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 ER α TRANS and ATG ERE CIS.

• Purpose of assay : Detection of chemicals that activate ER α 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 method s for conducting the assay. Components of assay are covered by patents and patent applicat ions filed in the USA and world wide by Attagene, Inc. ( http://www.attagene.com/technology.php )

• Primary journal re ference 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 commensurabl e 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 cleav ed 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 sing le reaction tube format, providing highly uniform conditions for detecting multiple transcription factors.

o ATG_ER α _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 ER α NM_000125) is transiently co

transfected into same cells for the ATG_ER α _cis assay.

 RTU for ATG_ER α _cis contains a 5X ERE synthetic response element.

o ATG_ER α _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 fo r 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 transfecte d full

- length, human ER α

 Trans assay uses transfected hER α
- 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 3x10 5

c ells/well or 0.8x10 5 cells/cm 2

.  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 ever y 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 TGF β 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 (PXR

cis, 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 micro molar (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

- diphenyltetra zolium 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 (Invitrog en). 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 p rimer (2 min at 95 1C, 20 s at 68 o C and 10 min at 72 o C) and then digested products with 5U of HpaI (New England Biolabs) for 2 h at 37 o C. Using Qiaquick PCR columns (Qiagen), purified the fragments and analyzed on an ABI 3130xL genetic analyzer4 (Applied Bi osystems). Peaks’ positions were identified by using a set of X

rhodamine (ROX)

- labeledMapMarker1000 molecular weight standards (BioVentures).

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

- ERE

- RTU or the ER α
- 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 17 β

- estradiol positive control compound r un 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 μ M sample treatment)

o Positive control : 0.2 μ M 17 β

- estradiol

o Use of controls :

 Co ncurrent 17 β

- 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 di luted 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 ar e 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

 Concentratio n
- 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 assessme nt 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 a mplification (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 belo w).

 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 as say 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 ce lls 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 sta bility 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 chem ical library. F .2 Estrogen Receptor Transactivation Assays (Tox21_ER α _BLA)

• Short name : Tox21 BLA

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

• Source of assay : Tox21_ ER α _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_ ER α _BLA: Wilkinson et al ., J Biomol Screen. 13(8):7 55

- 65 (2008).

• General description :
o Mammalian two

- hybrid system consisting of HEK293T cells stably transfected with 5X_UAS_ β

- lactamase and a human ER α ligand

- binding domain fused to a GAL4 DNA binding domain.

o Should only report ER α activity unless there is heterodimerization with endogenous ER β (in which case it could theoretically report ER α :ER β heterodimer activity if ER β is expressed in these cells)

o Expression of other steroid receptors not very relevant to two

- hybrid system7 o Repo rter 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 t he 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 describe d 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 ER α

. • How was the assay developed?
o Developed for commercial purposes by Invitrogen using standard HTS guidelines

o See accompanying data for re plication 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 miniat urized to 1536

- well format

o Assay not subject to luciferase inhibitors and stabilizers Assay protocol

• A ssay components and general procedure

o Source/characteristics of ER cell line : Tox21_ E R α _BLA: Recombinant human E R α
- 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 100 μ g/ml Streptomycin

• pH 7.0

 Antagonist mode: 0.5 nM 17 β

- 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 :

 Che micals are almost all 20 mM stocks in DMSO8  Concentration response series used with 15 concentrations, 2

- fold dilutions, concentration range = 92 μ M

– 6 nM

 Serial dilutions prepared in DMSO

 Compounds added from DMSO stocks by pin tool addition

 No solubilit y testing performed

o Cytotoxicity test : Cell lines were tested concurrent with the antagonist version of the assay using Tox21_ ER α _BLA: Promega CellTiter Glo (luminescence), an ATP

- dependent luciferase assay

o Detection system :

 Cells are loaded with an engi neered 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 resonan ce 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 r esults 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:fluoresce in ratio normalized to negative control (DMSO) and positive control (17 β

- estradiol).

• Reference estrogens and positive/negative /solvent controls
o Negative control : 0.5% DMSO

o Positive control : 17 β

- estradiol

 Tox21_ ER α _BLA concentration

- response curve (3.8

- 0 6, 1.3E

- 06, 4.3E

07, 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_ER α _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, 8.6E

14, 2.9E

- 14, 9.6E

- 15, 3.2E

- 15 M)

o Use of controls :

 Concurrent 17 β

- estradiol positive control tested in triplicate for each assay
run.

 Results for the positive controls, including, 17 β

- 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 r eplicates)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 eac h 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 ER α
- bla

• Compounds that q uench 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

• Solubil ity, 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 appropriat e 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 o r 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 concentration

response series12 F .3 Estrogen Receptor Transactivation Assays (Tox21_ER α _Luc_BG1)

• Short name : Tox21 BG1 LUC.

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

• Source of assay : Tox21_ER α _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_ER α _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 up stream 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 hi gher throughput than OECD validated bench top version Assay protocol

• Assay components and general procedure

o Source/characteristics of ER cell line : endogenous ER α and ER β

o Cell culture and plating conditions :

 Used between passages 8 and 10 from UC Davis.

 4 000 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 100 μ g/m L Streptomycin

• Media (p H) 7.0

 Antagonist mode: 0.5 nM 17 β

- estradiol

 Run as three independent replicate experiments13 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

 Con centration response series used with 15 concentrations, 2

- fold dilutions, concentration range = 92 μ M

– 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 repor ted: Luminescence normalized to negative control (DMSO) and positive control (17 β

- estradiol).

• Reference estrogens and positive/negative /solvent controls
o Negative control : 0.5% DMSO

o Positive control : 17 β

- estradiol

 Concentrations tested on each plate: (4.6 E

- 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 17 β

- estradiol positive control is tested in triplicate for each assay run.

 Results f or the positive controls, including, 17 β

- 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 Cri teria

 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 wells14 • Compounds that inhib it luciferase activity could be false negatives or false positives in ER α_ 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 o f 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 C hemical 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 ER α )

• Short name : OT LUC

• Purpose of Assay : Transactivation assay to identify ER α and ER β 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., Fr ame, 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 ER α and ER β TA assays are stable cell lines expressing full length human ER α or ER β 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 ER α and ER β . Other receptors present in cell line not characterized. The response with mock

- transfected cells to 17 β

- estradiol is more than 10

- fold less in maximal response compared to either ER α
- or ER β

transfected 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 w ith 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 E R α or E R β transiently transfected in bul k followed by freezing of aliquots of cells for subsequent assay. All chemicals screened from the same transfection batches.

 Endogenous hamster ER α and ER β

o Cell culture and plating conditions :

 5.5k cells/well in 384

- well plate

 Cells plated for 24 h rs befo re 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 di lutions, concentration range = 100 μ M

– 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: 17 β

- estradiol; antagonist: 4

- hydroxy tamoxifen).

• Reference estrogens and positive/negative /solvent controls
o Negative control : 0.5% DMSO

o Positive control : 17 β

- estradiol 3.00E

- 09 M with 6 replicates

o Use of controls :

 Concurrent 1 7 β

- 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

• Fa lse 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 cells17 • Cytotoxicity: Can result in false negatives (agonist) Can cause biphasic concentration

response 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 filte red 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 chemi stry 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 17 β

- estradiol.

• Source of assay : Caliper, a PerkinElmer company ( http://www.caliperls.com/products/contract

- research/in

- vitro/nuclear

- receptors/estrogen

h104161.htm ). Developed for pharmacological compound profiling. M odified 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 Resea rch. 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 funct ionally 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 [ 3 H]estradiol

o Buffer (molar concentrations, pH)

 10 mM TRIS

- HCI (pH 7.4)

 1.5 mM EDTA19  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 Z

score varying by at a minimum of 2.0 median absolute deviations from the median (MAD2).

 No solubility testing performed

 Serial dilutions pr epared in DMSO followed by intermediate dilution step in H 2 0 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 (17 β

- 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 17 β

- estradiol on each plate (3 wells total)

 0.25% DMSO negative controls on 3 wells per plate

o Use of contro ls :

 IC50 of reference estrogen, 17 β

- 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 we lls per plate

o Single conc entration tested in duplicate as initial screen

o Full c onc entration

- response screen conducted using single well per concentration

o Blinded chemical replicates included in each task order

• Acceptance criteria and/or procedure to deter mine 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 o ne 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 μ M, 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 selectio n 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 confi rmed 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 ch emicals 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

- 80 o C.
o Chemical identity, purity and concentration are determined from stock solutions by GC/M S 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 actu al 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 1 7 β

- 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 Ch anges 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 develope d 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 H 2 0 immediately prior to screening to yield a 10X solution for adding to assa y 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 curv es fit to generate IC50

• Reference estrogen and positive/negative /solvent controls

o Reference estrogen (17 β

- 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, 17 β

- estradiol, within 1⁄2 log unit of historical average for assay at Novascreen.

 Historical data available for controls a t plate level

o Plate format : 96

- well plates

o Replication :

 Three positive and three negative controls per plate

 Single conc entration tested in duplicate as initial screen

 Con centration

- response screen conducted using single well per
conc entration

 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 μ M which provides reasonable high concentration with minimal solubility problems

o Confirmation assays run in concentration

- response forma t starting at 50 μ M, the upper limit of testing concentration defined by the DMSO tolerance of the assay

• Protein denaturation, radioligand sequestration/depletion: No known issues23 • 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 10 00 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

- 80 o C.
o Chemical ide ntity, 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 ve ry 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 d enaturation 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 as say : Determine chemicals able to interact with ER ligand

- binding domain to displace radiolabeled 17 β

- estradiol.

• Source of assay : Caliper Life Sciences. Developed for pharmacological compound profiling. Modified from published radioligand binding assay prot ocols.

• 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 [ 3 H]

- 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 [ 3 H]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% fro m the vehicle (DMSO) control signal or the Z

score 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 H 2 0 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, 1 7 β

- estradiol, within 1⁄2 log unit of historical average for assay at Novascreen.

 Historical data available for co ntrols at plate level

• Plate format : 96

- well

• Replication :
o Three positive and three negative controls per plate

o Single conc entration tested in duplicate as initial screen

o Conc entration

- response conducted using a s ingle well per conc entration

o Blinded chemi cal 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 μ M which provides reasonable high concentration with minimal solubility problems

o Confirmation assays run in concentration

- re sponse format starting at 50 μ M, 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 t his 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

- 80 o 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 compo unds 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 positi ves 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.