from the same transfection batches.

 Endogenous hamster ERα and ERβ

o Cell culture and plating conditions: 

 5.5k cells/well in 384-well plate

 Cells plated for 24 hrs before treatment

 Chemical exposure for 19-24 hrs

 Medium:
16

• Phenol red-free DMEM

• 10% charcoal/dextran-stripped FBS

• Media (pH) 7.2-7.4

o Solvents: DMSO used as a universal solvent with final concentration of 0.5%; no 

interference with assay signal at this concentration. 

o Test chemical: 

 Chemicals are almost all 20 mM stocks in DMSO

 Concentration response series used with 6 concentrations, 3-fold dilutions, 

concentration range = 100 μM – 0.3 micromolar. 

 Serial dilutions prepared in DMSO

 No solubility testing performed

o Cytotoxicity test: 

 ONE-Glo luciferase reagent from Promega

 Endpoint reported: Luminescence normalized to negative control (DMSO) 

and positive control (agonist: 17β-estradiol; antagonist: 4-hydroxy

tamoxifen).

• Reference estrogens and positive/negative /solvent controls

o Negative control: 0.5% DMSO

o Positive control: 17β-estradiol 3.00E-09 M with 6 replicates

o Use of controls: 

 Concurrent 17β-estradiol positive control run in 6 replicates for each assay 

run at 3.00E-09 M.

 3.00E-09, 1.00E-08, 1.00E-09, 3.16E-10, 1.00E-10, 3.20E-11 M 

concentration response curve run on each plate

 8 replicates of DMSO negative control tested on each plate

• Plate format

o Assays have been optimized for 384-well.

o Replication 

 Chemicals tested in blinded format

 Many internal chemical replicates included in screening set

 Concentration-response format for all chemicals

 Triplicate points per chemical/concentration 

o Plate/Well Failure Criteria

 Well-to-well CV for RLU in vehicle controls <35%

 Z’ for reference compound on each plate >0.4 

 EC-50 for reference compound varies no more than 10-fold across all 

plates for a given assay

Known issues with assay performance 

• False negatives due to compound solubility issues

• Cytotoxicity can result in false negatives in agonist assay Compounds that inhibit 

luciferase activity can be false negatives in agonist format or false positives in antagonist 

format

• Compounds that stabilize luciferase enzyme can create false positives in agonist format

• Limits on concentration of test chemicals: Limited by DMSO tolerance of assay (0.5%)

• Metabolic capability of cell line: Considered low for CHO cells
17

• Cytotoxicity: Can result in false negatives (agonist) Can cause biphasic concentrationresponse curve and limit detection of true pharmacological Emax potential

• Stability of reagents: No known issues

• Solubility, volatility, physical properties, and stability of test chemicals

o Chemical library filtered by predicted solubility and volatility parameters. On 

average, there should be minimal problems. 

o Chemical structures are confirmed by GC/MS or other appropriate technique in 

the DMSO stock within one year of preparation. The available analytical 

chemistry will be included in the results.

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 
18

F.5 Estrogen Receptor: Cell-free competitive binding (human)

• Short name: NVS_NR_hER

• Purpose of assay: Identify chemicals able to interact with ER ligand-binding domain to 

displace radiolabeled 17β-estradiol.

• Source of assay: Caliper, a PerkinElmer company 

(http://www.caliperls.com/products/contract-research/in-vitro/nuclear-receptors/estrogenh104161.htm). Developed for pharmacological compound profiling. Modified from 

published radioligand binding assay protocols

• Primary journal reference for the assay: Haji, et al. Age-Related Changes in the 

Concentrations of Cytosol Receptors for Sex Steroids in the Hypothalamus and Pituitary 

Gland of the Rat. Brain Research. 204: 373-386 (1980) (with modifications).

• General description: 

o Human MCF7 breast cancer cell line lysate

o Full length, endogenous ERs

o [

3

H]-Estradiol

o Endpoint is displacement of [3H]-Estradiol from receptor prep

o This assay is mechanistically and functionally similar to a fully validated ER 

binding assay OPPTS 890.1250: Estrogen Receptor Binding Assay Using Rat 

Uterine Cytosol (ER-RUC)

• How was the assay developed: 

o Assay developed according to HTS industry standards 

(http://www.ncbi.nlm.nih.gov/books/NBK53196/)

o KD (binding affinity): 0.07 nM

o Bmax (receptor number): 3844 fmol/mg protein

o Reference Compound IC50 (nM)

 Estradiol (0.3 nM)

 R1881 (2200 nM)

 Reliability: Historic performance of positive control

o Similar attributes to OPPTS 890.1250 but with higher throughput

Assay protocol 

• Assay components and general procedure 

o Radiolabeled or fluorescent ER ligand (include concentration used in assay) 0.1 

nM [3H]estradiol

o Buffer (molar concentrations, pH) 

 10 mM TRIS-HCI (pH 7.4)

 1.5 mM EDTA
19

 1.0 mM DTT

 25 mM sodium molybdate

o Time, temperature, incubation period

 0-4°C

 18 hours

o Solvent: 0.25% DMSO

o Test chemical:

 Single concentration at 25 micromolar

 Retest chemicals that meet pre-defined threshold of the mean assay signal 

differing by at least 30% from the vehicle (DMSO) control signal or the Zscore varying by at a minimum of 2.0 median absolute deviations from the 

median (MAD2).

 No solubility testing performed

 Serial dilutions prepared in DMSO followed by intermediate dilution step 

in H20 immediately prior to screening to yield a 10X solution for adding 

to assay wells.

o Method for separation bound/free ER ligand; Detection system:

 Dextran-coated charcoal binding and centrifugation followed by 

scintillation counting of supernatant

o Endpoint reported:

 Single concentrations are % of maximum binding

 Concentration-response curves fit to generate AC50 

• Reference estrogen and positive/negative /solvent controls

o Reference estrogen (17β-estradiol, range of full concentration response curve)

 1X10-10 M, 3X10-10 M, 1 X 10-9 M on each plate

o Controls: 

 1X10-10 M, 3X10-10 M, 1 X 10-9 M 17β-estradiol on each plate (3 wells 

total)

 0.25% DMSO negative controls on 3 wells per plate

o Use of controls: 

 IC50 of reference estrogen, 17β-estradiol, within 1⁄2 log unit of historical 

average for assay at Novascreen.

 Historical data available for controls at plate level

o Plate format: 96-well plates 

o Replication: 

o Three positive and three negative control wells per plate

o Single concentration tested in duplicate as initial screen

o Full concentration-response screen conducted using single well per concentration 

o Blinded chemical replicates included in each task order

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid

o Specific binding at least 50% 

o Less than 10% of total radioligand bound

o No observable plate effects

o Wells pass if the covariance between replicates is 20% or less

o For AC50s, it is based on curve shape with one outlier in the middle allowed.
20

Known issues with assay performance 

• Interference issues likely to be encountered: Nuisance compounds (detergents, 

aggregators, reactive)

• Limits on concentration of test chemicals:

o Initial screening done at single concentration of 25 micromolar which provides 

reasonable high concentration with minimal solubility problems

o Confirmation assays run in concentration-response format starting at 50 μM, the 

upper limit of testing concentration defined by the DMSO tolerance of the assay

• Protein denaturation, radioligand sequestration/depletion: No known issues

• Stability of reagents: No known issues for screening format used

• Solubility, volatility, physical properties, and stability of test chemicals:

o Chemical library selection filtered by predicted solubility and volatility 

parameters. Approximately 66% passed all criteria; exceptions were permitted on 

a case by case basis.

 MW (50 to 1000 pass)

 logP (-2 to 6 pass)

 BP (>100 pass)

 VP (<10 pass)

o Chemical purity and identity confirmed by Certificate of Analysis with purity 

requirements of 90% or greater (limited exceptions for known mixtures). 

Approximately 97% of chemicals met this criteria, most > 95% pure.

o Chemicals dissolved in DMSO at a target 20 mM (this can be lowered for 

chemicals not soluble at 20 mM). Solubility determined by observation of 

preparer at this stage. 

o Chemicals are stored in sealed tubes or polypropylene plates at -80o

C. 

o Chemical identity, purity and concentration are determined from stock solutions 

by GC/MS or other appropriate technique within a year of preparation.

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 

o No actual solubility measurements made so this may have reduced intended 

concentration in some cases resulting in false negatives. 

o Nuisance compounds may produce false positives due to denaturation or 

aggregation of the receptor. 

o If chemical requires metabolic activation to be an ER agonist, it will not be 

detected in this assay.
21

F.6 Estrogen Receptor: Cell-free competitive binding (bovine)

• Short name: NVS_NR_bER

• Purpose of assay: Determine chemicals able to interact with ER ligand-binding domain 

to displace radiolabeled 17β-estradiol.

• Source of assay: Caliper Life Sciences. Developed for pharmacological compound 

profiling. Modified from published radioligand binding assay protocols

• Primary Journal Reference for the Assay: Haji, et al. Age-Related Changes in the 

Concentrations of Cytosol Receptors for Sex Steroids in the Hypothalamus and Pituitary 

Gland of the Rat. Brain Research. 204: 373-386 (1980) with modifications.

• General description: 

o Bovine uterine membranes 

o Full length, endogenous ERs

o [

3

H]-Estradiol

o Endpoint is displacement of [3

H]-Estradiol from receptor prep

o This assay is mechanistically and functionally similar to OPPTS 890.1250: 

Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC)

• How was the assay developed

o Assay developed according to HTS industry standards

o KD (binding affinity): 0.7 nM

o Bmax (receptor number): 406 fmol/mg protein

o Reference Compound IC50 (nM)

 Estradiol 0.2

o Reliability: Historic performance of positive control

o Similar attributes to OPPTS 890.1250 but with higher throughput

Assay protocol 

• Assay components and general procedure 

o Radiolabeled or fluorescent ER ligand: 0.7 nM [H3]estradiol

o Buffer: 10 mM TRIS-HCI (pH 7.4); 1.5 mM EDTA; 1.0 mM DTT; 25 mM 

sodium molybdate

o Time, temperature, incubation period:0-4°C, 18 hours

o Sovents: 0.25% DMSO

o Test chemical: 

 Single concentration at 25 micromolar

 Retest chemicals that meet pre-defined threshold of the mean assay signal 

differing by at least 30% from the vehicle (DMSO) control signal or the Z-
22

score varying by at a minimum of 2.0 median absolute deviations from the 

median (MAD2).

 No solubility testing performed

 Serial dilutions prepared in DMSO followed by intermediate dilution step 

in H20 immediately prior to screening to yield a 10X solution for adding 

to assay wells.

o Method for separation bound/free ER ligand, Detection system

 Dextran-coated charcoal binding and centrifugation followed by 

scintillation counting of supernatant

o Endpoint reported:

 Single concentrations are % of maximum binding

 Conc-response curves fit to generate IC50

• Reference estrogen and positive/negative /solvent controls

o Reference estrogen (17β-estradiol, range of full concentration response 

curve)1X10-10 M, 3X10-10 M, 1 X 10-9 M on each plate

o Controls:

 1X10-10 M, 3X10-10 M, 1 X 10-9 M 17B-estradiol on each plate (3 wells 

total)

 0.25% DMSO negative controls on 3 wells per plate

o Use of controls: 

 IC50 of reference estrogen, 17β-estradiol, within 1⁄2 log unit of historical 

average for assay at Novascreen.

 Historical data available for controls at plate level

o Plate format: 96-well plates

o Replication: 

 Three positive and three negative controls per plate

 Single concentration tested in duplicate as initial screen

 Concentration-response screen conducted using single well per 

concentration

 Blinded chemical replicates included in each task order

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid: 

o Specific binding at least 50% 

o Less than 10% of total radioligand bound

o No observable plate effects

o Wells pass if the covariance between replicates is 20% or less

o For AC50s it is based on curve shape with one outlier in the middle allowed.

Known issues with assay performance 

• List any assay interference issues likely to be encountered: Nuisance compounds 

(detergents, aggregators, reactive)

• Limits on concentration of test chemicals

o Initial screening done at single concentration of 25 μM which provides reasonable 

high concentration with minimal solubility problems

o Confirmation assays run in concentration-response format starting at 50 μM, the 

upper limit of testing concentration defined by the DMSO tolerance of the assay

• Protein denaturation, radioligand sequestration/depletion: No known issues
23

• Stability of reagents: No known issues for screening format used

• Solubility, volatility, physical properties, and stability of test chemicals:

o Chemical library selection filtered by predicted solubility and volatility 

parameters. Approximately 66% passed all criteria; exceptions were permitted on 

a case by case basis.

 MW (50 to 1000 pass)

 logP (-2 to 6 pass)

 BP (>100 pass)

 VP (<10 pass)

o Chemical purity and identity confirmed by Certificate of Analysis with purity 

requirements of 90% or greater (limited exceptions for known mixtures). 

Approximately 97% of chemicals met this criteria, most > 95%.

o Chemicals dissolved in DMSO at a target 20 mM (this can be lowered for 

chemicals not soluble at 20 mM). Solubility determined by observation at this 

stage. 

o Chemicals are stored in sealed tubes or polypropylene plates at -80o

C. 

o Chemical identity, purity and concentration are determined from stock solutions 

by GC/MS or other appropriate technique within a year of preparation. 

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 

o No actual solubility measurements made so this may have reduced intended 

concentration in some cases resulting in false negatives. 

o Nuisance compounds may produce false positives due to denaturation or 

aggregation of the receptor. 

o If chemical requires metabolic activation to be an ER agonist, it will not be 

detected in this assay.
24

F.7 Estrogen Receptor: Cell-free competitive binding (murine)

• Short name: NVS_NR_mER

• Purpose of assay: Determine chemicals able to interact with ER ligand-binding domain 

to displace radiolabeled 17β-estradiol.

• Source of assay: Caliper Life Sciences. Developed for pharmacological compound 

profiling. Modified from published radioligand binding assay protocols.

• Primary journal reference for the assay: Haji, et al. Age-Related Changes in the 

Concentrations of Cytosol Receptors for Sex Steroids in the Hypothalamus and Pituitary 

Gland of the Rat. Brain Research. 204: 373-386 (1980) with modifications.

• General description: 

o Recombinant murine ER alpha (Ligand Binding Domain)

o [

3

H]-Estradiol

o Endpoint is displacement of [3H]-Estradiol from receptor prep

o Mechanistically and functionally similar to OPPTS 890.1250: Estrogen Receptor 

Binding Assay Using Rat Uterine Cytosol (ER-RUC)

• How was the assay developed: 

o Assay developed according to HTS industry standards

o KD (binding affinity): 8 nM

o Bmax (receptor number): 710,000 fmol/mg protein

o Reference Compound IC50 (nM)

 Estradiol 5.0

o Reliability: Historic performance of positive control

o Similar attributes to OPPTS 890.1250 but with higher throughput

Assay protocol 

• Assay components and general procedure 

o Radiolabeled or fluorescent ER ligand: 1.0 nM [3H]estradiol

o Buffer: 25 mM TRIS-HCI (pH 7.4);1.5 mM EDTA; 1.0 mM DTT; 25 mM 

sodium molybdate

o Time, temperature, incubation period: 0-4°C; 18 hours

o Solvent: 0.25% DMSO

o Test chemical: 

 Single concentration at 25 micromolar

 Retest chemicals that meet pre-defined threshold of the mean assay signal 

differing by at least 30% from the vehicle (DMSO) control signal or the Zscore varying by at a minimum of 2.0 median absolute deviations from the 

median (MAD2).
25

 No solubility testing performed

 Serial dilutions prepared in DMSO followed by intermediate dilution step 

in H20 immediately prior to screening to yield a 10X solution for adding 

to assay wells.

o Method for separation bound/free ER ligand; Detection system:

 Dextran-coated charcoal binding and centrifugation followed by 

scintillation counting of supernatant

o Endpoint reported: 

 Single concentrations are % of maximum binding

 Conc-response curves fit to generate IC50

• Reference estrogen and positive/negative /solvent controls

o Reference estrogen: 1X10-10 M, 3X10-10 M, 1 X 10-9 M on each plate

o Controls: 

 1X10-10 M, 3X10-10 M, 1 X 10-9 M 17B-estradiol on each plate (3 wells 

total)

 0.25% DMSO negative controls on 3 wells per plate

o Use of controls: 

 IC50 of reference estrogen, 17β-estradiol, within 1⁄2 log unit of historical 

average for assay at Novascreen.

 Historical data available for controls at plate level

• Plate format: 96-well 

• Replication: 

o Three positive and three negative controls per plate

o Single concentration tested in duplicate as initial screen

o Concentration-response conducted using a single well per concentration

o Blinded chemical replicates included in each task order

• Acceptance criteria and/or procedure to determine if results from each well and 

plate valid: 

o Specific binding at least 50% 

o Small (10-15%) percentage of total radioligand bound

o No observable plate effects

o Wells pass if the covariance between replicates is 20% or less

o For IC50s it is based on curve shape with one outlier in the middle allowed.

Known issues with assay performance 

• List any assay interference issues likely to be encountered: Nuisance compounds 

(detergents, aggregators, reactive)

• Limits on concentration of test chemicals:

o Initial screening done at single concentration of 25 μM which provides reasonable 

high concentration with minimal solubility problems

o Confirmation assays run in concentration-response format starting at 50 μM, the 

upper limit of testing concentration defined by the DMSO tolerance of the assay

• Protein denaturation, radioligand sequestration/depletion: No known issues

• Stability of reagents: No known issues for screening format used

• Solubility, volatility, physical properties, and stability of test chemicals:
26

o Chemical library selection filtered by predicted solubility and volatility 

parameters. Approximately 66% passed all criteria; exceptions were permitted on 

a case by case basis.

 MW (50 to 1000 pass)

 logP (-2 to 6 pass)

 BP (>100 pass)

 VP (<10 pass)

o Chemical purity and identity confirmed by Certificate of Analysis with purity 

requirements of 90% or greater (limited exceptions for known mixtures). 

Approximately 97% of chemicals met this criteria, most > 95%.

o Chemicals dissolved in DMSO at a target 20 mM (this can be lowered for 

chemicals not soluble at 20 mM). Solubility determined by observation at this 

stage. 

o Chemicals are stored in sealed tubes or polypropylene plates at -80o

C. 

o Chemical identity, purity and concentration are determined from stock solutions 

by GC/MS or other appropriate technique within a year of preparation. 

• Physical properties of chemicals that likely are not amendable to this assay

o Predicted volatiles or compounds with very high or very low logP were not 

included in the chemical library. 

o No actual solubility measurements made so this may have reduced intended 

concentration in some cases resulting in false negatives. 

o Nuisance compounds may produce false positives due to denaturation or 

aggregation of the receptor. 

o If chemical requires metabolic activation to be an ER agonist, it will not be 

detected in this assay.