Document 1490623

ER Binding BRD: Table of Contents
October 2002
TABLE OF CONTENTS
Page Number
LIST OF TABLES ................................................................................................................... vi
LIST OF APPENDICES .........................................................................................................vii
LIST OF ACRONYMS AND ABBREVIATIONS................................................................. ix
ACKNOWLEDGEMENTS ...................................................................................................xiii
PREFACE .............................................................................................................................P-1
EXECUTIVE SUMMARY................................................................................................. ES-1
1.0
Introduction and Rationale for the Use of In Vitro ER Binding Assays ....................1-1
1.1
1.2
1.3
1.4
Introduction ....................................................................................................1-1
1.1.1
Historical Background of In Vitro Endocrine Disruptor Assays
and Rationale for Their Development .............................................1-1
1.1.2
Prior or Proposed Peer Reviews of In Vitro ER Binding Assays....1-5
Scientific Basis for the Proposed Tier 1 In Vitro ER Binding Assays ...........1-5
1.2.1
Purpose for Using In Vitro ER Binding Assays ..............................1-5
1.2.2
Development of In Vitro ER Binding Assays: Historical
Background .....................................................................................1-6
1.2.3
Mechanistic Basis of In Vitro ER Binding Assays .........................1-9
1.2.4
Relationship of Mechanisms of Action in In Vitro ER Binding
Assays Compared to the Species of Interest .................................1-11
Intended Uses of the Proposed In Vitro ER Binding Assays .......................1-12
1.3.1
Validation of In Vitro Assays........................................................1-12
1.3.2
Where Can In Vitro ER Binding Assays Substitute, Replace, or
Complement Existing Methods? ...................................................1-14
1.3.3
Similarities and Differences with Currently Used Methods .........1-14
1.3.4
Role of In Vitro ER Binding Assays in Hazard Assessment.........1-14
1.3.5
Intended Range of Substances Amenable to the ER Binding
Assay and/or Limits of In Vitro ER Binding Assays ....................1-15
Search Strategy and Selection of Citations for the In Vitro ER Binding
BRD……......................................................................................................1-16
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ER Binding BRD: Table of Contents
2.0
In Vitro ER Competitive Binding Assay Methods.....................................................2-1
2.1
Introduction ...................................................................................................2-1
2.2
General Overview of In Vitro Assays Used to Measure
Competitive ER Binding ................................................................................2-3
2.2.1
Mammalian Uterine Cytosol (Rat, Mouse, Rabbit)
as the ER Source..............................................................................2-3
2.2.2
MCF-7 Cells and Cytosol as ER Source .........................................2-5
2.2.3
2.2.2.1
Intact MCF-7 Cells........................................................2-5
2.2.2.2
MCF-7 Cytosol..............................................................2-6
Semi-Purified ERα and ERβ...........................................................2-6
2.2.3.1
Solid Phase Ligand Binding Assay using
ScintiStrip™ (Kuiper et al., 1998).................................2-7
2.2.4
GST-ERdef Fusion Proteins............................................................2-7
2.2.5
Fluorescent Polarization (FP)..........................................................2-8
2.2.6
3.0
October 2002
2.2.5.1
Theory of FP..................................................................2-8
2.2.5.2
Conduct of the Assay ....................................................2-9
Permutations of the Assays as Described in the Literature ...........2-10
Characterization of Substances Tested in ER Binding Assays ..................................3-1
3.1
Introduction ................................................................................................ …3-1
3.2
Rationale for Selection of Substances/Products Tested in In Vitro ER
Binding Assays...............................................................................................3-1
3.3
Chemical and Product Classes Tested............................................................3-2
4.0
Reference Data ...........................................................................................................4-1
5.0
Data on In Vitro ER Binding Assays .........................................................................5-1
6.0
5.1
Introduction ....................................................................................................5-1
5.2
Availability of Detailed In Vitro ER Binding Protocols ................................5-1
5.3
Availability of In Vitro ER Binding Data ......................................................5-2
5.4
In Vitro ER Binding Assay Results for Individual Substances ......................5-3
5.5
Use of Coded Chemicals and Compliance with GLP Guidelines ..................5-3
In Vitro ER Binding Test Method Performance Assessment.....................................6-1
6.1
Introduction ....................................................................................................6-1
6.2
Quantitative Assessments of Assay Performance ..........................................6-2
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ER Binding BRD: Table of Contents
7.0
8.0
9.0
October 2002
6.2.1
Measures of Intra-Class Correlation................................................6-3
6.2.2
Evaluation of Substances Tested in Nine or More In Vitro ER
Binding Assays................................................................................6-3
6.2.3
Variability in lnIC50 and lnRBA Values for Selected Substances...6-7
6.3
Qualitative Assessment of In Vitro ER Binding Assay Performance ............6-8
6.4
Performance of In Vitro ER Binding Assays ...............................................6-13
6.5
General Strengths and Limitations of In Vitro ER Binding Assays.............6-14
6.6
Conclusions and Recommendations.............................................................6-15
In Vitro ER Binding Test Method Reliability Assessment ........................................7-1
7.1
Introduction ....................................................................................................7-1
7.2
Quantitative Assessments of Interlaboratory Reproducibility .......................7-1
7.2.1
Measures of Intra-Class Correlation................................................7-2
7.2.2
Evaluation of Substances Tested in Nine or More In Vitro ER
Binding Assays................................................................................7-3
7.2.3
Variability in Standard Deviation for lnRBA and lnIC50 Values
by In Vitro ER Binding Assay.........................................................7-5
7.2.4
Variability in the IC50 Value for 17β-Estradiol...............................7-6
7.3
Reliability of In Vitro ER Binding Assays.....................................................7-7
7.4
Conclusions and Recommendations...............................................................7-8
Quality of Data Reviewed ..........................................................................................8-1
8.1
Extent of Adherence to GLP Guidelines........................................................8-1
8.2
Assessment of Data Quality ...........................................................................8-1
8.3
Quality Control Audit.....................................................................................8-2
8.4
Need for Data Quality ....................................................................................8-2
Other Scientific Reports and Reviews........................................................................9-1
9.1
Availability of Other In Vitro ER Binding Data ............................................9-1
9.2
Conclusions of Other Scientific Reviews of ER Binding Methods ...............9-2
9.2.1
1996 Endocrine Disruptor Screening Methods Workshop..............9-2
9.2.2
1997 Workshop on Screening Methods for Detecting Potential
(Anti-) Estrogenic/Androgenic Chemicals in Wildlife ..................................9-3
10.0
Animal Welfare Considerations ..............................................................................10-1
10.1
Refinement, Reduction, and Replacement Considerations ..........................10-1
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ER Binding BRD: Table of Contents
10.2
11.0
October 2002
Use of Animals in In Vitro ER Binding Assays...........................................10-1
Practical Considerations ...........................................................................................11-1
11.1 Test Method Transferability.........................................................................11-1
11.1.1
Facilities and Major Fixed Equipment ..........................................11-1
11.2
Training Considerations ...............................................................................11-2
11.3
Cost and Time Considerations .....................................................................11-3
12.0 Minimum Procedural Standards for In Vitro ER Binding Assays and
Recommendation of Substances for Use in Validation Studies ...............................12-1
12.1
Introduction ..................................................................................................12-1
12.2
Minimum Procedural Standards...................................................................12-1
12.2.1
Animal Studies ..............................................................................12-1
12.2.2
Dissociation Constant (Kd) of the Reference Estrogen .................12-1
12.2.3
Preparation of Test Substances......................................................12-2
12.2.4
Concentration Range of Test Substances ......................................12-2
12.2.5
Solvent and Positive Controls .......................................................12-3
12.2.6
Within-Test Replicates ..................................................................12-4
12.2.7
Dose Spacing.................................................................................12-4
12.2.8
Data Analysis ................................................................................12-4
12.2.9
Assay Acceptance Criteria ............................................................12-5
12.2.10 Evaluation and Interpretation of Results .......................................12-5
12.2.11 Test Report ....................................................................................12-5
12.2.12 Replicate Studies ...........................................................................12-7
12.3
Standardization of ER Binding Assays for Validation.................................12-7
12.3.1 Example Recommended General Protocol for Measuring ER
Binding using the RUC Assay.......................................................12-8
12.3.2
Preparation of Rat Uterine Cytosol ...............................................12-9
12.3.3
Standardization of In Vitro ER Binding Assays............................12-9
12.3.4
In Vitro ER Competitive Binding Assay Methodology ................12-9
12.4
Recommended List of Substances to be Used for Validation of In Vitro ER
Binding Assays........................................................................................12-11
12.5
Conclusions and Recommendations...........................................................12-16
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ER Binding BRD: Table of Contents
12.0 Annex
October 2002
Example Protocol for the In Vitro ER Competitive Binding Assay Using
RUC........................................................................................................... 12A-1
13.0
References ................................................................................................................13-1
14.0
Glossary....................................................................................................................14-1
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ER Binding BRD: List of Appendices
October 2002
LIST OF TABLES
Page Number
Table 3-1
Chemical Classes Tested in In Vitro ER Binding Assays .............................3-3
Table 3-2
Product Classes Tested in In Vitro ER Binding Assays.................................3-4
Table 5-1
Number of Substances Tested in Various In Vitro ER Binding Assays ........5-4
Table 5-2
Substances Tested in Ten or More In Vitro ER Binding Assays ...................5-4
Table 6-1
Number of Substances Tested in Multiple In Vitro ER Binding Assays .......6-2
Table 6-2
Components of Variance for Each Variable Adjusted for the Other
Two Variables – Performance Assessment ....................................................6-4
Table 6-3
Variance of lnRBA Values by Substance and Assay – Performance
Assessment .....................................................................................................6-5
Table 6-4
Variance for Y=lnRBA Values ......................................................................6-6
Table 6-5
Variability in Standard Deviations for lnRBA and lnIC50 Values For
Selected Substances........................................................................................6-8
Table 6-6
Qualitative Assessment of the Ability of Different ER Binding Assays
to Detect Substances with Different Relative Binding Affinities (RBA
Values) Compared to the RUC Assay..........................................................6-11
Table 6-7
Summary of In Vitro ER Binding Assay Performance ................................6-14
Table 7-1
Components of Variance for Each Variable Adjusted for the Other
Two Variables – Reliability Assessment........................................................7-3
Table 7-2
Variance of lnRBA by Substance and Assay – Reliability Assessment ........7-4
Table 7-3
Standard Deviation for lnRBA and lnIC50 Values for In Vitro ER
Binding Assays...............................................................................................7-6
Table 7-4
Standard Deviation for IC50 Values Obtained for 17β-Estradiol ...................7-7
Table 7-5
Summary of In Vitro ER Binding Assay Reliability......................................7-8
Table 11-1
Comparison of Costs, Time, and Special Equipment Needs of
Different ER Binding Assays .......................................................................11-4
Table 12-1
Recommended Substances for Validation of In Vitro ER Binding
Assays.........................................................................................................12-12
Table 12-2
List of Substances Being Tested in the In Vitro RUC Assay by Battelle ..12-15
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ER Binding BRD: List of Appendices
October 2002
LIST OF APPENDICES
Page Number
Appendix A
In Vitro ER Binding Assays ......................................................................... A-1
A1
Assays Using Rat Uterine Cytosol............................................................ A1-1
A2
Assays Using Mouse Uterine Cytosol ...................................................... A2-1
A3
Assays Using Human ERα and ERβ ........................................................ A3-1
A4
Assays Using Recombinant ERα and ERβ............................................... A4-1
A5
Assays Using Fluorescence Polarization .................................................. A5-1
A6
Assays Using GST-ERdef Constructs ...................................................... A6-1
A7
Assays Using MCF-7 Cells and Cytosol .................................................. A7-1
Appendix B
B1
In Vitro ER Binding Assay Protocols ........................................................... B-1
Protocol for the Competitive ER binding MCF-7 (Whole
Cell Assay) (Provided by Dr. Guy Leclercq, Clinique et
Laboratoire de Cancerologie Mammaire, Centre des
Tumeurs de l’Universite Libre de Bruxelles, Brussels,
Belgium ...................................................................................................... B1-1
B2
Protocol for the Fluorescence Polarization Assay of the
Competitive Binding of Ligands to Estrogen-Receptor
Complexes (Provided by Dr. William Allworth,
Department of Chemistry, University of New Orleans, New
Orleans, LA, USA) ..................................................................................... B2-1
B3
Protocol for the Competitive Ligand Binding Assay
(Provided by Dr. Timothy Zacharewski, Dept. of
Biochemistry, Michigan State University, Lansing, MI,
USA) ........................................................................................................... B3-1
B4
Standard Operating Procedure for the Rat Estrogen
Receptor Equilibrium Exchange Assay (Provided by Dr.
Weida Tong, Division of Genetic and Reproductive
Toxicology, National Center for Toxicological Research,
Jefferson AR, USA) ................................................................................... B4-1
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ER Binding BRD: List of Appendices
B5
October 2002
Protocol for the Estrogen Receptor Competitive Binding
Assay Using Rat Uterine Cytosol (Provided by Dr. Susan
Laws, U.S. EPA, NHEERL, RTP, NC and Mr. Gary Timm,
U.S. EPA, Washington, DC, USA) ............................................................ B5-1
Appendix C Chemical and Product Class Information for the Substances
Tested in the In Vitro ER Binding Assays .................................................... C-1
Appendix D
Substances Tested in the In Vitro ER Binding Assays..................................D-1
D1
Data Sorted by Substance Name and Assay................................................ D1-1
D2
References ................................................................................................... D2-1
Appendix E Assay Distribution of Substances Tested in In Vitro ER
Binding Assays.............................................................................................. E-1
Appendix F Median/Single RBA Values for Substances Tested in Two
or More In Vitro ER Binding Assays .............................................................F-1
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ER Binding BRD: List of Acronyms and Abbreviations
October 2002
LIST OF ACRONYMS AND ABBREVIATIONS
A
Observed anisotropy
Ao
Anistropy at 0% inhibition
A100
Anistropy at 100% inhibition
Ab
Anisotropy values of bound ligand
Af
Anisotropy values of free ligand
ACC
American Chemistry Council
AR
Androgen receptor
Bmax
The number of binding sites in a cytosolic preparation
BRD
Background Review Document
CASRN
Chemical Abstracts Service Registry Number
CMA
Chemical Manufacturers Association
cDNA
Complementary DNA
Ci
Curies
DDT
Dichlorodiphenyltrichloroethane
def
Ligand binding domain of the estrogen receptor protein
DES
Diethylstilbestrol
DMSO
Dimethyl sulfoxide
DPM
Disintegrations per minute
ED
Endocrine disruptor
EDSP
Endocrine Disruptor Screening Program
EDSTAC
Endocrine Disruptor Screening and Testing Advisory Committee
EDTA
Ethylenediamine tetraacetic acid
EPA
(U.S.) Environmental Protection Agency
ER
Estrogen receptor
ERα
Estrogen receptor alpha
ERβ
Estrogen receptor beta
ERb
Bound estrogen receptor
ERf
Free estrogen receptor
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ER Binding BRD: List of Acronyms and Abbreviations
October 2002
ERt
Total estrogen receptor
ERE
Estrogen response element
Fb
Fraction of ligand bound
FDA
(U.S.) Food and Drug Administration
FES1
Fluormone intrinsically fluorescent non-steroidal estrogen
FIFRA
Federal Insecticide, Fungicide and Rodenticide Act
FFDCA
Federal Food, Drug, and Cosmetic Act
fmol
Femtomole
FP
Fluorescent polarization
FQPA
Food Quality Protection Act
g
Gravity
GLM
General linear models
GLP
Good Laboratory Practices
GST
Glutathione-S-transferase
GST-aERdef
Glutathione-S-transferase fusion protein containing the ligand binding
domain of the lizard (anole) estrogen receptor
GST-cERdef
Glutathione-S-transferase fusion protein containing the ligand binding
domain of the chicken estrogen receptor
GST-ERdef
Glutathione-S-transferase fusion protein containing the ligand binding
domain of the estrogen receptor
GST-hERα def
Glutathione-S-transferase fusion protein containing the ligand binding
domain of human estrogen receptor alpha
GST-mERαdef
Glutathione-S-transferase fusion protein containing the ligand binding
domain of mouse estrogen receptor alpha
GST-rtERdef
Glutathione-S-transferase fusion protein containing the ligand binding
domain of the rainbow trout estrogen receptor
HAP
Hydroxyapatite
HDT
Highest dose tested
hERα
Human estrogen receptor alpha
hERα-FP
Human estrogen receptor alpha as measured by FP
hERβ
Human estrogen receptor beta
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ER Binding BRD: List of Acronyms and Abbreviations
October 2002
HPTE
2,2-Bis(p-hydroxyphenyl)-1,1,1-trichloroethane
I%
Percent inhibition
ICCVAM
Interagency Coordinating Committee on the Validation of Alternative
Methods
IC50
Concentration of test substance (inhibitor) that displaces 50% of the
reference estrogen from the receptor in a competitive binding assay
kDa
Kilodaltons
Kd
Dissociation or binding constant
Ki
Equilibrium dissociation constant of a receptor-ligand complex
Lb
Bound ligand
ln
Natural log
Lt
Total ligand
µg
Microgram
µL
Microliter
µM
Micromolar
M
Molar
MCF-7
Cell line derived from a human mammary adenocarcinoma
mL
Milliliters
mM
Millimolar
mmol
Millimoles
MUC
Mouse uterine cytosol
NAS
(U.S.) National Academy of Sciences
NCTR
National Center for Toxicological Research
NICEATM
National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods
NIEHS
National Institute of Environmental Health Sciences
nM
Nanomolar
NSB
Nonspecific binding
NTP
(U.S.) National Toxicology Program
OECD
Organisation for Economic Co-operation and Development
PCB
Polychlorinated biphenyl
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ER Binding BRD: List of Acronyms and Abbreviations
October 2002
pM
Picomolar
pmol
Picomoles
ppm
Parts per million
QA
Quality assurance
QSAR
Quantitative structure activity relationship
rI
Intra-class correlation
RBA
Relative binding affinity
RBC
Rabbit uterine cytosol
rERβ
Rat estrogen receptor beta
RUC
Rat uterine cytosol
SAB
Science Advisory Board
SAP
Scientific Advisory Panel
SDWA
Safe Drinking Water Act
SE
Standard error
SERM
Selective Estrogen Receptor Modulator
TEDG
Buffer containing Tris, EDTA, dithiothreitol, and glycerol buffer; used in
the isolation of ER from animal tissues
Tris
Tris(hydroxymethyl)aminomethane
TSCA
Toxic Substances Control Act
WWF
World Wildlife Fund
xii
ER Binding BRD: Acknowledgements
October 2002
ACKNOWLEDGEMENTS
The following individuals are acknowledged for their contributions to the In Vitro Endocrine
Disruptor Screening Assay review process:
Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Endocrine Disruptor Working Group (EDWG)
Agency for Toxic Substances and Disease
Registry (ATSDR)
FDA (cont’d)
Suzanne Fitzpatrick, Ph.D., D.A.B.T.
David Hattan, Ph.D. (EDWG, Co-Chair)
Abigail Jacobs, Ph.D.
Alexander Jordan, Ph.D.
David Morse, Ph.D.
Melvin Stratmeyer, Ph.D.
Thomas Umbreit, Ph.D.
Stephanie Miles-Richardson, Ph.D.
Consumer Product Safety Commission
(CPSC)
Marilyn Wind, Ph.D. (EDWG, Co-Chair)
Department of the Interior (DOI)
National Institute for Occupational
Safety & Health (NIOSH)
Timothy Gross, Ph.D.
Eisuke Murono, Ph.D.
Environmental Protection Agency (U.S.
EPA)
National Institute of Environmental
Health Sciences (NIEHS)
Angela Auletta, Ph.D.
Karen Hamernik, Ph.D.
Richard Hill, M.D., Ph.D.
Sally Perreault Darney, Ph.D.
Jerry Heindel, Ph.D.
Retha Newbold, M.A.
William Stokes, D.V.M., D.A.C.L.A.M.
(Executive Director, ICCVAM)
Julius Thigpen, Ph.D.
Food and Drug Administration (FDA)
Michael Bolger, Ph.D., D.A.B.T.
Paul Brown, Ph.D.
Kenneth Delclos, Ph.D.
Charles Eirkson, M.S.
Occupational Safety and Health
Administration (OSHA)
Surender Ahir, Ph.D.
xiii
ER Binding BRD: Acknowledgements
October 2002
National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
Bradley Blackard, M.S.P.H.
ILS, Inc.
Michael Paris
ILS, Inc.
Sue Brenzel
ILS, Inc.
Barbara Shane, Ph.D., D.A.B.T.
ILS, Inc.
Loretta Frye
NIEHS
William Stokes, D.V.M., Diplomate,
ACLAM (Director)
NIEHS
Christina Inhof, M.S.P.H.
ILS, Inc.
Judy Strickland, Ph.D., D.A.B.T.
ILS, Inc.
Linda Litchfield
ILS, Inc.
Raymond Tice, Ph.D.
ILS, Inc.
Deborah McCarley
NIEHS
Errol Zeiger, Ph.D., J.D.
ILS, Inc./Zeiger Consulting
Steven Myers
ILS, Inc.
xiv
ER Binding BRD: Acknowledgements
October 2002
Additional Reviewers for the In Vitro Endocrine Disruptor Screening Assay
Background Review Documents (BRD)
Neepa Choksi, Ph.D.
NIEHS
Susan Laws, Ph.D.
U.S. EPA
Ralph Cooper, Ph.D.
U.S. EPA
Gary Timm, M.S., M.A.
U.S. EPA
L. Earl Gray, Ph.D.
U.S. EPA
Gail Tudor, Ph.D.
University of North Carolina
Camille Jackson
North Carolina State University
Vickie Wilson, Ph.D.
U.S. EPA
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ER Binding BRD: Acknowledgements
October 2002
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