cc:

Jim Jones

Louise Wise

William Jordan

Margie Fehrenbach

Yu-Ting Guilaran

Robert McNally

Donald Brady

Jack Housenger

Susan Lewis

Lois Rossi

Jay Ellenberger

Richard Keigwin

Mary Manibusan

Jess Rowland

Don Bergfelt

Gregory Akerman

Catherine Aubee

Amy Blankinship

John Liccione

Tom Steeger,

Leslie Touart

Molly Hooven

Dale Kemery

Linda Strauss

Vanessa Vu

OPP Docket

FIFRA Scientific Advisory Panel Members

Daniel Schlenk, Ph.D.

Kenneth Delclos, Ph.D.

Marion F. Ehrich, Ph.D., D.A.B.T, A.T.S

Stephen Klaine, Ph.D.

James McManaman, Ph.D.

1

SAP Minutes No. 2013-03

A Set of Scientific Issues Being Considered by the

Environmental Protection Agency Regarding Endocrine 

Disruptor Screening Program (EDSP) Tier 1 Screening Assays 

and Battery Performance

May 21-23, 2013

FIFRA Scientific Advisory Panel Meeting

Held at the

Environmental Protection Agency Conference Center

Arlington, VA
2

NOTICE

These meeting minutes have been written as part of the activities of the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). 

The meeting minutes represent the views and recommendations of the FIFRA SAP, not 

the United States Environmental Protection Agency (Agency). The content of the 

meeting minutes does not represent information approved or disseminated by the Agency. 

The meeting minutes have not been reviewed for approval by the Agency and, hence, the 

contents of these meeting minutes do not necessarily represent the views and policies of 

the Agency, nor of other agencies in the Executive Branch of the Federal Government, 

nor does mention of trade names or commercial products constitute a recommendation 

for use.

The FIFRA SAP is a Federal advisory committee operating in accordance with the 

Federal Advisory Committee Act and established under the provisions of FIFRA as 

amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides 

advice, information, and recommendations to the Agency Administrator on pesticides and 

pesticide-related issues regarding the impact of regulatory actions on health and the 

environment. The Panel serves as the primary scientific peer review mechanism of the 

Environmental Protection Agency, Office of Pesticide Programs (OPP), and is structured 

to provide balanced expert assessment of pesticide and pesticide-related matters facing 

the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc 

basis to assist in reviews conducted by the FIFRA SAP. Further information about 

FIFRA SAP reports and activities can be obtained from its website at 

http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305-5805. Interested 

persons are invited to contact Fred Jenkins, Jr., Ph.D., SAP Designated Federal Official, 

via e-mail at jenkins.fred@epa.gov.

In preparing these meeting minutes, the Panel carefully considered all information 

provided and presented by EPA, as well as information presented by public commenters.
3

TABLE OF CONTENTS

PARTICIPANTS .......................................................................................................... 5

INTRODUCTION ........................................................................................................ 8

PUBLIC COMMENTS................................................................................................. 9

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS.................. 10

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE.............. 19

REFERENCES ............................................................................................................. 56

5

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)

Scientific Advisory Panel (SAP)

Endocrine Disruptor Screening Program (EDSP) Tier 1 Screening Assays and 

Battery Performance May 21-23, 2013

PARTICIPANTS

FIFRA SAP Chair

Daniel Schlenk, Ph.D.

Professor of Aquatic Ecotoxicology & Environmental Toxicology 

Department of Environmental Sciences 

University of California, Riverside 

Riverside, CA 

Designated Federal Official

Fred Jenkins, Jr., Ph.D.

U.S. Environmental Protection Agency

Office of Science Coordination & Policy

FIFRA Scientific Advisory Panel

EPA East Building, MC 7201M

1200 Pennsylvania Avenue, NW 

Washington, DC 20460

Phone: 202-564-3327

Fax: 202-564-8382

Email: jenkins.fred@epa.gov

FIFRA Scientific Advisory Panel Members

K. Barry Delclos, Ph.D.

Pharmacologist

National Center for Toxicological Research

U.S. Food and Drug Administration 

Jefferson, AR
6

Marion F. Ehrich, Ph.D., D.A.B.T, A.T.S

Co-director, Laboratory for Neurotoxicity Studies 

Professor 

Pharmacology and Toxicology

Department of Biomedical Sciences & Pathobiology

Virginia-Maryland Regional College of Veterinary Medicine

Blacksburg, VA

Stephen Klaine, Ph.D.

Professor & Director

Environmental Toxicology & Biological Sciences

Clemson Institute of Environmental Toxicology

Pendleton, SC 

James McManaman, Ph.D.

Professor & Chief

Section of Basic Reproductive Sciences

Department of Obstetrics & Gynecology, Physiology & Biophysics

University of Colorado, Denver

Aurora, CO

FQPA Science Review Board Members

Sherril Green, D.V.M, Ph.D.

Professor & Chair 

Department of Comparative Medicine 

Stanford University School of Medicine

Stanford, CA

Nelson D. Horseman, Ph.D.

Professor

Department of Molecular and Cellular Physiology

University of Cincinnati

Cincinnati, OH

Jack Leonard, Ph.D.

Professor

University of Massachusetts School of Medicine

Worcester, MA

Deborah MacLatchy, Ph.D.

Vice-President: Academic & Provost

Professor of Biology

Wilfrid Laurier University

Waterloo, Ontario, Canada
7

Reynaldo Patino, Ph.D.

Research Scientist/Professor

U.S. Geological Survey

Texas Tech University 

Lubbock, TX

Aldert H. Piersma, Ph.D.

Professor 

Center for Health Protection

National Institute for Public Health and the Environment

Bilthoven

The Netherlands 

Katherine F. Roby, Ph.D.

Associate Professor and Director

Reproductive Endocrinology Laboratory

Women’s Health Specialty Center

Division of Reproductive Endocrinology & Infertility of the Department of Obstetrics &

Gynecology 

University of Kansas Hospital

Kansas City, KS

Geoffrey Scott, Ph.D.

Director

Charleston Center for Coastal Environmental Health & Biomolecular Research

National Oceanic & Atmospheric Administration, National Ocean Services

Charleston, SC 

Daniel Selvage, Ph.D.

Associate Professor, Pharmacology

College of Pharmacy

University of New England

Portland, ME 

Terry Wayne Schultz, Ph.D.

Emeritus Professor 

Department of Comparative Medicine

University of Tennessee

Knoxville, TN

Donald Tillitt, Ph.D.

Branch Chief

Biochemistry and Physiology Branch 

Columbia Environmental Research Center, Biological Resources Division

U.S. Department of Interior, Geological Survey

Columbia, MO
8

INTRODUCTION

On May 21-23, 2013 the FIFRA Scientific Advisory Panel (SAP) met to address 

scientific issues associated with the “Endocrine Disruptor Screening Program (EDSP) 

Tier 1 Screening Assays and Battery Performance.” Under the Federal Food, Drug, and 

Cosmetic Act (FFDCA), section 408(p) and the Safe Drinking Water Act (SDWA), 

section 1457, the EPA is required to screen all pesticide chemicals (active and inert 

ingredients) and those drinking water contaminants to which a ‘‘substantial population’’ 

is exposed for the potential to interact with the endocrine system. As recommended by a 

Federal Advisory Committee, (Endocrine Disruptor Screening and Testing Advisory 

Committee, EDSTAC), the EPA Endocrine Disruptor Screening Program (EDSP) 

established a two tiered screening and testing program to address the potential of 

chemicals to perturb the estrogen, androgen or thyroid (E, A, or T) systems and elicit 

adverse human and ecological health outcomes. This FIFRA SAP review focused on a 

subset of the initial Tier 1 screening data received by the Agency in response to test 

orders issued for the first list of chemicals in 2009. The SAP provided advice and 

recommendations to the Agency regarding the performance of the 11 Tier 1 screening 

assays and the performance of the assays as a battery that was designed to detect the 

potential of a test chemical to interact with the E, A, or T, hormonal pathways. 

Opening remarks at the meeting were provided by: David Dix, Ph.D., Acting Director, 

Office of Science Coordination and Policy; Steven Bradbury, Ph.D., Director, Office of 

Pesticide Programs; and Mary Manibusan, Director, Exposure Assessment Coordination 

and Policy Division, Office of Science Coordination and Policy

US EPA presentations were provided by the following staff: 

Gregory Akerman, Ph.D

Catherine Aubee, M.P.A

Amy Blankinship, M.S

John Liccione, Ph.D

Tom Steeger, Ph.D,

Leslie Touart, Ph.D.
9

Public Commenters

Oral Public comments were provided by (provided in the order that they presented)

Steve Levine, Ph.D. of Monsanto on behalf of the Endocrine Policy Forum 

Sue Yi, Ph.D. of Syngenta Crop Protection on behalf of the Endocrine Policy Forum 

Sue Marty, Ph.D. of the Dow Chemical Company on behalf of the Endocrine Policy 

Forum 

Barb Neal, DABT of Exponent on behalf of the Endocrine Policy Forum 

John Brausch of BASF on behalf of the Endocrine Policy Forum 

Allen Olmstead, Ph.D. of Bayer CropScience on behalf of the Endocrine Policy Forum 

Ellen Mihaich, Ph.D., DABT of the Environmental and Regulatory Resources on behalf 

of the Endocrine Policy Forum 

Lisa Ortego, Ph.D., DABT of Bayer CropScience on behalf of the Endocrine Policy 

Forum 

Christopher Borgert, Ph.D. Applied Pharmacology and Toxicology, Inc. on behalf of the 

Endocrine Policy Forum

Clare Thorp, Ph.D. on behalf of CropLife America

Scott Slaughter on behalf of the Center for Regulatory Effectiveness

Catherine Willett, Ph.D. on behalf of the Humane Society of the United States

Patricia Bishop, M.S. on behalf of the People for Ethical Treatment of Animals

Colleen Toole, Ph.D. on behalf of CeeTox 

Leah Zorrilla, Ph.D. on behalf of Integrated Laboratory Systems

Michael L. Dourson, Ph.D., DABT, ATS on behalf of Toxicology Excellence for Risk 

Assessment (TERA)

Written Public Comments were provide by:

Will Davies on behalf of LSR Associates Ltd

Richard A. Becker, Ph.D., DABT and Emily V. Tipaldo, MA on behalf of the American 

Chemistry Council
10

SUMMARY OF PANEL RECOMMENDATIONS

Charge Question 1. Based on the analysis of the data presented in Section III, please 

comment on the proficiency of the contributing laboratories to execute each assay in 

accordance with the test guidelines and achieve the performance criteria.

Panel Summary

The Panel noted that there are several limitations which hampered their ability to 

determine how well the laboratories achieved the proficiency criteria. These limitations 

were based upon the many variations among the laboratories conducting the assays, 

including differences in their: 1) methodologies, 2) interpretation of the assay results, and 

3) adherence to the assay guidelines. The Panel also mentioned that the way in which the 

data were presented in the Agency’s White Paper made it difficult to identify which 

laboratory tested particular chemicals. Because of this lack of information the Panel was 

unable to compare the laboratories’ proficiencies in conducting the assays. 

Concerning the Fish Short Term Reproductive Bioassay (FSTRA) and the Amphibian 

Metamorphosis Assay (AMA), the Panel remarked that there are major deficiencies 

among the laboratories in the dosing conformance of these assays. The Panel specifically 

expressed the concern that if laboratories alter the rate of volume replacement, the total 

volume of chemical delivered per aquarium per day will be different. This could 

potentially cause a variation in the effects observed. Thus, the Panel recommended that 

the parameters of the protocol should assure that the initial target nominal dose is being 

delivered throughout the experiment. 

Lastly, the Panel pointed out two primary specific concerns associated with the 

Amphibian Metamorphosis Assay. Their first concern was the high rates of tail curvature 

observed in the controls and exposure groups. As noted by the Panel, this issue was 

obviously related to the organisms’ diet as well as their source of origin. Their second 

concern was how to use, or not use, the data which demonstrated potential effects from 

the solvents used to increase the test chemical’s solubility. The Panel recommended that 

the Agency address these concerns in the performance criteria.
11

Charge Question 2. The performance criteria for each in vitro assay are clearly stated in 

the test guidelines for the ER binding (OCSPP 890.1250), AR binding (OCSPP 

890.1150), ERα Transcriptional Activation (OCSPP 890.1300, OECD 455), H295R 

Steroidogenesis (OCSPP 890.1550) and aromatase human recombinant (OCSPP 

890.1200) assays. Although contributing laboratories did not always demonstrate that 

results were within the specified boundaries of the performance criteria, the majority of 

the deviations were still close to the performance criteria. In this regard, the EPA 

concluded that the data were still adequate for use. Please comment on the EPA’s 

conclusion. Please comment on when a deviation from the recommended performance 

criteria would render the study unreliable.

Panel Summary

The Panel was in general agreement with the Agency’s determination that, even though 

there were circumstances in which the performance criteria were not met, these 

performance criteria deviations were deemed as minor and did not impact the 

interpretation or reliability of the data. Nevertheless, the Panel’s verdict encompassed 

two caveats/concerns including: 1) a determination based on the limited data set of 21 

chemicals, and 2) a limited ability to interpret the data because of the manner in which 

the data were presented in the White Paper (i.e., the lack of direction and magnitude of 

the measured values as compared to control values).

The Panel recommended that Tier I guidance be developed to indicate the point at which 

deviation from the norm is appropriate for an individual run to be considered acceptable. 

Such guidance should alleviate the inclusion of cases in which there are questions about 

assay parameters. The Panel noted that this guidance could be in the form of a decision 

tree that would assist laboratories in deciding when to redo individual runs that have not 

met the criteria.

The Panel questioned the redundancy of the Estrogen Receptor (ER) binding and ERα 

Transcriptional Activation (ERTA) assays used in Tier I in the context of the Rainbow 

Trout Estrogen Receptor (rtER) binding and liver slice gene activation assays used in the 

EDSP development of the Computational Toxicology Tools. Specifically, the Panel 

advised the Agency to consider eliminating the Tier I in vitro assays related to the ERpathways based on the premise that “the hormone binding domain of the ER is highly 

conserved across species.” The Panel’s recommendation to eliminate the ER-binding and 

ERTA assays from the battery of Tier 1 assays is further supported by the Agency’s 

interest in using high throughput screening data to expand the structural domain used in 

the prescreening activities. The Panel recommended that the Agency consider replacing 

the current Tier I in vitro assays related to the AR-pathways (i.e., one based on rat 

prostate cytosol) with one based on a recombinant cell line. Replacing the current ARbinding assay bypasses addressing the major shortcomings of the protocol, namely the 

limitations imposed by the preparation, attaining performance criteria, and storage life for 

the prostate cytosolic preparations. Moreover, replacement of AR-binding assay

decreases the numbers of animals required to complete the Tier 1 testing.
12

Charge Question 3: A positive control is not required for the male and female pubertal 

assays. For these in vivo assays with rats, the coefficient of variation limits are specified

in the test guidelines for most endpoints. Submissions from different laboratories 

sometimes fell short of meeting all the test guideline-recommended Coefficient of 

Variation (CV) limits for the endpoints evaluated. However, in most cases these 

shortcomings were considered of minor importance to the overall results, and the EPA 

concluded that the data are still adequate for endocrine screening. Please comment on 

when a deviation from the recommended CV limits would render the study unreliable. 

Panel Summary

The Panel generally concurred that although different laboratories fell short of meeting 

all the guideline-recommended CV values for endpoints evaluated, the data from the 

assays were adequate for endocrine screening. Nevertheless, the Panel cautioned that 

there were many challenges in accepting data that comes short of meeting the 

performance criterion for CVs. For example, the Panel believed that accepting large CVs, 

weakens the statistical power of the tests. This can make the assays undependable for the 

detection of a biologically significant effect.

The Panel agreed with EPA’s conclusion that data generated using the male and female 

pubertal assays are useful. However, the Panel advised that the guidelines be carefully 

modified and cautioned that significant care be applied in interpreting the results of these 

assays.
13

Charge Question 4: The test guidelines for the six in vivo assays (Hershberger assay -

OCSPP 890.1400, OECD 441; Uterotrophic assay- OCSPP 890.1600, OECD 440; Male 

Pubertal assay- OCSPP 890.1500; Female Pubertal assay - OCSPP 890.1450; FSTRA -

OCSPP 890.1350, OECD 229 and AMA - OCSPP 890.1100) offer some guidance on 

setting the dose/concentration range when testing for specific effects on the E, A, or T 

signaling pathways. In some of the in vivo assays, overt toxicity was noted based on 

effects on growth, other sublethal effects, and even mortality at the highest 

dose/concentration tested. Positive Tier 1 findings indicating the potential for endocrine 

activity can be difficult to interpret in the presence of overt toxicity.

Please comment on the nature and severity of overt toxicity that would render study 

results unreliable for the purpose of Tier 1 screening of a chemical for endocrine activity. 

Please comment on what, if any, additional guidance is needed to minimize overt toxicity 

while ensuring adequate dose selection for these screening assays. Also, specifically 

comment on the validity of including treatment concentrations with apparent body weight 

effects in the analysis for potential endocrine interaction for FSTRA.

Panel Summary

There is much utility in using a broad dose range to evaluate the potential endocrine 

disrupting effects of test compounds. Nevertheless, overt toxicity seen during testing 

obscures the interpretation of data from the EDSP Tier I in vivo assays. For instance, if 

effects are observed only within a dose group with overt toxicity and in the only screen 

that shows a positive effect, it will be complicated to determine whether the compound 

should be evaluated via Tier 2 testing without a repeat experiment at a lower dose. Thus, 

the Panel concurred that it is necessary to reduce the test compound to sub-toxic levels to 

enable an independent evaluation of the target agent on endocrine function.

For specific criteria in defining maximum-tolerated-doses (MTD) in the EDSP assays, the 

Panel suggested that the common standard of practice/animal welfare benchmark for 

short-term toxicity studies is a 10% upper limit of body weight loss. This cut off is 

considered to be adequate to determine maximum-tolerated-doses, and it is a commonly 

accepted standard by most institutional research animal use and care committees 

(Chapman et al., 2013). The Panel recommends that this 10% benchmark be used as 

general guideline only for mammalian assays and not the FSTRA or AMA.

The Panel made several general recommendations regarding dose selection for the in vivo

assays in the EDSP. When overt toxicity occurs it was suggested that the testing labs 

employ two additional lower doses below the original MTD used to generate the dataset, 

thus increasing the probability of using concentrations that do not illicit overt toxicity. If 

effects on A, E or T endpoints are still observed in the absence of overt toxicity, then 

there is a higher degree of confidence that the effects on A, E, and T are directly related 

to the mode of action of the chemical as opposed to a more generalized stress response. 

If, on the other hand, effects on A, E and T are not observed in the lower dosing regimen, 

then the original dataset obtained at toxic MTD levels should not be considered to be 

definitive for direct effects via alterations in hormonal pathways. 
14

Charge Question 5. Spinal curvature, usually manifesting as “bent tail” in X. laevis 

tadpoles, was reported in 15 of 18 AMA studies reviewed thus far. The anomaly appears 

to be first observed several days after study initiation, and prevalence increases with time. 

Overall, the prevalence of spinal curvature in these studies ranged from “a few per 

replicate” to 92% of a given treatment group by test termination. Experimental work by 

the EPA Office of Research and Development suggests that overfeeding can be a primary 

cause of spinal curvature in their Xenopus test populations; however, spinal curvature 

remained prevalent (range: 16-92%) in the five industry AMA studies in which feed was 

reduced by 50% compared to guideline recommendations.

Overall, the incidence of spinal curvature appears to be highly variable. From a 

qualitative review of the data, there appear to be no consistent differences in the 

incidence or variability of spinal curvature when studies using guideline versus reduced 

feeding regimes are compared. Please comment on whether the presence or prevalence 

of spinal curvature in test specimens, including controls, compromises the utility or 

validity of an AMA submission. If so, when does the prevalence of spinal curvature 

render the study unreliable? What technical guidance may be useful for laboratories in 

reducing the occurrence of spinal curvature and determining if, or at what point within 

the study, a study may be compromised because of this phenomenon?

Panel Summary 

The Panel advised that the Agency keep track of the clutch performance (including 

number and quality of embryos from each spawn) and that the Agency select high 

quality, healthy breeding pairs. Eliminating breeding pairs that produce bent tail should 

minimize its occurrence. Specifically minimizing the use of wild-caught breeding stock 

may be beneficial.

The Panel also recommended that there should be a careful review of water quality 

conditions under which bent tail tadpoles are kept. Temperature, pH, and nutritional 

deficiencies (vitamin C and Ca) are reported to cause bent tail in laboratory reared 

American bullfrogs, Leopard frogs (Marshall et al, 1980; Leibovitz et al, 1992; Martinez 

et al, 1992), and in several other amphibian species. One panel member noted that, based 

on personal observation, temperature and pH have appeared to be associated with the 

condition in Xenopus tropicalis and water softness (Ca and K) associated with the 

condition in Xenopus laevis. Over-feeding can alter the water biochemistry and uneaten

food and excess waste causes changes in ammonia and pH. These changes are 

detrimental to the health of the test animals; thus, they could potentially explain the bent 

tail seen in the laboratories that reported a decrease in the occurrence of the condition

when food was decreased. Feed and water sources should be screened for herbicides, 

biological toxins and pesticides currently known to cause bent tail in Xenopus laevis, 

nominally. The Panel recommended further monitoring the portion of each clutch not 

used in a given study. It would be potentially useful to report these results. The Panel 

acknowledged that some information on this concept is in the guidance document, but 

recommended that it be elaborated further. The Panel further recommended that for 

acceptance of the AMA test results overall, the clutch should not have a rate of mortality 

and morbidity > 20%.
15

Charge Question 6. With the exception of thyroid gross pathology findings (thyroid 

gland atrophy and hypertrophy) in the AMA, severity grades are generally assigned based 

on comparison to “normal” X. laevis thyroid findings depicted in the guidance or based 

on the professional opinion of the pathologist conducting the assessment; they are not 

assigned in comparison to concurrent control findings from a given study. (Please refer 

to Section III.2.f in the document entitled “Interpreting Amphibian Thyroid 

Histopathology Diagnoses” and supporting documents, OECD Guidance Document on 

Amphibian Thyroid Histology No. 82, 2007 and Grim et al., 2009).

a. In one study, the pathologist’s report identified a lower incidence and 

severity of follicular cell hypertrophy when compared to the incidence and 

severity of this trait in control specimens. Similar trends have been 

observed in other studies. In this case, the pathologist concluded that the 

finding was potentially consistent with treatment-related delay of 

metamorphosis because thyroid follicular cells normally increase in 

height during tadpole development. Please comment on the validity of this 

conclusion.

Panel Summary

Even though thyroid hormone agonistic activity could provide a justification for the 

pathologist’s observation of a lower incidence and severity of thyrocyte hypertrophy via 

negative feedback inhibition of the hypothalamic-pituitary-thyroidal (HPT) axis, this 

circumstance would also have to be complemented by signs of accelerated development 

in the experimental population. However, since accelerated development did not appear 

to occur, the Panel concurred with the pathologist’s conclusion that the finding is 

consistent with a treatment-related retardation of metamorphosis via disruption of the 

HPT axis. As demonstrated in the public literature, this case may represent an unusual 

disruption of the axis that is not based on classical anti-thyroid mechanisms such as those 

observed after exposure to thyroid synthesis inhibitors (goitrogens), but on a failure of the 

axis to activate normally or fully (Sharma and Patiño 2008).

b. What guidance may be given to better distinguish between histological 

changes in the thyroid associated with the normal progression of 

metamorphosis and treatment-related effects? Are there certain lesions 

or diagnoses which may, by their absence or lessened severity as 

compared to controls, be indicative of treatment-related HPT effects such 

as delayed metamorphosis?
16

Panel Summary

The Panel acknowledged that thyroid activity normally intensifies during metamorphosis, 

and consequently it is relatively challenging to perform this activity as a control 

condition. The Panel believed that a marker of normal thyroid activity that does not 

change during metamorphosis would be ideally useful. The Panel was unaware of any 

standard histological feature of the thyroid that does not normally change during tadpole 

development. However, the Panel noted that the T4-immunoreactivity has been 

demonstrated to concentrate in a ring at the periphery of the colloid and that the intensity 

of this ring remains fairly constant during metamorphosis (Hu et al., 2006). Thus, the 

Panel recommended that the Agency explore the T4-immunoreactive ring as a potential 

marker. They noted, however, that the T4-immunoreactive ring has not been validated for 

standard testing. They also noted that the T4-immunoreactive ring could possibly provide 

a benefit to the thyroid assessments in the rat pubertal assays.

Lastly, the Panel recommended that the Agency consider the use of quantitative thyroid 

activity measurements as opposed to the semiquantitative grading scheme currently used 

which may be prone to problems with inconsistencies.

Charge Question 7. In 2008, the SAP acknowledged that the in vivo assays included in 

the Tier 1 battery provide both redundancy and complementarity for evaluating 

interactions with the E, A, or T signaling pathways. The panel also noted that all of the 

Tier 1 assays and the broad range of endpoints appeared to be necessary to “discriminate 

positive and negative results”.

a. Please comment on the battery performance with respect to the 

anticipated complementary nature of the more complex, multi-parameter 

in vivo assays in the context of the observed responses with the case 

studies. Please comment separately on the E-, A-, and T-related assays 

and endpoints.

Panel Summary

The Panel observed that the Tier I assays did not provide information on the magnitude 

or the direction of the effects. Thus, they noted that it was difficult for them to determine 

how useful the observed complementary effects will be when applied in the weight-ofevidence (WoE) analysis to strengthen or weaken the case for the E, A, or T activity of 

these test compounds and determine the need for further testing. Specifically, in regard to 

the E,A, and T-individual assays and their endpoints, the Panel noted that the prevalence 

of overt toxicity in each of these assays appeared to confound the interpretation of the 

results for many of the chemicals tested.

b. Please comment on the battery performance with respect to the anticipated 

redundancy across the 11 assays in the context of the observed responses 

with the case studies. Please comment separately on the E-, A-, and Trelated assays and endpoints.
17

Panel Summary

The Panel concluded that due to the manner in which the data was presented, they could 

only provide limited input on the performance of the battery. Particularly, without 

information on the magnitude or direction of change, the Panel determined that it did not 

seem possible to provide a thorough judgment on how the battery performed.

c. The EPA concluded that the battery has performed as anticipated by the 

2008 SAP. Please comment on this conclusion.

Panel Summary

The Panel generally agreed that the battery performed as expected with the subset of 

chemicals on which data summaries were provided. However, the Panel emphasized that 

only limited conclusions can be drawn at this time based on the narrow amount of 

information that has been provided.

Many panel members strongly expressed their belief that positive controls that are well 

known to interact with E, A, and T pathways should have been used to evaluate the entire 

battery just as what was done in the validation of each individual assay. The ability of 

the battery to clearly detect such known compounds would have been useful in providing 

confidence that the battery functioned as intended and serve as a means for determining 

which assays might be considered for removal or replacement.
18

Charge Question 8. The EPA is committed to minimizing animal usage in the screening 

battery while maintaining the effectiveness of the battery to answer the question of 

whether a chemical has the “potential” to interact with the endocrine system.

a. In 1998, the EDSTAC described the conceptual framework for Tier 1 

assays and recommended the strategy to “require the minimal number of 

screens and tests necessary to make sound decisions, thereby reducing 

the time needed to make these decisions”, and that the screens should be 

conducted at a minimal cost necessary to make decisions. Based on the 

preliminary battery performance evaluation, to what extent can the 

current Tier 1 battery of 11 assays be modified to reduce animal usage 

and/or lower cost while adequately ensuring the EPA’s ability to answer 

the question of “whether a chemical has the potential to interact with the 

endocrine system?” More specifically, please comment on whether the 

Uterotrophic and Hershberger assays provide necessary redundancies in 

the Tier 1 battery based on this preliminary analysis. Please include in 

your comments what information may be lost and what uncertainties may 

be introduced by absence of either or both of these assays.

Panel Summary

While the Uterotrophic or Hershberger assays are limited in what they are able to detect, 

both of these assays are well-accepted tests of Gonadal hormone action on peripheral 

tissues. Although the reduction of animal usage and unnecessary testing are big concerns, 

the Panel thought that the assays should be maintained since they provide for specific 

testing of the actions of potential endocrine disrupting agents and their metabolites in a 

mammalian system. Such testing can provide more definitive information regarding the 

mode of action (MOA) of the test compound than what can be learned from the more 

comprehensive pubertal assays. At a minimum, a decision to remove these assays from 

the battery should be postponed until the completion of the Agency’s WoE analysis of 

these compounds to determine what role these assays play in the decision process.

b. Please comment on the scientific criteria the Agency should consider in 

evaluating necessary redundancies and eliminating assays from the 

current battery.

Panel Summary

The Panel advised that the decision to remove an assay from the battery could likely be 

made after an exhaustive review of all the data generated from the complete set of 

compounds that have been run through the Tier I screens. As a consequence, an assay 

should be removed if it consistently produces results that are not clearly interpretable. 

Concerning the potential of modifying the current battery, the Panel strongly stressed that 

the Agency should do a more extensive data evaluation in order to best determine the 

range of possibilities for modification. 
19

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE

Individual EDSP Tier 1 Assay Performance

Charge Question 1. The results of the EPA’s evaluation to determine how well each Tier 

1 assay was executed relative to defined performance criteria in the respective test 

guideline are presented in Section III of the white paper. This evaluation was based on a 

subset of pesticide active ingredients that represent a broad range of physical-chemical 

properties (e.g., water solubility, octanol-water partition coefficients, volatility), as well 

as a range of chemical classes and pesticidal activities. This preliminary analysis 

generally demonstrates that the laboratories executing the assays can meet the 

performance criteria defined in the test guidelines and the results provide useful 

information to determine whether a chemical has the potential interact with the endocrine 

system. During this initial analysis of individual study data, however, EPA identified a 

few issues with some assays. The following questions seek comment from the FIFRA 

SAP on assay (laboratory) performance and advice on how to address technical 

deviations and other issues encountered with specific assays. Based on the analysis of the 

data presented in Section III, please comment on the proficiency of the contributing 

laboratories to execute each assay in accordance with the test guidelines and achieve the 

performance criteria.

Panel Response

Established guidelines for each test in the Tier 1 battery define test parameters, criteria 

for assessing testing proficiency, and criteria for acceptable limits for various positive 

and negative controls to be included in most tests. Thus, general proficiency of the testing 

laboratories can be monitored by several end-points.

Based on the information presented in Section III of the White Paper, a general 

assessment indicates that the testing laboratories demonstrate proficiency in carrying out 

the battery of tests. However, there were variations across laboratories in their ability to 

achieve proficiency criteria, raising some concerns.

The overall proficiency of the contributing laboratories to execute each assay in 

accordance with the test guidelines and achieve the performance criteria was variable 

according to each in vitro and in vivo test. These differences in proficiency across testing 

laboratories were primarily attributable to slight variations in methodology, 

interpretation, or follow-through of testing guidance. For example, when solubility issues 

were encountered one laboratory did not adhere to the guidance to reset with the 

recommended half-log increment dilution; instead, they omitted the higher log dose and 

added an additional lower dose at the end of the dose series. Utilization of the mid-log 

approach at the upper end of the concentration range would better characterize the 

response curve and provide more confidence in the interpretation of the results. 

Additional variation may also be introduced due to an individual laboratory’s conditions; 

such as an inability to maintain water temperature requirements to the +/- 1°C in the fish 

tests. Further, variation within a given laboratory may occur based on the proficiency of
20

different analysts performing the test and thus, individual performance criteria for each

analyst should be documented and recorded over time.

Given the manner in which the data are presented in Section III, it is difficult to 

determine what laboratory tested what chemical. In addition, within the description of a 

given test, there are places in the text where the number of testing laboratories and the 

numbers of chemicals tested do not agree, further complicating the assessment of 

laboratory proficiency. Further, it appears that each test chemical was assessed by only 

one laboratory. Having more than one laboratory test the set of chemicals, or even a 

subset of chemicals, would provide an additional monitor for proficiency. Based on this 

lack of multi-laboratory testing for chemicals, the Panel was only able to comment on the 

proficiency for an individual laboratory to perform the battery.

In the evaluation of results from individual assays in Section III, especially with regard to 

the in vitro assays, multiple statements were made indicating the testing laboratories did 

not provide complete proficiency data with reports submitted to the Agency. The 

exclusion of proficiency data from some datasets weakens the ability of the Panel to 

carefully assess the proficiency in these instances. Examples include results presented for 

the Steroidogenesis and Aromatase assays. The test guidance should clearly indicate the 

requirement of the testing laboratory to include the complete proficiency data set with 

submission of the test report to the Agency. If this is not clearly stated, the guidance 

should be amended. 

Similar to the data description for the in vitro tests, in evaluating the summary of the in 

vivo assays it was not clear how many laboratories participated in each test and thus 

conclusions about multiple laboratory proficiency cannot be made. In addition, Section 

III generally provided limited data for analysis. The lack of data providing direction and 

magnitude for test endpoints including controls, proficiency measures, and test chemicals 

limit the Panel’s ability to carefully assess laboratory proficiency. 

In regard to the aquatic tests data, again, the laboratories conducting each of the tests 

were not respectively identified. Consequently, variation in laboratory proficiency cannot 

be judged. As with the other Tier 1 tests, the aquatic tests had variation around 

performance criteria and, similar to the other tests, clear definition of acceptable limits of 

deviation around the performance criteria are needed. It is understood that the acceptable 

deviation may differ for each test and endpoint measure. 

In many cases, including both the in vitro and in vivo tests, the established performance 

criteria were not met. As described in Section III, deviation from the performance criteria 

was most often considered to be ‘slight’ or ‘minor’. The Agency concluded these 

instances of slight deviation from the established criteria did not affect interpretation of 

study outcome. The Panel was in agreement with the conclusion by the Agency and also 

believed these slight variations did not impact interpretation of the data. However, 

describing the deviation around performance criteria as “slight” or “minor” should be 

avoided in favor of quantitative definitions for the variation. An example might be 

defining minor and major non-conformities and then define quantitatively what these two
21

ranges of deviation from norm include, such as : (1) based upon a certain percentile 

deviation from each performance criteria (e.g., minor < 10% different and major > 10% 

different); or (2) a statistical based estimation (such as +/- 1 or 2 standard deviations). 

Specific criteria should be established to aid in the identification of unreliable test runs 

and the interpretation of successful runs. In addition, more specifically in regard to CVs 

reported in in vivo studies, criteria defining acceptable performance CVs may need to be 

revisited to account for the variations existing in control populations. 

In addition to further defining non-conformities, it was considered that a decision-tree 

approach would allow for clear assessment of when test criteria have or have not been 

met and when a given test can be accepted or rejected, or should be rerun. For example, 

when negative controls included in a test run are read as positive, as indicated in at least 

one instance in the data summary provided, the run should likely be rejected. In addition, 

guidance on how to utilize the data in the presence of cytotoxicity and toxicity should be 

included.

Although the majority of the deviation from the proficiency criteria was not considered to 

impact the interpretation of the data, there were some deviations that may be cause for 

concern. In regard to the FSTRA and the AMA, there are major deficiencies in dosing 

conformance. If this was due to analytical limitations, absorption or biodegradation, these 

processes need to be characterized. There was some concern that if laboratories alter the 

rate of volume replacement, the total volume of chemical delivered per aquarium per day 

will be different and this will be a source of variation, in effect. The parameters of the 

protocol should assure that the initial target nominal dose is being delivered. An 

additional measurement in aquaria not containing test animals is recommended, although 

this will add additional cost. As mentioned by EPA, there are routine stock concentration 

measurement requirements as well as daily flow rate determinations. This combination of 

measurements should be reported and could be used to predict the estimated nominal 

concentration being delivered throughout each bioassay. In addition, this would at least 

provide an accurate estimate of the delivered nominal dose, without having to measure it. 

Also, it is important to try to use only one stock for dosing purposes. A second concern 

with the FSTRA relates to the cumulative effects of several non-conformities, including 

low dissolved oxygen levels, elevated exposure temperatures, and elevated levels of 

chlorine. The fish will respond to these changes with increased ventilation rate that may 

damage the fish gills and may also result in increased uptake of the chemical being tested. 

An additional concern is that the accumulation of multiple non-conformities may increase 

the potential for false positives. These three mentioned non-conformities for performance 

criteria are easily corrected and participating laboratories should have taken steps to 

better assure compliance with these criteria. The Agency may consider evaluating and 

identifying combinations of cumulative non-conformities which may rise to a level of 

non-conformity requiring re-testing.

There were two primary concerns related to the performance of the AMA. The first 

concern relates to the high rates of tail curvatures observed in controls and exposure 

groups. This high incidence raises the question as to the health of the animals being used 

in this bioassay. There are obvious issues with this abnormality being tied to diet as well
22

as the source of the test organisms. EPA should examine the underlying health of frogs 

post metamorphosis since the tail is reabsorbed during the developmental process. If this 

abnormality does not affect growth, health, and survival later in life, this issue may not be 

as significant as it would be if the animals were affected later in life. Also, maintaining 

accurate records of the source of frogs is a critical performance criterion that should be 

included. The second concern is related to the potential effects of carriers used to increase 

chemical solubility. The performance criteria presented clearly demonstrated that it is 

possible to discern this effect with the current assay. The question is how to use or not 

use the data when a carrier effect is observed. EPA should formally address this in the 

performance criteria, if they have not already done so. For example, if carrier effects are 

observed, an end point analysis between carrier control and chemical should be 

conducted. This should be clearly informed within the guidance.

Charge Question 2. The performance criteria for each in vitro assay are clearly stated in 

the test guidelines for the ER binding (OCSPP 890.1250), AR binding (OCSPP 

890.1150), ERα Transcriptional Activation (OCSPP 890.1300, OECD 455), H295R 

Steroidogenesis (OCSPP 890.1550) and Aromatase Human Recombinant (OCSPP 

890.1200) assays. Although contributing laboratories did not always demonstrate that 

results were within the specified boundaries of the performance criteria, the majority of 

the deviations were still close to the performance criteria. In this regard, the EPA 

concluded that the data were still adequate for use. Please comment on the EPA’s 

conclusion. Please comment on when a deviation from the recommended performance 

criteria would render the study unreliable.

Panel Response

Charge Question 2 is specifically related to evaluating how well each Tier 1 assay test 

guideline can be consistently executed based on defined performance criteria and to 

highlighting any issues associated with interpretation of responses within each assay.

As noted in the Endocrine Disruptor Screening Program (EDSP) White Paper, the EDSP 

Tier 1 screening consists of a battery of eleven assays (see White Paper Table 1 “Current 

EDSP Tier 1 Screening Assays”). However, only the five in vitro assays are addressed in 

Question 2 which include the: 1) Estrogen Receptor (ER) binding assay, 2) ER 

Transcriptional Activation assay (ERTA), 3) Androgen Receptor (AR) binding assay, 4) 

Aromatase assay, and 5) Steriodogenesis assay. 

Tier 1 screening consists of a battery of less complex in vitro and in vivo assays designed 

to effectively and efficiently detect the potential of a chemical to interact with E, A, or T 

hormonal pathways. Tier I assays are also intended to determine, based on a WoE 

analysis, whether a chemical is a candidate for Tier 2 testing. In contrast, Tier 2 testing 

involves more complex in vivo studies to clarify the potential interaction with the 

endocrine or non-endocrine systems and to establish dose-response relationships that may 

be needed for risk assessment.
23

As such, Tier 1 screening lies between: 1) prescreening or prioritization with in silico 

tools, which identify chemicals with the potential to be hazardous via select endocrine 

disruption mechanisms, and 2) more resource-intensive testing for risk assessment. 

Generally, Tier 1 assays are: 1) shorter duration and lower cost, 2) designed to minimize 

false negative, 3) report the potential to interact in a yes/no format, and 4) use limited 

dose regimes, focused on the higher end.

Each of the Tier 1 assays, which form the basis for this SAP was previously validated 

with a known set of reference compounds for endocrine screening specific to either the E, 

A, or T signaling pathways through a process of test method development, pre-validation, 

and inter-laboratory validation (US EPA, 2009). These efforts culminated in an 

evaluation by a 2008 FIFRA SAP (i.e. March 25-28, 2008 Session of the FIFRA SAP 

entitled “Review of the Endocrine Disruptor Screening Program (EDSP) Proposed Tier 1 

Screening Battery”), which supported using the eleven assays to initiate Tier 1 screening

(US EPA, 2008).

The Panel understood that one aspect of the current SAP focuses on current assay 

performance analysis, specifically the proficiency of the contributing laboratories to 

execute each Tier 1 assay in accordance with the test guidelines. This includes the proper 

execution of the performance criteria and the generation of the expected results for those 

assays utilizing positive controls. Additionally, the Panel understood that while Tier 1 

screening is linked to a WoE analysis, WoE analysis is not part of this SAP. 

The Panel congratulated the EPA on providing a summary of the results for the 21 

compounds brought forth as part of this SAP for the eleven Tier I assays (see section III 

“Evaluation of Results from Individual Assays” of the White Paper). The Panel also 

appreciated the public commenters' input, which in many cases, the Panel found to be 

highly informative.

The Panel acknowledged that the 21 test compounds evaluated as part of this SAP 

represent the fifty active and two inert ingredients that have been screened in the Tier 1 

battery as an intermediate step toward the 67 pesticides ordered for testing by the Agency 

in 2009 in follow up to the recommendation by the 2008 SAP. While the Agency stated

in the White Paper that the 21 chemicals are representative of the 52 chemical set 

selected for Tier 1 testing, especially in regard to physico-chemical properties and modes 

of toxic action, the ability of the Panel to address this and other charge questions is 

impacted by this limited data set. Moreover, because of a variety of issues, not all 21 

compounds were assessed in each in vitro assay associated with the Tier 1 battery.

The Panel recognized that while the assays in toto were designed to identify whether a 

chemical has the potential to interact with estrogen, androgen or thyroid (E, A and T) 

hormone systems, the in vitro assays, however, focus primarily on the E and A systems

due to the characteristics of their endpoints. Furthermore, the Panel understood that not 

all in vitro and in vivo data are of equal value, and the in vitro data may be or may not be 

informative to results from the in vivo assays. Specifically, the Tier 1 in vitro assays are
24

intended to provide some mechanistic data for single known pathways, while the in vivo

Tier 1 assays are more amenable to detecting multiple mechanisms of action.

As noted in Table 1 and in the White Paper, the in vitro assays in the current EDSP Tier 1 

screening battery address only the E and A systems by quantifying the key endpoints of 

the most likely mode of action (e.g., receptor binding as well as steriodogenesis).
25

Table 1. Endocrine Effect Assessment by the Tier 1 Assays

Steroid Synthesis

E E- A A- A E HPG HPT

In vitro

ER Binding X X

ER Transcriptional Activation X

AR Binding X X

Steroidogenesis (H295R) X X

Aromatase (Recombinant) X

In vivo

Uterotrophic X

Hershberger X X

Pubertal male X X X X X

Pubertal female X X X X X

Fish Reproductive Screen X X X X X X X

Amphibian Metamorphosis X
26

Specific to the estrogen system are four in vitro assays. The ER binding assay OCSPP 

890.1250 is a rat uterine cytosol assay that has the potential to detect ERα or ERβ binding 

but cannot distinguish between estrogen agonists and antagonists. The ERTA assay 

OCSPP 890.1300 is a human cervical tumor cell (Hela 9903) assay that has the potential 

to detect estrogen agonists through ERα binding and subsequent gene transcription. The 

aromatase assay OCSPP 890.1200 is a human microsome assay that has the potential to 

detect the inhibition of the enzyme aromatase that converts androgen to estrogen. The 

Steroidogenesis assay OCSPP 8980.1550, is a human cell line H295R assay that has the 

potential to detect a change in production (i.e., increase or decrease) of testosterone and 

estradiol.

There are three in vitro assays specific to the androgen system. The AR Binding assay 

OCSPP 890.1150 is a rat prostate cytosol assay that has the potential to detect androgen 

receptor binding but cannot distinguish between androgen agonists and antagonists. The 

previously noted Aromatase Assay has the potential to detect the inhibition of conversion 

of androgen to estrogen and the previously noted Steroidogenesis assay has the potential 

to detect an increase or decrease in production of testosterone and estradiol.

The Panel recognized that there are no in vitro assays associated with the T pathway in 

the Tier I battery. Moreover, the Panel perceived that, based on the outcome of FIFRA 

SAPs in August 2009 and January 2013 related to the EDSP, the Agency has a much 

more experience with screening the ER mediated pathway than the AR mediated 

pathway.

The Panel acknowledged that an earlier FIFRA SAP (USEPA, 2008) reviewed the 

combination of EDSP Tier 1 screening assays of the current battery and concluded that 

the battery of assays considered the different key events (e.g., receptor binding, cell 

signaling, organ response) along the most common pathways for E, A, and T disruption. 

In addition, the degree of redundancy (i.e., concordance of endpoints across the assays 

that are intended to evaluate an interaction within a particular pathway, E, A or T) and 

complementarity (i.e., concordance of endpoints within an assay) of the assays and 

endpoints in the Tier I battery were likely to minimize the potential for "false negatives" 

and "false positives" and did not result in unnecessary redundancies for a mode of action. 

While the 2008 review was based on individual assay validation results with selected 

reference chemicals known to interact with the E, A, and T hormonal pathways, the 

current review focuses on the application of the entire battery of eleven assays with 

chemicals not necessarily known to interact with the endocrine system.

In answering Charge Question 2, the Panel has elected to address each of the five in vitro

assays separately and then provide other comments of a more general nature.
27

ER-Binding Assay

As noted in the White Paper, 19-norethindrone was used by all laboratories as a 

replacement for the recommended weak positive control ligand, norethynodrel. Since the 

guideline does not contain performance criteria for 19-norethindrone the Panel 

recommended that the Agency establish performance criteria for 19-norethindrone.

The Panel agreed that ER-binding assay performance was by and large, consistent across 

all 18 test compounds and that, in general, issues were with individual runs. In addition, 

while a number of the evaluated assays did not meet all of the performance criteria, 

overall, the laboratories’ performance of the ER binding assays was acceptable.

One Panel member noted that the most problematic aspect of this assay is that it uses a 

highly complicated and labor-intensive protocol involving extracted uterine cytosol. This 

assay was conducted by different labs with varying success. It is recommended that the 

Agency standardize this parameter of the assay if they are to continue to use this assay. 

The Panel believed the results and conclusions for this assay are supported in a WoE by 

the Agency’s work with the Rainbow Trout ER-binding assay.

ERα Transcriptional Activation (ERTA) Assay

As noted earlier the ERTA is used to determine if a chemical interacts with the endocrine 

system by functioning as an ERα ligand and activating transcription of an estrogendependent reporter gene. This protocol is validated for the detection of estrogen agonists.

As noted in the White Paper the response curve parameters for the weak estrogen agonist 

(17α-estradiol) were met for only 5 of the 19 chemicals. Since the out-of-range values 

were very close to the guideline ranges for the majority of the outliers, the Panel 

recommended the Agency look into revising the guideline ranges for this control 

concomitant with reviewing whether the issues are with laboratory performance 

(minimizing the need to revise the guidelines and focusing more on laboratory 

compliance with the guidelines).

The Panel generally agreed that the majority of the deviations in the ERTA can be 

considered minor. However, some panel members raised concern about the cytotoxicity 

observed with corticosterone (Laboratory 1) and 17α-methyltestosterone (Laboratories 1, 

4 and 5) as they impact interpretation of the results.

Androgen Receptor (AR) Binding Assay

As noted earlier, the AR binding assay consists of a saturation binding experiment and a 

competitive binding experiment using rat prostate cytosol as such it is analogous to the 

ER-binding assay. Specifically, the assay measures the ability of radiolabeled [3H]-

R1881 to interact with the AR in the presence of increasing concentrations of a test

chemical. While the assay cannot distinguish between AR agonists and AR antagonists, 
28

the fact that the AR is conserved across species means that a chemical capable of binding 

in this assay is highly likely to affect different species.

The Panel agreed with the Agency in their findings that, overall, the laboratories 

performance of the AR binding assays was generally acceptable and the data for the 17 

tested chemicals were reliable. The Panel agreed that these results demonstrate that the 

AR binding assay as performed can distinguish between chemicals that bind or do not 

bind to the AR in vitro. The robustness of this assay is demonstrated by the result that the 

control compounds tested appropriately in the assay, even if the assay preparations did 

not fully meet all the assay criteria.

Steroidogenesis Assay

As noted, the Steriodogenesis assay is an in vitro screen intended to identify chemicals 

that affect the steroidogenic pathway beginning with the sequence of reactions involved 

in the production of testosterone and estradiol/estrone occurring after gonadotropin 

hormone receptors are activated. As such, the assay is not intended to identify chemicals 

that affect steroidogenesis due to alterations in the hypothalamus or pituitary gland. The 

assay endpoints measure changes in estradiol and testosterone levels.

The Panel agreed with the Agency that the laboratories’ performance of the assay was 

mainly consistent across all 18 test compounds, and the performance criteria were by and 

large generally met for all compounds. In the majority of cases where the performance 

criteria were not met, the values only slightly exceeded the expected values and these 

slight deviations did not impact the interpretation or reliability of the studies. The Agency 

reported in the White Paper that proficiency testing for the ERTA and Steroidogenesis in 

vitro assays was often omitted from the laboratory reports. The Panel suggested that if 

these proficiency compounds were routinely run and reported, the Agency would have 

greater confidence in the results of these tests, even if all of the performance criteria were 

not met. The Panel agreed that the overall results demonstrate that the Steroidogenesis 

assay as performed can distinguish between chemicals that alter or do not alter 

testosterone and/or estrogen levels in vitro.

Aromatase Assay

The Aromatase assay determines if a chemical could affect the endocrine system by 

inhibiting the catalytic activity of the aromatase enzyme, which is responsible for the 

conversion of androgens to estrogens. Specifically, the assay uses recombinant human 

microsomes containing aromatase (CYP19) and cytochrome P450 reductase, and 

measures the release of tritiated water during the conversion of 3H-androstenedione to 

estrone. As such, it measures competitive inhibition of aromatase by the tested chemical.

The Panel concurred with the Agency that, overall, the Aromatase assay as performed by 

the testing laboratories was able to distinguish between inhibitors and non-inhibitors of 

activity and the performance of the Aromatase assay was mainly consistent across all 18 

test compounds. Moreover, the Panel agreed with the Agency that the performance
29

criteria for the assay were generally met in each study and the deviations noted were 

minor. Specifically, the failure, by small amounts, to stay within performance criteria 

ranges did not adversely impact interpretation of the results. The Agency should consider 

requiring assay proficiency data when drawing conclusions on the results of an assay as it 

pertains to the Tier 1 screen.

General Comments

As noted in the White Paper, the laboratory performance of each Tier 1 assay was 

evaluated across 15 to 21 chemicals. Although there were situations where performance 

criteria were not met, the Agency considers those deviations to be “minor” and they did 

not impact the interpretation or reliability of the data. As noted above, in summary the 

Panel was in basic agreement with the Agency’s findings. However, most panel members 

agreed that their enthusiasm for this endorsement is tempered by two important factors:

1) limiting the reported chemicals to 21 rather than all 52 chemicals tested and 2) the 

manner in which the data was presented in the White Paper (i.e., the lack of direction and 

magnitudes of the measured values as compared to control values).

The Panel noted that the number of test chemicals reported, 21 or less, is small. The 

Agency should look at adjusting the performance criteria for the individual assays to 

better reflect the experience gained since 2008. Specifically, the Panel recommended that 

Tier I guidance be developed that indicates how much deviation from the norm is 

appropriate for an individual run to be considered acceptable. This type of guidance

should minimize cases where there are questions about assay parameters. The Panel 

further noted that this guidance could take the form of a decision tree that would assist 

labs in deciding when to redo individual runs that have not met the criteria.

Additionally, the Panel recommended that in the new guidance the Agency should refrain 

from using terms such as “slight” and rather report allowable percent difference from 

performance criteria. The Panel felt that small deviations (i.e., < 10%) are likely to be 

insignificant. Generally, the Panel felt that deviation from the recommended performance 

criteria that would render a study unreliable are likely to vary between specific assays. 

Moreover, the Panel generally thought that, since data for less than 21 chemicals are 

reported for most of the in vitro assays, in some cases it may be too early to set more 

specific reliable criteria. However, after the data for all 52 chemicals are evaluated this 

task should be easier.

The Panel understood that the protocols used in the Tier 1 assays represented the state of 

the science in 2008 when the initial Tier 1 testing order was issued. The Panel agreed

with the Agency’s decision to not alter the protocol until the first round of testing was 

completed. While, this SAP is based on data for only 21 of the 52 chemicals being tested 

in this order, the Panel recommended that the Agency make all data available on the 

remaining 31 chemicals as soon as possible. The Panel felt strongly that having data sets 

for all Tier 1 tested chemicals will have a positive impact on WoE deliberations.
30

With that said, the Panel believed that there is sufficient evidence that a reevaluation of 

selected test protocols may be in order. Based on both the report from the January 2013 

FIFRA SAP on the EDSP prioritization scheme and the public comments for the present 

SAP, the Panel questioned the redundancy of the ER-binding and ERTA assays used in 

Tier I in the context of the rainbow trout rtER binding and liver slice gene activation 

assays used in the EDSP development of the Computational Toxicology Tools. 

Specifically, the Panel recommended that the Agency look at eliminating the Tier I in 

vitro assays related to the ER-pathways in light of the fact that “the hormone binding 

domain of the ER is highly conserved across species.”

The recommendation for the Agency to eliminate the ER-binding and ERTA assays from 

the battery of Tier 1 assays is reinforced by the Agency’s interest in using high 

throughput screening data to expand the structural domain used in the prescreening 

activities. Moreover, eliminating the binding assay eliminates the need to address the 

major shortcomings of the protocol, namely the limitations imposed by the preparation, 

attaining performance criteria and storage life for the uterine cytosolic preparations. 

Moreover, elimination of the ER-binding assay will reduce the numbers of animals 

required to complete the Tier 1 testing.

The Panel recommended that the Agency look at replacing the current Tier I in vitro

assays related to the AR-pathways (i.e., one based on rat prostate cytosol) with one based 

on a recombinant cell line. Replacing the current AR-binding assay bypasses addressing 

the major shortcomings of the protocol, namely the limitations imposed by the 

preparation, attaining performance criteria, and storage life for the prostate cytosolic 

preparations. Moreover, replacement of AR-binding assay will reduce the numbers of 

animals required to complete the Tier 1 testing.

Charge Question 3: A positive control is not required for the male and female pubertal 

assays. For these in vivo assays with rats, the coefficient of variation limits are specified 

in the test guidelines for most endpoints. Submissions from different laboratories 

sometimes fell short of meeting all the test guideline-recommended Coefficient of 

Variation (CV) limits for the endpoints evaluated. However, in most cases these 

shortcomings were considered of minor importance to the overall results, and the EPA 

concluded that the data are still adequate for endocrine screening. Please comment on 

when a deviation from the recommended CV limits would render the study unreliable. 

Panel Response

The male and female pubertal assays use rats undergoing puberty to test for potential 

endocrine disrupting effects by various compounds designated by the EPA for initial Tier 

I testing. These assays have the potential to yield valuable information, as this period is 

highly dependent on normal endocrine function for the successful progression of the HPG 

axis to sexual maturity. Moreover, the assays have an advantage over others in the EDSP 

in that they utilize a relatively realistic route of administration (oral gavage) to determine 

the potential endocrine disrupting effects of test chemicals (see White Paper for 

description). The Panel noted that the assays also use a significant number of animals in 
31

order to ensure proper study populations for both the preliminary range of finding studies 

and for the assays themselves.

Regarding the acceptability of the data for use, in general, the Panel agreed that, although 

different laboratories sometimes fell short of meeting all the guideline-recommended CV 

for endpoints evaluated, the data from the assays were adequate for endocrine screening. 

However, it was noted that there are many difficulties with accepting the evidence from 

experiments that fall short of meeting the performance criterion for CVs, and that 

additional guidance is needed to clearly establish criteria defining acceptable / 

unacceptable tests. Ultimately, the acceptable level of variance in the screening assay 

needs to be sufficiently broad to capture the range of biological variability, but not so 

large as to allow for careless execution of the assays. 

Specifically, regarding the CVs for the different endpoints measured in the assays, the 

Panel concurred in general with the EPA that most of those that were marginally outside 

of the criteria in the guidelines likely did not impact the interpretation of the data. 

However, it was cautioned that higher CVs in the test controls will tend to increase the 

likelihood of committing a type II error (that is, failing to detect a true difference, a false 

negative), and that decisions regarding the value of these assays are difficult to make 

without considering larger issues, some of which relate directly to considerations of the 

“weight of evidence” determinations that will be the subject of a future Panel. It was also 

noted that in Tier 1 screening, if a bias were to be acceptable, one might suggest that the 

bias would be to accept type I errors over type II errors. While doing so would likely 

increase the number of compounds moving forward to Tier 2, it would minimize the 

chances of failing to move compounds that are, in fact, endocrine disruptors. The 

application of redundant testing in the Tier 1 battery should be sufficient to take care of 

the potential type I problem, to the extent that it exists. On the other hand, there is the 

potential for piling multiple type II errors on top of one another if the Tier 1 battery 

admits multiple assays that do not meet the performance criteria.

Additionally, the Panel believed that by accepting large CVs, the extent of variability 

weakens the statistical power of the test and can make assays unreliable for the detection 

of biologically significant effects. In the event that other assays (Uterotrophic assay or 

Hershberger assay) that evaluate estrogenic or androgenic activity are removed from the 

battery, perhaps the inclusion of positive controls, or at least periodic laboratory 

proficiency assays with controls, should be considered, although this is not a favorable 

option from the standpoint of animal use reduction or cost. It was also noted that 

performance consistency within and between testing laboratories is important, and that 

this can be achieved by increasing communication among the laboratories performing the 

tests and the EPA. Potential laboratory-specific confounding factors contributing to high 

CVs that were mentioned include diet, genetics, daily care protocols, and stress 

responses. Further, while Sprague-Dawley rats were used in most of the pubertal assays 

reported, it was not clear that these rats were from the same supplier, and factors such as 

substrain or diet could contribute to variability in means and variances among 

laboratories. Consideration for comparison to laboratory-specific historical CVs for the 

measured endpoints may reduce deviation from the guidance criteria and provide a more 
32

meaningful comparison for determination of the significance of chemical exposure. 

Moreover, assays for female rats likely exceeded CV levels due to the effects of the 

estrous cycle, which causes large cyclic changes in many of the endpoints being 

measured, particularly uterine and ovarian weights. Determination of estrous cycling was 

mentioned in the guidelines as a parameter to be measured, but was not addressed in the 

White Paper provided by the EPA. 

One panel member expressed the opinion that in order to accurately identify potential 

endocrine disruptors rather than agents that alter the parameters tested via other pathways 

such as stress, potential interactions between the hypothalamic-pituitary-adrenal (HPA), 

hypothalamic-pituitary-thyroidal (HPT), and hypothalamic-pituitary-gonadal (HPG) axes 

need to be taken into account when interpreting the data from each assay. In particular, 

when a CV for a test chemical is high, low-level chronic stress can alter HPG axis 

development and function. Specifically, it was noted that the female pubertal assay is a 

useful tool that has the potential to indicate if an agent disrupts HPG function at a variety 

of levels. It is also likely the most difficult assay to interpret, as many findings could be 

due to factors not related to the test chemicals, including experiment-induced stress. This 

is especially of concern given the frequency of overt toxicity reported in the White Paper. 

Potential problems from stress and the chronic, low level activation of the HPA or 

‘stress’ axis by some of these chemicals include decreased or altered GnRH secretion at 

the level of the hypothalamus, decreased or altered LH or FSH secretion from the 

pituitary, and increased estradiol production at the level of the ovaries. In the studies 

where doses of test chemicals were high enough to elicit overt toxicity, HPA axis 

activation nearly certainly would have played a role in the results; even in experiments 

where overt toxicity did not occur, the likelihood that the chemicals used provided 

enough of a homeostatic challenge to either acutely or chronically alter HPA function is 

high. Because relatively large CVs are allowable in this assay in order to account for the 

effects of the estrous cycle on parameters like estrogen secretion and ovarian/uterine 

weight, effects of HPA activation on these and related measures have the potential to be 

masked (leading to false negatives), particularly when secondary histological analysis is 

not performed. For instance, chronic stress has been shown to increase a number of assay 

endpoints influenced by estrogen, including uterine weight (Guinn, 1996). 

In order to take into account stress parameters in the female pubertal assay, it was 

suggested that the EPA consider monitoring HPA axis parameters other than simple overt 

toxicity and adrenal weight in order to rule out potential interplay between this axis and 

the HPG axis. Since measures of the stress hormones corticosterone or 

adrenocorticotropin hormone (ACTH) at sacrifice are unlikely to provide useful 

information regarding stress in the experimental animals over the course of the assay, 

these measures could be taken during the range-finding phase during which doses are 

decided. Alternatively, a non-invasive approach would be to measure corticosteroid 

levels in the fecal matter of test animals in order to identify whether a generalized stress 

response is a confounding factor in the responses elicited by various test chemicals 

(Cavigelli et al., 2005). (References for a test kit that can be used for this is: 

http://www.enzolifesciences.com/ADI-900-097/corticosterone-elisa-kit/).
33

In addition to suggesting that the EPA consider taking measures of HPA axis activation 

in the female pubertal assay in order to account for the potential confounding effects of 

stress on HPG axis function, ways to decrease variability in results due to the estrous 

cycle were also suggested. These effects could be accounted for experimentally while 

still assaying rats at the same age by synchronizing the estrous cycles of the rats using 

GnRH analogue injections in the days prior to the end of the assay. Doing this would 

allow the rats to be examined at the same age and on the same day of the cycle, plus 

effectively test the responsiveness of the HPG axis at the levels of the pituitary (GnRH 

challenge) and ovaries (LH and FSH challenge), assuming these parameters aren’t 

disrupted by test chemicals. Given that the 42 – 43 day period is ‘standard’ but still rather 

arbitrary, this approach could potentially yield important information currently lost due to 

estrous cycle variations. Alternatively, since the parameters being investigated are 

reproductive, instead of using absolute age as the deciding factor for taking tissue 

samples, the EPA could use “reproductive age” (i.e., the day of first estrus of each 

animal) as the comparison point.

In specific comments regarding the male pubertal assay, one panel member stated that as 

occurred in the female pubertal assay, a fairly large number of test chemical CVs were 

out of range, but accepted as interpretable by the EPA. The panel member reiterated that, 

due to the nature of these studies, there was a chance that the effects of HPA axis 

activation on the parameters being tested could have contributed to the high CVs seen in 

the assays. It was noted that even low levels of chronic stress can significantly inhibit 

testosterone secretion, and that this was a potentially confounding factor because most of 

the measures taken are a direct reflection of the actions of this hormone. Further, such an 

effect would unlikely be seen in single measures of testosterone, as the secretion of this 

hormone fluctuates widely. As such, it was suggested that HPA axis activation needed to 

be ruled out as the cause for effects seen, and that to do this would require the 

measurement of endpoints related to this axis in a similar manner as suggested for the 

female pubertal assay. Finally, it was suggested that in order to more accurately test the 

integrity of the HPG axis and the responsiveness of Leydig cells in the testes to 

luteinizing hormone receptor stimulation and the synthesis and secretion of androgens, 

the EPA should consider using an human chorionic gonadotropin (hCG) challenge to 

stimulate testosterone. In addition to testing testicular responsiveness to LH receptor 

activation, an hCG challenge would give the Agency a potentially more stable measure of 

testosterone secretion, and given the short-term nature of the test, would be highly 

unlikely to influence parameters being measured other than testosterone secretion. A 

reference for the use of hCG to stimulate testosterone secretion can be found here: 

http://www.ncbi.nlm.nih.gov/pubmed/14684600.

In conclusion, the Panel generally agreed with the EPA that the data generated using the 

male and female pubertal assays are useful, but advocated for careful modification of the 

guidelines, and cautioned that significant care needs to be taken when interpreting results 

from the assays.
34

Charge Question 4: The test guidelines for the six in vivo assays (Hershberger assay -

OCSPP 890.1400, OECD 441; Uterotrophic assay- OCSPP 890.1600, OECD 440; Male 

Pubertal assay- OCSPP 890.1500; Female Pubertal assay - OCSPP 890.1450; FSTRA -

OCSPP 890.1350, OECD 229 and AMA - OCSPP 890.1100) offer some guidance on 

setting the dose/concentration range when testing for specific effects on the E, A, or T 

signaling pathways. In some of the in vivo assays, overt toxicity was noted based on 

effects on growth, other sublethal effects, and even mortality at the highest 

dose/concentration tested. Positive Tier 1 findings indicating the potential for endocrine 

activity can be difficult to interpret in the presence of overt toxicity.

Please comment on the nature and severity of overt toxicity that would render study 

results unreliable for the purpose of Tier 1 screening of a chemical for endocrine activity. 

Please comment on what, if any, additional guidance is needed to minimize overt toxicity 

while ensuring adequate dose selection for these screening assays. Also, specifically 

comment on the validity of including treatment concentrations with apparent body weight 

effects in the analysis for potential endocrine interaction for FSTRA?

Panel Response

The use of a range of doses when investigating the potential environmental endocrine 

disrupting effects of test compounds is obviously of great importance. However, the overt 

toxicity seen during testing complicates the interpretation of data from the in vivo assays 

employed by the EPA for the EDSP. Several difficulties are encountered when overtly 

toxic doses are used in the screening program. For instance, toxic compounds impact the 

basic cell machinery essential for endocrine function: energy utilization, metabolic 

precursors, genome stability, etc. Further, energy disruptors (mitochondrial targets) alter 

energy dynamics. This can lead to well characterized changes in signal transduction 

pathways (i.e. Protien Kinase B (AKT), Autophagyrelated (ATGs), Mammalian target of 

rapamycin (mTOR), or c-Jun amino-terminal kinase (JNKs)) that directly impact 

hormone responses that are required for transcriptional activity and for cell progression 

through the cell cycle. Because of these and other physiological changes associated with 

overtly toxic doses, there is a high potential for confounding effects to be elicited by test 

substances, and these can render findings potentially unreliable. If effects are observed 

only in a dose group with overt toxicity and in the only screen that shows a positive 

effect, it will be difficult to decide whether the compound should undergo Tier 2 testing 

without a repeat experiment at a lower dose. As such, the Panel in general agreed that 

efforts to reduce the test compound doses to sub-toxic levels is required to allow the 

impact of the target agent on endocrine function to be evaluated independent of its 

general impact of organismal well-being.

Suggested criteria to define the Maximum-Tolerated-Doses (MTD)

In suggesting criteria for defining MTD in the EDSP assays, the Panel noted that the 

common standard of practice/animal welfare benchmark for short-term toxicity studies is 

a 10% upper limit of body weight loss. This cutoff is deemed to be sufficient to 

determine maximum-tolerated doses, and is a common standard accepted by most 

Institutional Research Animal Use and Care Committees (NC3R2

, 2013). However, in 
35

discussing this parameter further panel members recommended the 10% weight loss 

benchmark be used as a general guideline for the mammalian assays, but not the FSTRA 

or AMA (see below). As an alternative, it was suggested the EPA better define MTD 

estimates derived from range finding studies by increasing the number and dose spacing 

in these bioassays. One way to do this would be to evaluate the 95% Confidence Limit 

for the MTD and if this variance is high (>20%), it would trigger additional range finding 

studies to better define the MTD. If these determinations are made for several 

compounds, it may be possible to compare the “better defined” MTD estimate with the 

original maximum MTD estimate statistics, such as the lower 95% Confidence Limit of 

that estimate. If these two values are close, it may be possible to simply use the lower 

95% Confidence Limit in lieu of having to conduct additional range finding tests to 

define the MTD. 

Suggestions regarding test dose selection and reducing overt toxicity:

The Panel made several general recommendations regarding dose selection for the in vivo 

assays in the EDSP. When overt toxicity is present it was suggested that the testing labs 

employ two additional lower doses below the original MTD used to generate the dataset, 

using concentrations that do not illicit overt toxicity. If effects on A, E or T endpoints are 

still observed in the absence of overt toxicity, then there is a higher degree of confidence 

that the effects on A, E, and T are directly related to the mode of action of the chemical 

as opposed to a more generalized stress response. If, on the other hand, effects on A, E 

and T are not observed in the lower dosing regimen, then the original dataset obtained at 

toxic MTD levels should not be considered to be definitive for direct effects via 

alterations in hormonal pathways. However, the effects of a generalized stress response 

altering A, E, and T should be noted in the event that more information about this effect 

of overt toxicity can be further used in future risk assessments. 

An alternative method for reducing overt toxicity proposed by the Panel included the 

following steps: 1) reduce the initial highest test concentration; 2) add another 

concentration group (i.e., four test concentrations); 3) consider an approach similar to the 

in vitro binding assays in which solubility becomes a problem and run test concentrations

in half-log unit steps as opposed to whole-log units with additional concentration groups; 

4) change the criteria for evaluation such that concentration-related changes are not

required to indicate potential endocrine-related effects (i.e., effects at a single 

concentration indicate an effect); or 5) accept the overt toxicity, loss/obscurity of 

information, and reduced power of the test for detecting of endocrine-related effects. 

Finally, it was suggested by a panel member that the EPA use toxicology data that 

already exists from the previous testing of chemicals initially chosen for the EDSP. 

Briefly, concentrations of pesticides and other chemicals (xenobiotic may be a better 

choice for the generic “chemicals”) that are overtly toxic can already be subject to 

regulation based on non-endocrine disrupting criteria, so there is no value added when 

these compounds are retested for endocrine disrupting activities at those concentrations. 

It would, therefore, be important for the Agency to analyze how its Tier 1 testing can 

encompass dose/concentration ranges that allow for maintaining the necessary
36

physiological integrity of the systems, while providing the sensitivity needed for Tier 1 

screening.

Mammal specific comments:

For the mammalian in vivo assays, overt toxicity will include the activation of the HPA 

axis, which in turn affects all endocrine systems, including those that are the focus of the 

EDSP. Many of the potentially confounding stress axis-related factors in the mammalian 

studies are discussed in the Panel’s reply to Charge Question #3, which addresses the 

variability found in the data obtained in the Male and Female Pubertal Assays. These are 

also true with the Hershberger and Uterotrophic Assays, although with these two 

protocols, the potential confounding direct and indirect gonadal effects of stress-related 

parameters are not in play. Also in the Panel’s response to Charge Question #3 are 

recommendations for testing markers of HPA stress-response axis activation such as 

corticosteroid production, which could be assayed in the range finding studies. In terms 

of dose selection for the mammalian assays, it was suggested that screening dosage 

should be chosen as the highest concentration of the test compound that does not alter 

body weight, or general physiological tone beyond expected changes. Alternatively, if 

doses at or near the MTD are used to set the high dose, the Panel suggested that at least 3 

doses, or more, be used in the screening assay to increase the likelihood that a dose 

without overt toxicity is tested. In this regard, the MTD should be not relied upon in 

every case in dose setting. If exposure data were available, dose setting could be based on 

a margin of exposure approach. Other existing toxicity data, even if not using the same 

administration protocol, should also be considered. Alternatively, when overt toxicity is 

observed, it is essential to run effects studies at lower doses that do not elicit overt 

toxicity. The general approach of lowering the overall dose response curve to lower log 

doses or half-log doses around the mid-point of the exposure range and then excluding 

the higher, toxic doses seems like a reasonable compromise to address this issue. This 

approach avoids biasing the data analysis with a dose that may only have more resistant 

and less affected individuals. Finally, it was noted that initiation of oral gavage dosing is 

expected to cause a transient drop in total body weight. Thus, examination of historical 

data from each of the laboratories performing the assays will likely provide insight as to 

the extent of transient change in weight with dosing and thus, inform as to an anticipated 

weight loss unrelated to overt toxicity.

FSTRA-specific comments

In comments specific to the FSTRA, the Panel agreed with the Agency that the FSTRA 

test protocol is robust with study metrics (endpoints) at multiple levels of biological 

organization. This is a real strength of the test and greatly enhances the utility of FSTRA 

testing as an integral part of the EDSP Tier 1 Panel. However, the complexity of the 

protocol and the variety of expertise required to conduct and evaluate all of the endpoints 

renders this a challenging assay for most laboratories to conduct, while meeting all of the 

criteria. That said, evaluation of individual endpoints and their relevance toward the 

determination of the potential of a xenobiotic to interact with endocrine function is 

greatly enhanced in the FSTRA due to the fact that consideration of other test metrics can 
37

be evaluated simultaneously. At several times in the EPA report it was correctly stated 

that the validity of a test was not lost when performance criteria for a single endpoint was 

not met. Consideration of the results within the context of other endpoints is required and 

is a major strength of the FSTRA.

Regarding dose selection for the FSTRA, it was noted that the greatest of the three test 

concentrations is one-third of the 96-hr LC50 for the compound in fathead minnow. The 

96-hr LC50, by definition will cause 50% lethality to the test organism in 96-hr.

Depending on the slope of the dose-response curve, a concentration that is one-third of 

the 96-hr LC50 would likely be associated with some degree of mortality within a fourday period. Thus, it seems likely that there would be some degree of lethality when fish 

were exposed to this same concentration for 21-days. Moreover, some other overt toxic 

effects of this concentration (one-third 96-hr LC50) during the course of a 21-day 

exposure that is more than five-times longer in duration is highly likely. Therefore, it is 

not surprising at all that survival was affected in one-third (7 of 21) of the assays, weight 

reductions were apparent in nearly half (10 of 21) of the FSTRA evaluated, and over onethird of the test chemicals resulted in sub-lethal clinical signs of toxicity. Overt toxicity, 

including reduced survival and body weight loss can have a confounding impact on the 

results of the FSTRA at the exposure concentrations that express the toxicity. It may not 

be possible to discern if changes in endocrine-related endpoints (e.g., GSI) are due to 

direct effects on endocrine functions or secondary effects of toxicity. There are certainly 

cases where a clear endocrine-related effect of a chemical could be evident, even in the 

presence of clinical signs of overt toxicity (e.g., ovo-testes, secondary sexual traits of the 

other sex, etc.); yet all too often, those exposure groups that exhibit an overt toxicity 

response will be lost to the final dataset. The end result is an assay with fewer test 

concentration data, and ultimately a test with reduced power of detection of endocrinerelated effects. Results would be rendered unusable when: 1) high levels of actual 

mortality (> 10-25%) occur at a given exposure treatment; 2) overt toxicity effects 

commonly occurring throughout a large portion of the exposed individuals (> 50%) 

within a given treatment; or 3) there are multiple evident stress effects (altered behavior, 

reduced feeding, etc.) that will alter physiology and may affect hormonal pathways. 

Specifically regarding body weight in the FSTRA, the Panel noted that these 

determinations are easy to measure and may be directly related to altered behavior and 

reduced feeding rates that may be contributory in causing overt toxicity. Body weight 

effects are directly measurable and are not subjective as opposed to observations of 

behavior that by their very nature are more subjective. The percentage of feeding 

response is also more directly quantified (presence (feeding)/absence (not feeding) 

criteria) and are not subjective. In the fish bioassay, only 1 compound of the 21 (< 5%) 

compounds tested resulted in significantly reduced length and weight determinations. 

Changes in body weight co-occurred with other sub-lethal effects in 5/21 compounds 

tested (23%) as significant differences in male or female body weight were observed at 

doses where other sub-lethal effects occurred. Changes in body weight also occurred in 

some compounds in the absence of other sub-lethal effect in 4 out of the 21 compounds 

tested (19%). Thus changes in body weight in the fish bioassay were not always found to 

co-exist with overt toxicity effects. It was also noted that changes in body weight of 
38

females were problematic and would lead to exclusion of some data at higher doses and 

thus, reliance on data at 10 and 100 times lower than the higher dose. Females are more 

apt to see changes in body weight due to the energy diverted into egg production when 

compared to males. Despite these complications, tracking of body weight is still an 

important endpoint for test compliance guidelines as it is a generalized measure of fish 

health. Given that length and weight changes were noted in the FSTRA it may be useful 

to use a condition index approach (Bolger and Conolly, 1989) that integrates weight and 

length (e.g., CI = Weight/Length 3

) as a more integrated measure of response changes in 

the presence of overt toxicity .

Finally regarding the FSTRA, it was noted that overt toxicity may affect the potential to 

discern endocrine disrupting chemical (EDC) effects. It is the most sensitive and most 

affected individuals that are highly stressed, thus compromising interpretation of EDC 

effects using the different bioassays in the Tier 1 Screening Battery. If there is actual 

mortality, this further complicates results, as test end points in the Tier 1 battery cannot 

be measured in what may be the most sensitive portion of the population of individuals 

exposed.

AMA-specific comments

Regarding the AMA, the Panel noted that 10% weight loss is not recommended as a 

parameter to set MTD, as tadpoles may grow considerably during the 21-day test period, 

and under certain conditions they can exhibit wide individual variability in development 

and growth rates. Growth and development during late premetamorphosis and 

prometamorphosis in Xenopus laevis are concurrent but not necessarily associated events. 

For example, tadpole development can be inhibited by a number of well-known 

goitrogens without significant effects on growth (Degitz et al., 2005); or growth can be 

accelerated by compounds such as triclosan without effects on development (Fort et al., 

2007); or, for a more complicated example, tadpole growth and certain aspects of 

development such as hindlimb length can be inhibited by environmentally relevant 

concentrations of cadmium without detectable effects on endpoints of thyroid activity 

such as epithelial cell height (Sharma and Patiño, 2010). Although it is important to 

measure and differentiate effects on growth from effects on development, given the 

known variety in the specific responses of growth and development to different chemicals 

there is no a priori reason to believe that overt toxicity as defined by effects on growth 

would render the assay results unreliable for the purpose of Tier I screening. 

Nonetheless, the overarching goal of the EDSP and the Tier 1 battery is to identify 

chemicals/xenobiotics that impact the endocrine system in the absence of overt toxicity. 

As such, all efforts should be made to eliminate toxicity as a confounding response when 

screening of endocrine disruptors.

A relevant issue in regards to growth measurements in the AMA is the method for 

normalizing hindlimb length (HLL) for tadpoles of different size. The Agency 

recommends normalizing HLL based on snout-vent length (SVL); namely, by dividing 

HLL by SVL. This method, however, assumes that the relationship between the two 

variables is isometric. Generally speaking, an isometric scale relationship can be defined 
39

as one where the slope of the regression line on a double-log plot is equal to 1. However, 

if the slope is different than one, higher or lower, this normalization method could lead to 

errors depending on the magnitude of the deviation from a slope of 1 and the size range 

of individuals in the population. Most size variables do not scale isometrically and it is 

likely that neither do HLL and SVL or HLL and any other size scale such as body mass 

or whole body length. An alternative method to correct for tadpole size that would 

eliminate the potential problem of allometric scaling would be to use an appropriate 

regression method for HLL and SVL and calculate the residuals, and to use these 

residuals for statistical analyses instead of raw values or ratios. The Agency was advised 

to consult with a biometrician or morphometrician to find appropriate ways to address 

this issue.

Charge Question 5. Spinal curvature, usually manifesting as “bent tail” in X. laevis

tadpoles, was reported in 15 of 18 AMA studies reviewed thus far. The anomaly appears 

to be first observed several days after study initiation, and prevalence increases with time. 

Overall, the prevalence of spinal curvature in these studies ranged from “a few per 

replicate” to 92% of a given treatment group by test termination. Experimental work by 

the EPA Office of Research and Development suggests that overfeeding can be a primary 

cause of spinal curvature in their Xenopus test populations; however, spinal curvature 

remained prevalent (range: 16-92%) in the five industry AMA studies in which feed was 

reduced by 50% compared to guideline recommendations. Overall, the incidence of 

spinal curvature appears to be highly variable. From a qualitative review of the data, 

there appear to be no consistent differences in the incidence or variability of spinal 

curvature when studies using guideline versus reduced feeding regimes are compared. 

Please comment on whether the presence or prevalence of spinal curvature in test 

specimens, including controls, compromises the utility or validity of an AMA submission. 

If so, when does the prevalence of spinal curvature render the study unreliable? What 

technical guidance may be useful for laboratories in reducing the occurrence of spinal 

curvature and determining if, or at what point within the study, a study may be 

compromised because of this phenomenon?

Panel Response

Summary Recommendations for Technical Guidance:

The Panel recognized that the AMA is a screening assay in the EDSP Tier 1 battery 

intended to empirically identify substances that may interfere with the normal function of 

the HPT axis. The AMA is a general vertebrate model to the extent that it is based on the 

conserved structures and functions of the HPT axis. As such it is important as an ex vivo

model for thyroid-dependent process which respond via the HPT axis.

The Panel further recognized the AMA is related to altered hypothalamic-pituitary 

function, anti-thyroid activity and thyromimetic activity. In the case of altered 

hypothalamic-pituitary function, the AMA is complimentary to the rat pubertal assays and 

the fish reproductive screen. In the case of altered anti-thyroid activity, the AMA is 

complimentary to the rat pubertal assays. In the case of thyromimetic activity, the AMA 

has no complimentary assays within the Tier 1 battery.
40

The general experimental design was described by Fort et al. (2007). Briefly, 

Nieuwkoop-Faber (NF) stage 51 African clawed frog (Xenopus laevis) tadpoles, are 

exposed in separate treatment groups, to a minimum of three concentrations of a test 

chemical or a negative control (clean water) for 21 days. There are four replicate tanks 

within each test treatment. Larval density at test initiation is 20 tadpoles per test tank 

(replicate) for all treatment groups (80 larvae per treatment). The observational endpoints 

include: 1) hind limb length (HLL); 2) snout-to-vent length (SVL); 3) NF developmental 

stage; 4) body weight; 5) thyroid histopathology, and 6) daily observations of mortality 

and clinical signs.

As noted in the White Paper the occurrence of “bent tail” or “spinal curvature” in 

developing frogs during the AMA test appears to be very common place in most of the 

studies reported; specifically 83% (15 of the 18) compounds tested reported this effect 

and the other studies did not indicate whether bent tail was observed in those tests. The 

bent tail syndrome was present in both controls and treatment in almost all tests and was 

unrelated to the mode-of-toxic-action of the chemical tested.

As further noted in the White paper, within the AMA Test Guidelines, attempts have 

been made to reduce feeding in order to control development within the frogs to produce 

appropriate life stages at the end of the test. EPA has investigated this reduction in 

feeding and in particular the reduction in iodide within the diet as a result, which may in 

part play a role in the occurrence of bent tail. Results reported in Table 32, (see pages 

120-121 in the White Paper) indicate three sources of food were used including Sera 

Micron (Compounds B, C, E, F, H, I, J, N, O, P, S, U, and V), Xenopus Express 

(Compounds A, G, K, and Q), and Nasco Frog Brittle (Compound M) and all food 

sources were used in studies with high levels of reported bent tail. Levels of iodide were 

only reported for Sera Micron (range 40-54 ug/g) and Nasco Frog Brittle (48 ug/g). The 

levels of iodide in these two feed stocks are very similar. Water levels of iodide reported

were also quite similar in most tests ranging from 3.6 - < 50 ug/L for all studies and 

ranging from 3.6 - < 10 ug/L in 16/18 studies. Therefore, differences in iodide in food 

and water do not seem to explain this syndrome per se. An examination of the data 

presented in the White Paper suggests that the response seems to be somewhat laboratory 

dependent suggesting the importance of husbandry conditions within different 

laboratories. Thus, the Panel specifically recommended that it will be important to:

1. Keep track of clutch performance (number and quality of embryos from each 

spawn) and select high quality, healthy breeding pairs. Eliminating breeding pairs 

that produce bent tail should minimize its occurrence. Minimizing the use of wildcaught breeding stock may be beneficial.

2. Closely review the water quality conditions under which the bent tail tad poles 

were kept. Temperature, pH and nutritional deficiencies (vitamin C and Ca) are 

reported to cause bent tail in laboratory reared American bullfrogs and the

Leopard frog (Marshall et al, 1990; Leibovitz et al, 1992; Martinez et al, 1992), 

and in several other amphibian species. Temperature and pH have been associated 

with the condition in Xenopus tropicalis and water softness (Ca and K) associated 
41

with the condition in Xenopus laevis. Over- feeding can alter the water 

biochemistry and uneaten food and excess waste causes changes in ammonia and 

pH that are detrimental to the health of the animals and could potentially explain 

the bent tail seen in the laboratories which reported decreased occurrence of the 

condition with a decrease in feed.

3. Feed and water sources should be screened for herbicides, biological toxins and 

pesticides currently known to cause bent tail in Xenopus laevis, nominally. 

Biological toxins would include both potential Harmful Algal Bloom (HAB) and 

microbial toxins.

4. The Panel recommended further monitoring the portion of each clutch not used in 

a given study. It would be potentially useful if these results were reported. The 

Panel understood that some information on this concept is in the guidance 

document, but it needs to be elaborated further.

5. The Panel further recommended that for acceptance of the AMA test results 

overall, the clutch should not have a rate of mortality and morbidity > 20%.

The Panel understood that the Fort protocol represented the state of the science in 2008 

when the initial Tier 1 testing order was issued. The Panel agreed with the Agency in not 

altering the protocol until the first round of testing was completed. While, this SAP is 

based on only 21 of the 52 chemicals being tested in this order, the Panel believed there is 

sufficient evidence with the AMA that a reevaluation of the test protocol may be in order. 

The Panel expressed the opinion that without an evaluation of WoE, the AMA must 

currently be considered important to the Tier 1 screening battery, but the prevalence and 

variability in percent of clutch expressing this tail malformation does affect confidence in 

the utility of the assay. However, the Panel recommended not changing the experimental 

design of the actual study outcomes until all 52 chemicals are evaluated.

Since SVL is a key measurement in the AMA, the Panel felt the “bent tail” issue has the 

potential to impact both the performance criteria for acceptance of individual test results 

and the scientific confidence in the utility and validity of the AMA. Therefore, the Panel 

recommended that the Agency determine the cause and possibly mode of action for the 

bent tail syndrome so guidance can be given on how to eliminate or at least reduce this 

morbidity response to < 10% of the populations.

The Panel understood that the assay typically begins with stage 51 larvae that do not 

show tail curvature. However, curvature is typically not obvious until the larvae are 

further into prometamorphosis (i.e., the window of susceptibility in the AMA is stage 51-

55). Therefore, laboratories cannot simply select the effect out of the test. However, the 

Panel thought that there may be signs in the stage 51 embryo that can be used to do a 

better job of selecting against this response.
42

Evidence presented in the White Paper and in the oral presentations at the SAP does not 

allow for a definitive understanding of the bent tail syndrome (i.e., there is no 

histopathology or biochemical data presented for the syndrome). The Panel understood

that from a developmental point of view, the bent tail syndrome does not seem to have an 

effect on the actual resorption of the tail, in most cases. An examination of the AMA data 

suggests that the occurrence of bent tail seems to be laboratory dependent. The Panel 

believed the potential causes for bent tail are: 1) genetics (as described above), 2) nutrient 

deficiency, 3) poor egg quality, and/or 4) poor water quality (possibly due to 

overfeeding). There was a high probability that one or more of these factors may all have 

a role in the development of bent tail. The Panel believed that a pathological examination 

of the tadpoles may reveal information that may be helpful.

The Panel benefited from having members that have extensive experience with Xenopus 

husbandry. There is antidotal information that leads to suggesting the syndrome is 

related to the ionic imbalance, poor water quality or a combination of factors that are 

affected by both the composition of the water and the feeding scheme.

One Panel member has extensive experience in performing the Frog Embryo 

Teratogenesis assay: Xenopus (FETAX). While the assay is different in its protocol from 

the AMA, the test species is the same. Unpublished work by this panel member found 

that softer water (water with less Ca and K) caused a greater incidence of lateral tail 

curvature and other general “toxic” effects such as edema and abnormal gut coiling. 

Moreover, experience by some panel members has shown that by keeping track of clutch 

performance (number and quality of embryos from each spawn) high quality breeding 

pairs could be identified. Breeding these paired frogs on a regular schedule, regardless of 

the need for embryos, improved clutch quality. 

The Panel noted that in fish, nutritional deficiencies (micronutrients, such as vitamin C) 

can lead to developmental curvatures of the spine. Also, excess of certain nutrients (e.g.,

selenium and copper) can also cause this lesion. Lastly, low dissolved oxygen can lead to 

spinal curvatures in fish embryos.

While not directly asked in Charge Question 5, the Panel strongly noted that the issues of 

bent tail (Charge Question 5), and thyroid histology (Charge Question 6) and their 

interactions with other AMA endpoints (i.e., HHL, SVL, final NF stage and body weight) 

leads to a recommendation that the Agency look into changing the experimental design of 

the AMA study. It is further recommended that the Agency specifically look into 

developing a stage dependent rather than a time dependent protocol.

Since neither the White Paper nor the Agency’s oral presentation described the 

histopathology of the “bent tail” lesion or presented photomicrographs of the condition, 

the Panel found it difficult to provide meaningful guidance. Those members of the Panel 

with experience with Xenopus testing and issues associated with tail malformations 

generally realized that lateral tail flexion is typically considered a non-specific effect, 

which is more of a muscular phenomena than a connective tissue or skeletal phenomena.
43

In contrast, if curvature of the spine in the AMA test is a dorsal to ventral kinking which 

occurs primarily in the anterior region of the tail, the Panel was concerned that the lesion 

is in the notochord and related to connective tissue elements. The latter anatomical 

pathological description is very similar to that noted for an osteolathyrogenic response 

where the alteration in elastin and collagen formation leads to a weak notochord which 

kinks or bulges in response to swimming movement (Schultz et al., 1985).

Schultz et al. (1985) examined the osteolathyrogenic effects of semicarbizide using the 

FETAX. Osteolathyrism is a connective tissue lesion associated with decreased 

intermolecular bonding in collagen and elastin. It is manifested in early embryos as gross 

anatomical changes in the long axis of the animal and kinking of the tail. Histopathology 

examination, including electron microscopy, reveals that gross effects were produced by 

changes in connective tissue fibers of the notochordal sheath, specifically a marked 

reduction in elastic fibers and a concomitant disorganization of collagen fibers.

Lysyl oxidase (Lox) is a copper-dependent amine oxidase that catalyzes the cross-linking 

of collagen and elastin fibers. Geach and Dale. (2005) characterized Xenopus laevis

cDNAs for Lox, Loxl-1 and Loxl-3, and showed that they are expressed during early 

embryonic development. Using RT-PCR they detected maternal transcripts for Xloxl-1, 

but levels remained low until tailbud stages. Transcripts for Xlox and Xloxl-3 were not 

detected until early neurulae, although transcripts for Xlox remained at low levels until 

tailbud stages. Whole mount in situ hybridization showed that transcripts for Xloxl-1 and 

Xloxl-3 are localized in the notochord, while transcripts for Xlox are found in the 

notochord, somites, and head. X. laevis Lox-like enzymes were inhibited by incubating 

embryos, from cleavage stages to tadpole stages, in β-aminopropionitrile, a specific 

inhibitor of the catalytic domain. The resulting embryos appeared to differentiate 

normally but suffered from poor collagen fiber formation. Defects included kinks in the 

notochord. These results suggest that Lox-related enzymes are required for the proper 

formation of the collagen and elastin fibers during X. laevis development.

The Panel suggested that other factors to consider in examining the bent tail syndrome 

may be to include examining other ingredients in food, such as anti-oxidants. For 

example, Ellenberg (2000) found that TetraMin fish food contained high levels of the 

antioxidant ethoxyquin which increased the production of P Glycoproteins (PGP) and 

Multi-Drug Resistant (MDR) Proteins in estuarine fish, Fundulus heteroclitus. This 

effect was very pronounced as fish from pristine habitats had elevations in PGP and 

MDR to levels comparable to fish which were collected from highly polluted sites (e.g.,

EPA Superfund Sites). Elevated PGP and MDR in the fish has an effect on overall energy 

metabolism, diverting energy away from growth and development, which might render 

fish more susceptible to disease. Also this increase in PGP and MDR will also affect 

uptake/depuration rate kinetics for some compounds and may result in possible 

underestimation of toxicity since these proteins are generally associated with reduced 

drug uptake. In the case of the frogs and bent tail, examining other contents of the food 

may be warranted to systematically eliminate these as a contributing factor in “bent tail”. 

Of particular concern are inert food ingredients that may increase the costs of 
44

maintenance metabolism and possibly increase susceptibility to disease, if bent tail is 

found to be associated with a disease process as more is learned about it.

The Panel noted that frogs are not the only toxicity test species to have altered 

development or abnormalities. For example, the Eastern Oyster, Crassostrea virginica, 

which has been extensively used in toxicity testing, may commonly be infected with the 

protistan parasite Perkinsus marinus, which may produce annual mortalities in excess of 

40% in adult oysters. Aquatic toxicologist simply avoided periods of high temperature 

and salinity during the summer months (July-September) and confined toxicity testing to 

the other times of the year (October-June) so that high control mortality rates could be 

avoided. Similarly, crustaceans such as grass shrimp, used extensively in marine testing 

of pesticides, may have diseases (e.g., black spot) which are parasitic in origin that have 

been well studied and have not compromised toxicity test results. In other words, there 

have been instances with other toxicity testing where confounding factors have been 

addressed and tests have been adapted accordingly, so as to minimize impacts of the 

confounding factor. 

As noted above one factor in the bent tail syndrome may be the source or suppliers of 

frogs as there appeared to be some differences in the rates of this syndrome observed in 

comparisons of the different supplier. One of the panel members mentioned the need to 

possibly conduct some molecular sequencing to discern if there is an underlying 

molecular basis for this defect, which could enhance our knowledge of this problem.

Given that the tail reabsorption as the frog matures is a critical factor in this assay, 

occurrence of bent tail may affect overall health, growth, development, reproduction and 

survival over the life time of the organism. Thus, the phenotype may not be a highly 

confounding factor. If it does affect any of these variables, then the significance of bent 

tail will have to be further examined. In terms of its affect on E, A, and T outcome 

measurements within this portion of the life cycle of the frog is the most important thing 

for the Agency to focus on. Resolving this along with determining the overall health 

effects of this syndrome will be important.

This is a vertebral/muscular deformity, which has been reported with certain pesticides, 

which raises the significance of concern regarding this syndrome. The fact that this 

occurs in all control and exposure treatments clearly indicates this is not mode-of-action 

related to compounds being tested; however, if a compound were to illicit this effect, with 

a high rate of effects in controls, the ability to discriminate this effect will be reduced and 

possibly obscured. This is a concern. It may be prudent to test a known pesticide such as 

endosulfan, or another chemical known to illicit tail deformities in fish, and examine 

whether the assay can discern this effect in the AMA, assuming it would exert this effect 

in frogs. This approach would at least allow analysis of the degree of impediment this 

syndrome may cause and help possibly develop a positive control step for addressing 

bent tail. This would be an aid in discerning when a test is unreliable.
45

Charge Question 6. With the exception of thyroid gross pathology findings (thyroid 

gland atrophy and hypertrophy) in the AMA, severity grades are generally assigned based 

on comparison to “normal” X. laevis thyroid findings depicted in the guidance or based 

on the professional opinion of the pathologist conducting the assessment; they are not 

assigned in comparison to concurrent control findings from a given study. (Please refer 

to Section III.2.f in the document entitled “Interpreting Amphibian Thyroid 

Histopathology Diagnoses” and supporting documents, OECD Guidance Document on 

Amphibian Thyroid Histology No. 82, 2007 and Grim et al., 2009).

a. In one study, the pathologist’s report identified a lower incidence and 

severity of follicular cell hypertrophy when compared to the incidence and 

severity of this trait in control specimens. Similar trends have been 

observed in other studies. In this case, the pathologist concluded that the 

finding was potentially consistent with treatment-related delay of 

metamorphosis because thyroid follicular cells normally increase in 

height during tadpole development. Please comment on the validity of this 

conclusion.

Panel Response

The AMA test guideline states that the assay should begin with Nieuwkoop-Faber (NF) 

stage 51 African clawed frog (Xenopus laevis) tadpoles and that the median NF stage in 

control tadpoles at test termination (21 days) should be ≥ 57 with a difference between 

the 10th and 90th percentile of ≤ 4 NF stages. It is therefore important to avoid or 

minimize the occurrence of NF stage ≥ 60 in control tadpoles at test termination.

An assessment of HPT axis activity at test termination is at the core of the AMA test 

guideline. Because thyrocyte height is directly dependent on TSH stimulation and is 

relatively easy to measure, an evaluation of thyrocyte appearance is a primary component 

of the test guideline. The test guideline calls for the use of a standard scheme to grade 

thyroid “lesion severity” that is based on normal changes that occur during 

metamorphosis in X. laevis (Grim et al. 2009). The lack of quantitative endpoints of 

thyroid activity, however, was one of the concerns expressed in some of the written 

statements provided by public commenters. As explained in more detail in the paragraphs 

that follow, this concern was shared by the Panel. 

Although thyroid hormone agonistic activity could explain the pathologist’s observation 

of a lower incidence and severity of thyrocyte hypertrophy via negative feedback 

inhibition of the HPT axis. However, accelerated development in the experimental 

population would also have resulted in similar histological findings. Because accelerated 

development appears not to have occurred, the Panel agreed with the pathologist’s 

conclusion that the finding is consistent with a treatment-related retardation of 

metamorphosis via disruption of the HPT axis. This case may represent an unusual 

disruption of the axis that is not based on classical anti-thyroid mechanisms such as those 

observed after exposure to thyroid synthesis inhibitors (goitrogens), but on a failure of the 

axis to activate normally or fully. A similar finding by the laboratory of a panel member 
46

was reported in Xenopus tadpoles exposed to a high concentration of cadmium where 

metamorphosis and development were suppressed along with the apparent failure of the 

HPT axis to activate (Sharma and Patiño 2008). Possible interpretations of these findings 

(pathologist’s finding; Sharma and Patiño 2008) include a reduced ability of thyrocytes to 

respond to TSH stimulation (thyroid lesion) and/or a reduced ability of the pituitary to 

produce TSH (hypothalamus/pituitary lesion). A better understanding of the pathologist’s 

finding would require not only consideration of the actual effects on tadpole development 

recorded during the study (HLL or NF stage), but also clarification of the mechanisms 

responsible for the failure of the HPT axis to activate properly, if in fact this is what 

happened. It may be appropriate to consider these questions in more detail during the 

assessment of Tier 2 assay performances.

b. What guidance may be given to better distinguish between histological 

changes in the thyroid associated with the normal progression of 

metamorphosis and treatment-related effects? Are there certain lesions 

or diagnoses which may, by their absence or lessened severity as 

compared to controls, be indicative of treatment-related HPT effects such 

as delayed metamorphosis?

Because thyroid activity normally increases during metamorphosis, the assessment of 

treatment effects is relatively difficult to perform as the control condition is, literally, a 

moving target. To partially compensate for this situation, the AMA test guideline 

recommends the use of stage-matched individuals for histological analyses at study 

termination. Specifically, the guideline states that 5 tadpoles per treatment replicate (20 

total) should be matched to the median stage of pooled controls tadpoles, whenever 

possible. However, comments provided by the Endocrine Policy Forum indicate that 

studies conducted in response to the test orders reported variation in the median stage of 

development of control tadpoles at test termination and raised concern about the 

inconsistency among studies with respect to the developmental stage at which treatment 

effects are being evaluated. It would appear that a marker of normal thyroid activity that 

does not change during metamorphosis would minimize concerns associated with Charge 

Question 6 and, therefore, be preferable over changing reference markers. Although the

Panel was unaware of any standard histological feature of the thyroid that does not 

normally change during tadpole development, there is one immunohistochemical feature 

of the Xenopus thyroid that may be independent of development. 

A study of X. laevis reported that T4-immunoreactivity is concentrated in a ring at the 

periphery of the colloid and that the intensity of this ring remains fairly constant during 

metamorphosis (Hu et al. 2006). Moreover, changes in the intensity of this ring following 

exposure to a goitrogen (perchlorate) showed better sensitivity to detect anti-thyroid 

activity than changes in standard histological features such as thyrocyte height. The T4-

immunoreactive ring has not been validated for standard testing and its value for 

situations like the one presented in Charge Question 6, which may represent failure of the 

HPT axis to activate, is also uncertain. Nevertheless, the availability of developmentindependent markers of thyroid activity would offer clear advantages over the current 

scheme. The Panel noted that there are additional assays that may also be useful, such as 
47

iodide uptake assays. The Panel encouraged the Agency to consider the possibility of 

future refinements to the AMA test guideline by incorporating some of these assays if 

they can be validated and confirmed to be development-independent. The Panel 

recommended that the T4-immunoreactive ring marker, if properly validated, could 

potentially also benefit thyroid assessments in the rat pubertal assays.

The Panel offered an additional comment of relevance to HPT activity assessments in the 

AMA. Although the current AMA test guideline relies on a semiquantitative grading 

scheme to assess thyroid effects, rank variables typically do not yield the same statistical 

power that continuous variables do. A second limitation of grading or ranking schemes is 

that they are subject to technician biases and therefore may yield inconsistent results 

among studies as well as among laboratories. Therefore, the Agency was advised to 

consider the use of quantitative thyroid activity measurements to further standardize the 

AMA test guideline. There are a number of computer-assisted image analysis procedures 

that could be used for quantitative measurements of thyrocyte height, T4-immunoreactive 

ring intensity, and possibly other histological features in a fairly automated manner. 

Software packages for image analyses are publicly available at no cost to users.

Charge Question 7. In 2008, the SAP acknowledged that the in vivo assays included in 

the Tier 1 battery provide both redundancy and complementarity for evaluating 

interactions with the E, A, or T signaling pathways. The panel also noted that all of the 

Tier 1 assays and the broad range of endpoints appeared to be necessary to “discriminate 

positive and negative results”.

a. Please comment on the battery performance with respect to the 

anticipated complementary nature of the more complex, multi-parameter 

in vivo assays in the context of the observed responses with the case 

studies. Please comment separately on the E-, A-, and T-related assays 

and endpoints.

Panel Response

General Comments

A total of 18 chemicals for E, 17 chemicals for A and 16 chemicals for T were evaluated 

in this battery of assays. For the E and A pathways, there were apparent complementary 

responses with two or more endpoints responding in the pubertal assays and in the 

FSTRA. In some case studies, the male and female pubertal assays and/or the FSTRA 

identified endpoint responses along with endpoint responses in the relevant in vitro

assays for both the E and A pathways. As anticipated from the battery design, there was 

less complementarity evident for the T pathway effects. 

One panel member noted that to increase the chances that complementary results are 

provided by the male and female pubertal assays, the interplay between the HPA, HPT, 

and HPG axes need to be taken into account. In particular, accounting for HPA 

activation, for instance by assaying for corticosteroids in rat feces, could be an important 
48

addition to the batteries. While this question focused on the in vivo assays, it was also 

pointed out that the ER and AR binding assays had many or all of the attendant problems 

associated with in vivo assays in that they use rat tissues to provide receptors. It was 

suggested that more complementary results would likely be provided by alternative 

assays using recombinant receptors; this suggestion was made repeatedly during the 

meeting. It was noted that there were examples in which bioassay concordance was not 

observed between the in vivo and in vitro tests. Overall, findings suggest the importance 

of multi-parameter and multi-taxa among in vivo assays as a component of the battery to 

evaluate E, A, and T alterations.

There was a general feeling expressed among the Panel that since the present exercise 

does not provide information on the magnitude or the direction of the effects, it is 

difficult to say how useful the observed complementary effects will be in the WoE 

analysis to strengthen or weaken the case for the E, A, or T activity of these test 

compounds and determine the need for further testing. The fact that multiple endpoints 

were reported to respond is encouraging.

Estrogen Pathway

The current Tier 1 battery contains 3 in vivo assays that are designed to detect estrogenic 

activity. The endpoints and reported responses measured in each were as follows:

(1) Uterotrophic (U) - The uterotrophic assay includes only one required endpoint, 

uterine weight, but is a well-established and reliable assay for the detection of estrogenic 

activity. There were no significant effects reported for this assay (0/17). Since there is 

only one end point, no complementary analysis could be conducted. (2) Female Pubertal 

Rat (fPR) - The Female pubertal assay includes vaginal opening, time to first estrus, 

reproductive organ weights, and histology of reproductive organs as complementary 

endpoints. The guideline also includes monitoring the estrous cycle, but this was not 

discussed and the contribution of this endpoint to the Female pubertal assay is not clear. 

Significant effects in at least one or more of these end points were observed for 7/17 

chemicals tested (42%). Three tested chemicals gave a response for only one endpoint 

(3/17 = 17.6%). Complementary responses were observed as follows: two responsive end 

points (4/17 chemicals tested = 23.5%), three responsive endpoints (2/17 chemicals tested 

= 11.8%), and four responsive endpoints (0/17 chemicals tested = 0%). (3) Fish Short 

Term Reproduction Assay (FSTRA) - The FSTRA assay potentially has seven 

complementary endpoints (fecundity, fertilization, secondary sex characteristics, gonadosomatic index, gonad histopathology, plasma concentrations of vitellogenin in both sexes, 

and an optional endpoint of plasma sex hormones). The optional endpoint of plasma 

concentrations of sex steroids was stated by EPA to be useful and potentially should be 

added as a requirement. Significant effects in at least one or more of these end points 

were observed for 16/17 chemicals tested (95%). One tested chemical gave a response for 

only one endpoint (1/17 chemicals tested = 5.9%). Complementary responses among the 

seven different test endpoints were observed as follows: two responsive end points (3/17 

chemicals tested = 17.6%), three responsive endpoints (7/17 chemicals tested = 41.2%); 

four responsive endpoints (3/17 chemicals tested = 17.6%); five responsive endpoints 

(3/17 chemicals tested = 17.6%); six responsive endpoints (2/17 chemicals tested = 
49

11.8%); and seven endpoints (0/17 chemicals tested = 0%). Overt toxicity confounded the 

interpretation of results for some (or many) of the chemicals tested and to avoid this 

serious problem, potential remedies in the dose selection procedure were discussed in 

responses to earlier Charge Questions.

The 2008 SAP indicated that the Tier 1 assays have good coverage for the detection of 

estrogenic responses mediated through Estrogen Receptor 1 (ESR1), and relatively less 

ability to detect anti-estrogenic effects or effects that might be mediated through other 

potential mechanisms Estrogen Receptor 2 (ESR2, membrane receptors, etc.). This 

situation remains the same. The 2008 SAP encouraged pursuit of assays for such 

potential mechanisms, and pursuit of the modification of the uterotrophic assay to 

improve the ability to detect antiestrogens. It is unclear at this time whether such efforts 

are warranted, although at least one panel member believed that the extension of the 

uterotrophic assay for detection of antiestrogenic activity would have added value.

Androgen Pathway

Three in vivo assays in the battery including the Hershberger assay, the Male pubertal

assay and the FSTRA are able to detect effects on the androgen pathway. The endpoints 

and reported responses measured in each were as follows:

(1) Hershberger - The Hershberger assay measures changes (increase or decrease) in 

weights of androgen-dependent tissues and is a well-established and reliable assay. 

Complementary responses among different androgen- dependent tissues were observed 

for 7/17 chemicals tested (41.2%). (2) Male pubertal assay (MPR) - The male pubertal 

assay evaluates sexual development (preputial separation), androgen-dependent organ 

weights and histology, and single point (sacrifice) serum testosterone. Significant effects 

in at least one or more of these end points were observed for 10/17 chemicals tested 

(58.8%). Four of the 17 chemicals tested (23.5%) gave responses in only one endpoint. 

Complementarity responses among the four endpoints were observed as follows: two 

responsive end points (4/17 chemicals tested = 23.5%), three responsive endpoints (2/17 

chemicals tested = 11.8%), and four responsive endpoints (0/17 chemicals tested = 0%). 

(3) Fish Short Term Reproduction Assay (FSTRA) – The FSTRA contains seven 

potential androgen-sensitive endpoints (fecundity, fertilization success, secondary sex 

characteristics, gonad-somatic index, histopathology of androgen-dependent reproductive 

tissues, plasma vitellogenin, and the optional measurement of plasma testosterone). 

Significant effects in at least one or more of these end points were observed for 15 of the 

17 chemicals tested (88.2%). One of the chemicals gave a response for only one endpoint 

(1/17 = 5.9%). Complementary responses among the seven different test endpoints were 

observed as follows: two responsive end points (4/17 chemicals tested = 23.5%); three 

responsive endpoints (8/17 chemicals tested = 47%); four responsive endpoints (1/17 

chemicals tested = 5.9%); five responsive endpoints (4/17 chemicals tested = 23.5%); six 

responsive endpoints (1/17 chemicals tested = 5.9%); and seven responsive endpoints 

(0/17 chemicals tested = 0%). As was the case for estrogen pathway endpoints, overt 

toxicity appeared to confound interpretation of the results for some (many) chemicals 

tested.
50

Thyroid Hormone Pathway

Three in vivo bioassays included endpoints to evaluate the thyroid hormone pathway 

(Male and Female pubertal assays and the AMA). Of the 16 chemicals evaluated, there 

were significant effects on T noted in 7 of 16 (43.8%) chemicals tested in the male 

pubertal assay, 9 of 19 (56.3%) chemicals in the Female pubertal assay and 7 of 16 

chemicals (43.8%) in the AMA. The Female and Male puberty assays include 

measurements of T4, TSH, and thyroid gland weight, and thyroid gland histopathology as 

endpoints to evaluate the HPT axis. A variety of chemicals affected one or more of these 

endpoints with no consistent pattern and with no endpoint clearly being the most affected. 

In some cases these were sex-specific. The AMA assay contains evaluations of tadpole 

development (NF stage and normalized hind limb length) and thyroid histology. EPA has 

provided a clear decision logic for evaluating potential thyroid axis activity. In 4 cases of 

the positive AMA assays, both thyroid endpoints were altered while in 3 cases there was 

only one altered. Overt toxicity may confound the interpretation of the effects in several 

cases.

b. Please comment on the battery performance with respect to the anticipated 

redundancy across the 11 assays in the context of the observed responses 

with the case studies. Please comment separately on the E-, A-, and Trelated assays and endpoints.

Panel Response

The Panel noted, given the manner in which the data are presented, only limited 

statements on battery performance can be provided. Without knowledge of the magnitude 

or direction of the change, it does not seem possible to thoroughly judge how the battery 

has performed.

Estrogen Pathway

There were no clear ER binders and no test chemicals active in the uterotrophic assay. On 

the other hand, 4 of the 21 test chemicals gave positive results in the ERTA. It seems 

somewhat surprising that these compounds did not show activity in one or both of those 

assays. One panel member noted that the variability in the ER transcriptional activation 

assay evident in one of the public commenters’ presentations suggested that this may in 

part explain the lack of concordance between the ER binding and ER transcriptional 

activation assays presented in the report. As shown in the Agency presentations, there 

were no compounds that showed only in vitro effects, although in 9 of the 10 cases in 

which both in vitro and in vivo effects were seen, there was a positive in only one in vivo

assay, with the FSTRA being the positive in 8 of those 9 cases. There were some 

potential issues of overt toxicity in several of the positive in vivo studies, so that it 

appeared that there was only one compound (Q) that showed clear positives in two in 

vivo assays. In short, there is redundancy across assays for the estrogen pathway 

endpoints, but it remains to be determined how clear the interpretation of these 
51

redundancies will be. There seem to be a high proportion of equivocals in the aromatase 

assay and it is not clear how important that assay will be in the overall evaluations.

The results indicated that at least one of the three E bioassays (Uterotrophic, Female 

pubertal and FSTRA) detected an effect in 17/17 chemicals tested (100%). As noted 

above, positive results were not observed in the Uterotrophic assays, but only in the 

Female pubertal and FSTRA tests. Thus, there were no positive results (0%) in all 3 

bioassays for the 17 compounds tested. Positive results in two of the three bioassays were 

observed for 5 of the 17 (29.4%) chemicals tested in the Female pubertal assay and 

FSTRA. Overt toxicity may confound the interpretation some of these results. In terms of 

redundancy between in vitro and in vivo E tests, highest rates of redundancy were 

observed for the steroidogenesis assay and the FSTRA, as concordance was observed for 

7/17 (41.2%) of the chemicals tested. Redundancy was also observed between the in vitro

aromatase assay and FSTRA, as concordance was observed for 5/17 (29.4%) of the 

chemicals tested.

Androgen Pathway

The data presented clearly indicates that there is redundancy across the in vitro and in 

vivo assays designed to detect effects on the androgen pathway in the sense that 13 of the 

17 compounds presented as having been tested in the six androgen pathway assays appear 

to show effects in multiple assays. As with the estrogen pathways discussed above, the 

value of these redundancies will be better determined after the WoE evaluation.

Results indicated that at least one of the three A bioassays (Hershberger, male pubertal, 

and FSTRA) detected an effect in 17/17 chemicals tested (100%). Positive results in all 3 

bioassays were observed in 3/17 chemicals tested (17.6%). Positive results in two of the 

three bioassays were observed in 1/17 (5.9%) of the chemicals tested for Hershberger and 

male pubertal assays; 3/17 (17.6 %) of the chemicals for Hershberger and FSTRA; and 

5/17 (29.4%) of the chemicals for Male pubertal and FSTRA tests. Redundancy was 

observed in at least 2 or more of the 3 assays for 12/17 (70.6%) chemicals tested, with 

Male pubertal and FSTRA accounting for 8/12 (67%) of the redundancy, Hershberger 

and FSTRA accounting for 3/12 (25%) of the redundancy, and Hershberger and male 

pubertal accounting for 1/12 (8%) of the redundancy. Overt toxicity may have 

confounded some of these results.

Thyroid Hormone Pathway

As indicated in the 2008 SAP report, in the current White Paper and the EPA 

presentations, there is less redundancy in the assays for effects on the thyroid axis. The 

2008 SAP suggested exploring additional endpoints, such as thyroid hormone-sensitive 

gene expression and transcriptional activation assays, but it is not clear if additional 

endpoints to strengthen the evaluation of this pathway are being considered. Three of the 

16 chemicals tested in this battery showed some thyroid effect in all three thyroid axis 

assays, three showed effects in both rat assays but not the frog assay, and three showed 

effects in the frog but not the rat. Four chemicals showed effects in only one sex in the 
52

rat assays. The Panel agreed with the EPA conclusion that all three assays are necessary 

to evaluate this axis. Dose selection and overt toxicity were issues associated with 

performance of these in vivo T pathway bioassays.

There were responses in all three in vivo T assays for 3 of 16 (19%) chemicals 

(Chemicals C, F, and G) tested. For the AMA, thyroid histopathology and developmental 

alterations were both affected in 2 of the 3 cases. In the male and female pubertal assays, 

T4 was affected in both sexes for all 3 compounds. Thyroid histology was affected in 

both the female rat and AMA for one of the 3 compounds (G).

There were responses in only the male and female pubertal assays, but not in the AMA, 

for 3 of 16 (19%) tested chemicals (Chemicals E, K, and P). Thyroid histopathology, 

TSH, and T4 were responsive for these 3 compounds, but with different patterns based on 

chemical and sex. There were responses in only the AMA for 3 of 16 (19%) chemicals 

(Chemicals H, M, and V) tested, with both thyroid histology and development affected in 

2 cases and development alone affected in the third case. Potential solvent interference in 

the tadpole development assessment was noted in 2 of the cases. There were responses in 

only the AMA and female pubertal assays for 1 of the 16 (6.4%) tested chemicals 

(Chemical B), with thyroid histology affected in both assays. There were responses in 

only one bioassay for 3 of the 16 (19%) chemicals tested (Chemicals A, J and S). There 

were negative responses in all three T pathway bioassays for 3 of 16 (19%) chemicals 

tested (Chemicals I, N, and T).

Overall concordance for the T Pathway was:

Positive Responses all three T Pathway Bioassays = 19% (i.e. of the chemicals tested)

Negative Responses all three T Pathway Bioassays = 19%

Positive Responses in both rat pubertal assays but not AMA = 19%

Positive Responses in only the AMA =19%

Positive Response in the female pubertal and AMA = 6.7%

Positive Response in only one of the 3 bioassays = 19%

c. The EPA concluded that the battery has performed as anticipated by the 

2008 SAP. Please comment on this conclusion.

The 2008 SAP concluded that the Tier 1 suite of assays should be able to detect agents 

that alter E, A, and T pathways. Although there was general agreement that the battery 

appears to have performed as expected with the subset of chemicals on which data 

summaries were provided, there can be limited conclusions drawn at this time given the 

amount of information that has been provided. The assays were able to be performed by 

several laboratories and showed changes in the assay critical endpoints that will be used 

to determine if a compound should be required to undergo further testing to establish 

definitively the ability of the compound to alter the E, A, or T pathways. Without 

information on the direction, magnitude and consistency of the observed effects, it is 

difficult to fully assess how the assays performed as a group. No compounds were clearly 

positive in the estrogen receptor-binding assay or in the uterotrophic assay, but several 

chemicals were positive in the ER transcriptional activation assay and also in the pubertal 
53

female and/or FSTRA. It might be expected that positives in the transcription assay 

would give positives in the ER binding and/or uterotrophic assays. With respect to the in 

vitro assays, one point that has been made repeatedly is that there have been significant 

scientific advancement in in vitro assay technology that may allow for improved testing, 

and the EPA should take this into account. In any case, the battery of assays did show 

responses in one or more assays and/or endpoints for E, A, and T pathways. As 

mentioned above and in previous charge questions, overt toxicity overlapping potential 

hormonal effects is a concern in several of the in vivo assays, particularly the FSTRA and 

the pubertal assays. Thus, one might question whether the degree of selectivity 

envisioned by the 2008 SAP was achieved. The WoE analysis may address this.

Several panel members indicated strongly that the use of positive controls that are well 

known to interact with E, A, and T pathways should have been used to evaluate the entire 

battery as was done in the validation of each individual assay. The ability of the battery to 

clearly detect such known compounds would have been helpful in providing confidence 

that the battery functioned as intended and serve as a means for determining which assays 

might be considered for removal or replacement.

Charge Question 8. The EPA is committed to minimizing animal usage in the screening 

battery while maintaining the effectiveness of the battery to answer the question of 

whether a chemical has the “potential” to interact with the endocrine system.

a. In 1998, the EDSTAC described the conceptual framework for Tier 1 

assays and recommended the strategy to “require the minimal number of 

screens and tests necessary to make sound decisions, thereby reducing the 

time needed to make these decisions”, and that the screens should be 

conducted at a minimal cost necessary to make decisions. Based on the 

preliminary battery performance evaluation, to what extent can the 

current Tier 1 battery of 11 assays be modified to reduce animal usage 

and/or lower cost while adequately ensuring the EPA’s ability to answer 

the question of “whether a chemical has the potential to interact with the 

endocrine system?” More specifically, please comment on whether the 

Uterotrophic and Hershberger assays provide necessary redundancies in 

the Tier 1 battery based on this preliminary analysis. Please include in 

your comments what information may be lost and what uncertainties may 

be introduced by absence of either or both of these assays.

Panel Response

Based on the data presented, the majority of the Panel believed that a definitive decision 

to discard the Uterotrophic assay or Hershberger assays from the battery cannot be made 

at this time. While the tests are limited in what they can detect, and are considered by 

some as “in vivo test tube assays,” agents with estrogen agonist or anti-androgenic 

activities have been of particular concern up to this point. Both the Hershberger and 

uterotrophic assays are well-accepted tests of gonadal hormone action on peripheral 

tissues. These assays have the ability to very specifically detect androgenic- or 
54

estrogenic- receptor mediated effects of test compounds using in vivo models that (when 

gavage is used) involve important parameters such as a realistic method of test substance 

administration and metabolism. When expanded they also have the ability to test for 

anti-androgenic and anti-estrogenic effects. They are simple to run and have easy to 

measure endpoints. In comparison to the male and female pubertal assays, they have the 

advantage of testing the potential androgenic or estrogenic effects of various compounds 

using model systems where possible indirect, non-HPG related effects (i.e. stress 

response parameters) on gonadal androgen or estrogen secretion are not directly related 

to the effects on HPG function. While reduction of animal usage and unnecessary testing 

are major concerns, the majority of the panel members believed that these assays should 

be retained as they allow for specifically testing the actions of potential endocrine 

disrupting agents and their metabolites in a mammalian system, and may provide more 

definitive information than that which may be gleaned from the more comprehensive 

Pubertal assays.

At the very least, a decision to remove these assays from the battery should await the 

completion of the WoE analysis on these compounds to determine what role these assays 

play in the decision process. Several members of the Panel indicated strongly that the 

WoE analysis should include all of the compounds on which data have been collected. In 

the case of the Hershberger assay, for example, the Panel observed from the data 

presented that a response in the Hershberger assay was accompanied by a response in the 

male pubertal or the FSTRA assay, but it was not indicated if these effects were in the 

same direction. In the case of the Uterotrophic assay, it is not clear at this point if the 

negative response for the 21 chemicals evaluated in the set of chemicals under review 

could be factored in to a decision on a compound that showed an effect in the FSTRA 

assay only in the presence of overt toxicity. Even if they are removed from the battery as 

absolute requirements, they should be maintained as options if the other assays do not 

provide clear results. While, as indicated above, reduction of animal use and cost are 

important, it is also important that the screening assays serve their intended function to 

limit the number of compounds that go to Tier 2 while minimizing false negatives. A 

potential problem indicated by the Tier 1 results from the first set of 21 compounds is the 

potential confounding of endocrine effects with overt toxicity in the FSTRA and to a 

lesser extent, the pubertal assays. 

The advantages of moving away from the current ER and AR binding assays that use rat 

tissue cytosols were discussed at multiple points during the meeting. One advantage of 

cell line based assays would be to reduce animal usage, another would be increased 

robustness and reproducibility. The Panel recommended that the Agency give serious 

consideration to replacing the binding assays. 

b. Please comment on the scientific criteria the Agency should consider in 

evaluating necessary redundancies and eliminating assays from the 

current battery.
55

Panel Response

A decision to remove a battery from the assay could potentially be made after a thorough 

review of all data from the complete set of compounds that have been run through the 

Tier 1 screens. If an assay consistently produces results that are not clearly interpretable, 

removal could be considered. Likewise, if assays produce data that always only confirm 

the results of other assays and are not helpful in resolving questionable data, they could 

be considered for removal.

With regard to evaluating potential modifications to the current battery, the need for more 

extensive data evaluation was stressed. The need to increase the database, chemical and 

endocrine space, reveal compound identities and compare with existing data on 

mechanisms of action, and consider any potency data available was stressed by one panel 

member. Several panel members recommend a reanalysis of the data using the entire data 

set of 52 chemicals to provide an assessment of complementary and redundancy 

relationships. This more robust data set would also be amenable to analysis to determine 

if a particular assay or assays could be eliminated without increasing the probability of 

finding false negatives. Further, after completion of this assessment it would also be 

useful to consider splitting the Tier 1 assay battery into two sub-tiers using the in vivo

assays as a prescreen followed by targeted in vitro tests to explore mechanisms.
56

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