USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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U.S. Environmental Protection Agency

Endocrine Disruptor Screening Program

SAP Review of EDSP Tier 1 

Screening: Assay and Battery 

Performance

May 21-23, 2013

This document was developed by the EPA to provide general background and technical 

information to support and facilitate SAP review of the performance of EDSP Tier 1 

screening assays and battery.

This document does not create or confer legal rights or impose any legally binding 

requirements on the EPA or any other party. This document is distributed solely for the 

purpose of sharing this information with the public, consistent with the EPA 

transparency objectives. It is not intended to serve any other purpose, and should not 

be construed to represent formal dissemination of any agency determination or policy. 

As such, the information correction process under the agency's Information Quality 

Guidelines does not apply to this document.
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Table of Contents 

SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance

List of Acronyms ............................................................................................................................ 6

Executive Summary ........................................................................................................................ 9

I. Introduction ........................................................................................................................... 13

II. Methods ................................................................................................................................. 20

III. Evaluation of Results from Individual Assays ...................................................................... 24

1. In vitro ................................................................................................................................ 24

a. ER Binding .................................................................................................................................. 24

b. ERα Transcriptional Activation .................................................................................................. 32

c. AR Binding ................................................................................................................................. 37

d. Steroidogenesis ........................................................................................................................... 45

e. Aromatase (Human Recombinant) Assay ................................................................................... 53

2. In vivo................................................................................................................................. 59

a. Uterotrophic ................................................................................................................................ 59

b. Hershberger ................................................................................................................................. 64

c. Pubertal Female .......................................................................................................................... 70

d. Pubertal Male .............................................................................................................................. 78

e. Fish short-term reproduction ....................................................................................................... 86

f. Amphibian Metamorphosis Assay ............................................................................................ 110

IV. Performance of Battery ....................................................................................................... 130

1. Interaction with the HPT Axis ......................................................................................... 131

2. Interaction with the Androgen Pathway .......................................................................... 141

3. Interaction with the Estrogen Hormonal Pathway ........................................................... 147

V. Overall Summary and Conclusions ..................................................................................... 152

VI. References ........................................................................................................................... 156
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List of Tables

Table 1. Current EDSP Tier 1 Screening Assays. ...................................................................................... 16

Table 2. Number of Chemicals Included in the Assay and Battery Performance Analysis ....................... 22

Table 3. Overview of Case Study Chemical Product Types and Chemical Classes. ................................. 23

Table 4. ER Binding Classification Results for the 18 Chemicals Evaluated. ........................................... 27

Table 5. Performance Criteria for Saturation Binding ............................................................................... 27

Table 6. Cytosolic Preparations Used for More than One Chemical. ........................................................ 28

Table 7. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Positive 

Control. ..................................................................................................................................... 29

Table 8. Classification of the 19 Chemicals Evaluated in the ERTA ........................................................ 35

Table 9. AR Binding Classification Results for the 17 Chemicals Evaluated ........................................... 39

Table 10. Performance Criteria for Saturation Binding ............................................................................. 40

Table 11. Prostate Cytosol Preparations Used for More Than One Chemical. .......................................... 41

Table 12. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Strong and 

Weak Positive Controls. ........................................................................................................... 42

Table 13. Performance Criteria to be Met on Each QC Plate .................................................................... 49

Table 14. Summary of Fold-Change in Estradiol and Testosterone Levels............................................... 51

Table 15. Results from the 18 Steroidogenesis Studies. ............................................................................ 52

Table 16. Summary of the High Test Concentration for the 18 Aromatase Assays. ................................. 55

Table 17. Classification of the 18 Chemicals Evaluated in the Aromatase Assay. .................................... 56

Table 18. Performance Criteria for Aromatase Assay ............................................................................... 56

Table 19. Summary of Overt Toxicity Observed in the Uterotrophic Assay ............................................. 60

Table 20. Summary of Overt Toxicity Observed in the Hershberger Assay. ............................................ 66

Table 21. Maximum recommended % CVs for the Mandatory Sex Accessory Tissues. .......................... 68

Table 22. Summary of Overt Toxicity Observed in the Pubertal Female Assay ....................................... 73

Table 23. Performance Criteria for Controls (Sprague-Dawley Strain). .................................................... 77

Table 24. Summary of Overt Toxicity Observed in the Pubertal Male Assay. .......................................... 81

Table 25. Performance Criteria for Controls (Sprague-Dawley and Wistar Strains). ................................ 85

Table 26. Test Validity Elements for the FSTRA ...................................................................................... 91

Table 27. Identification of the Only Test Validity Criteria Not Achieved in 11 Studies ........................... 92

Table 28. Summary of the Types of Overt Toxicity Observed in the 20 Assays ....................................... 98

Table 29: Validity Elements for the Amphibian Metamorphosis Assay. ................................................ 115

Table 30: General Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted 

Under Flow-Through or Static-Renewal Conditions .............................................................. 115

Table 31: Additional Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted 

Under Static-Renewal Conditions .......................................................................................... 116

Table 32: Prevalence of Spinal Curvature in the Amphibian Metamorphosis Assay. ............................. 120

Table 33: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints,

 in the AMA with 

Chemical F. ............................................................................................................................. 123

Table 34: Growth Endpointsa,b in the AMA with Chemical F. ................................................................ 123

Table 35. Endpoints for Detecting Potential Effects on the Thyroid Hormonal Pathway According to the 

2008 FIFRA SAP. ................................................................................................................... 132

Table 36. Preliminary Results of Tier 1 Assays Relevant to the HPT Axis or Thyroid Hormonal Pathway, 

by Case Study Chemical. ........................................................................................................ 134
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Table 37. Associations between Endpoint Responses in Tier 1 Assays Relevant to the HPT and Thyroid 

Hormonal Pathway, by Case Study Chemical (Preliminary).................................................. 136

Table 38. Endpoints for Detecting Potential Effects on the Androgen Hormonal Pathway. ................... 142

Table 39. Summary of Results in Tier 1 Assays Relevant to the Androgen Hormonal Pathway by 

Chemical. ................................................................................................................................ 145

Table 40. Endpoints for Detecting Potential Effects on the Estrogen Hormonal System. ....................... 148

Table 41. Summary of Results in Tier 1 Assays Relevant to the Estrogen Hormonal Pathway, by 

Chemical. ................................................................................................................................ 151
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List of Figures

Figure 1. Saturation Binding Curves. ..................................................................................................... 28

Figure 2. Performance Criteria Results for the Saturation Binding for Cytosolic Preparations. ............ 29

Figure 3. ER Competitive Binding Curves ............................................................................................. 30

Figure 4. Performance Criteria Results for the Strong Positive Control. ............................................... 31

Figure 5. Performance Criteria Results for the Saturation Binding for Cytosolic Preparations. ............ 41

Figure 6. Saturation Binding Curves. ..................................................................................................... 42

Figure 7. Competitive Binding Curves ................................................................................................... 43

Figure 8. Performance Criteria Results for Strong (R1881) Positive Control. ....................................... 44

Figure 9. Performance Criteria Results for Weak (Dexamethasone) Positive Control. .......................... 44

Figure 10. Steroidogenic Pathway in H295R cellsa

. ................................................................................. 46

Figure 11. Quality Control Plate Performance for the 18 Steroidogenesis Studies. ................................. 50

Figure 12. Concentration Response Curve for Positive Control 4-OH ASDN. ........................................ 54

Figure 13. Aromatase Performance Criteria Results. ............................................................................... 58

Figure 14. Summary of Number of Studies Achieving Each Individual Test Validity Criteria ............... 91

Figure 15. Summary of the Number of Individual Test Validity Criteria Achieved by Each Study ........ 92

Figure 16. Summary of the Range of Reported Coefficient of Variation (CV) for the Studies .................. 96

Figure 17. Male VTG data ...................................................................................................................... 106

Figure 18. The majority of AMA study submissions reviewed to date have met the validity

elements and performance criteria outlined in the test guideline. ......................................... 116

Figure 19. Suggested Decision Logic for the Conduct and Interpretation of the AMA (OCSPP 890.1100, 

OECD 2009). ......................................................................................................................... 133
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List of Acronyms

Abbreviation Terminology 

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

AMA Amphibian Metamorphosis Assay

ANOVA Analysis of Variance

AR Androgen Receptor

ATCC American Type Culture Collection

Bmax Binding at maximum

CG Cowper’s Gland

ChE Cholinesterase

CV Coefficient of Variation

DER Data Evaluation Record

DMEM Dulbecco's Modified Eagle Medium

DMF Dimethyl-Formamide

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

DRP Detailed Review Paper

EACPD Exposure Assessment Coordination and Policy

ECVAM European Centre for the Validation of Alternative Methods

EDMVAC Endocrine Disruptor Methods Validation Advisory Committee

EDMVS Endocrine Disruptor Methods Validation Sub-committee

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

E Estrogen (hormonal pathway)

ER Estrogen Receptor

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol

FACA Federal Advisory Committee Act

FIFRA Federal Insecticide, Fungicide, Rodenticide Act

FFDCA Federal Food, Drug, and Cosmetic Act

FQPA Food Quality Protection Act

FRN Federal Register Notice

FSHR Follicle-Stimulating Hormone Receptor

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide

GLP Good Laboratory Practices

GP Glans Penis

GSI Gonado-Somatic Index

HBD Hormone Binding Domain

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis
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Abbreviation Terminology 

HPLC/MS/MS High Pressure Liquid Chromatography/tandem Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods

ISR Integrated Summary Report

Kd Equilibrium Dissociation Constant

LABC Levator Ani-Bulbocavernosus

LC50 Lethal Concentration in 50% of test organisms

LHR Luteinizing Hormone Receptor

MDL Minimum Detection Level

MoA Mode of Action

MRID Master Record Identifier

MTC Maximum Tolerated Concentration

MTD Maximum Tolerated Dose

NADPH Nicotinamide Adenine Dinucleotide Phosphate 

NF stage Nieukoop and Faber’s Staging Atlas

NIEHS National Institute of Environmental Health Sciences

NOAEC No Observed Adverse Effect Concentration

NSB Non-Specific Binding

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organisation for Economic Co-Operation and Development

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

PC Positive Control

PC10 Positive Control at 10% of response

PC50 Positive Control at 50% of response

PND Post-Natal Day

QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells

RPCmax Relative to Positive Control at maximum

SAB Science Advisory Board

SAP Scientific Advisory Panel

SC Solvent Control

s.c Subcutaneous

SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T3 3,3’,5 triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Propionate

TR Thyroid Receptor
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Abbreviation Terminology 

TSH Thyroid Stimulating Hormone

VC Vehicle Control

VO Vaginal Opening

VP Ventral Prostate

VTG Vitellogenin

WoE Weight-of-Evidence
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Executive Summary

The EPA developed the Endocrine Disruptor Screening Program in response to the statutory 

mandate in the Federal Food, Drug, and Cosmetic Act (FFDCA) to “develop a screening 

program...to determine whether certain substances may have an effect in humans that is similar 

to an effect produced by a naturally occurring estrogen, or such other endocrine effects as the 

Administrator may designate.” In addition to FFDCA, the Safe Drinking Water Act (SDWA) 

provides the EPA with authority to provide for testing “of any other substances that may be 

found in sources of drinking water if the Administrator determines that a substantial population 

may be exposed to such substance.” To begin meeting this statutory mandate, the EPA in 1996 

chartered a Federal Advisory Committee to address endocrine disruption, the Endocrine 

Disruptor Screening and Testing Advisory Committee (EDSTAC). One of the key 

recommendations made by the EDSTAC in 1998 was to develop a two-tiered screening and 

testing strategy, now known as the EDSP. The two-tiered screening and testing process is 

intended to ensure that only those chemicals that were screened to have potential endocrine 

activity would be advanced for further Tier 2 testing to determine whether the chemical interacts 

with the endocrine system. The purpose of the Tier 1 screening is to identify chemicals that have 

the potential to interact with the estrogen, androgen or thyroid hormone systems. 

EPA issued the first test orders of the Endocrine Disruptor Screening Program (EDSP) Tier 1 

screening on 67 chemicals (List 1) between October 29, 2009 and February 26, 2010 

(http://www.epa.gov/endo/). Of these, there were eight voluntary cancellations and seven inert 

ingredient manufacturers that chose to "opt out" of the pesticide market. Consequently, data 

were submitted on 50 pesticide active ingredients and 2 inert ingredients. For some test orders, 

EPA accepted “other scientifically relevant information” (OSRI) in lieu of specific study data 

(http://www.epa.gov/endo/). 

A subsample of up to 21 of the 52 chemicals on List 1 provided case studies to evaluate the 

performance of the EDSP Tier 1 individual assays and the entire battery. The 21 chemicals were 

comprised of 20 compounds representing a range of chemical classes (e.g., azoles, imides, 

organophosphates) and pesticidal activities (e.g., insecticides, herbicide and fungicides) and one 

inert ingredient. The case studies provide a robust chemical and biological domain to evaluate 

each of the Tier 1 assays and overall battery performance given the breadth of physico-chemical 

properties and given that all but one of these chemicals are designed to be biologically active. 

The EDSP Tier 1 screening consists of a battery of 11 assays: five in vitro (Estrogen Receptor 

binding, ER transcriptional activation assay, Androgen Receptor binding, Aromatase, and 

Steroidogenesis) and six in vivo (Uterotrophic, Hershberger, Female Pubertal, Male Pubertal, 

Fish Short-term Reproduction, and Amphibian Metamorphosis). The assays were to identify 

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

hormone systems. Understanding that not all in vitro and in vivo data are equal, the in vitro data 

may be quite informative to the in vivo assay results. Tier 1 in vitro assays were intended to 
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provide some mechanistic data for single known pathways, whereas the in vivo assays would 

capture multiple modes of action including those not yet known mechanisms and effects in an 

intact organism, accounting for both toxicokinetic and toxicodynamics parameters. 

The purpose of this review of the Tier 1 assays was to: (i) evaluate how well each Tier 1 assay 

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

highlight any issues associated with interpretation of responses within each assay (discussed in 

Section III), (ii) evaluate the complementary relationships among responses within the assays 

(discussed in Section IV), and to (iii) evaluate the redundancy of responses across the Tier 1 

battery (discussed in Section IV).

Additionally, this review considered whether or not a modified battery of assays, could reduce 

animal usage and lower costs, while adequately ensuring the identification of potential 

interactions with the E, A or T hormonal pathways (as discussed in Section IV). No 

determinations concerning chemical interactions with the E, A, or T signaling pathways are 

made in the current effort. A more detailed analysis of chemical endocrine interaction will be 

presented in a future SAP on the Agency’s proposed weight-of-evidence (WoE) approach during 

July 30 through August 2, 2013.

Each of the Tier 1 assays was previously validated with a known set of reference compounds (FR 

Notice, October 21, 2009) for endocrine screening specific to E, A, and T signaling pathways 

through an extensive process of test method development, prevalidation and interlaboratory 

validation that culminated in an evaluation by a 2008 FIFRA SAP1

, which supported using the 

11 assays to initiate Tier 1 screening. Thus, one aspect the focus of current assay performance 

analysis is on the proficiency of the contributing laboratories to execute each Tier 1 assay in 

accordance with the test guidelines, including the proper execution of the performance criteria 

and to obtain the expected results for those assays utilizing positive controls. The laboratory 

performance of each Tier 1 assay was typically consistent across the 21 chemicals evaluated. 

Although there were situations where performance criteria were not met, those deviations were 

considered minor and did not impact the interpretation or reliability of the data. Overall, 

laboratory performance for each Tier 1 assay met expectations, and the submitted Tier 1 assay 

data provide the expected useful information to evaluate potential interactions of test chemicals 

with the E, A and T signaling pathways.

Another purpose of the current review of assay performance was to identify any issues associated 

with the interpretation of Tier 1 assays. The most noteworthy issue encountered was associated 

with dose selection and the presence of overt toxicity (e.g., body weight reductions >10%, 

clinical signs, abnormal behavior, etc.) in the execution of some submitted Hershberger, Rat 

Pubertals and the Fish Short-Term Reproduction Assays. When positive Tier 1 findings are 

observed in the presence of overt toxicity there is uncertainty as to whether or not the responses 

are mediated through an endocrine pathway. A more detailed analysis of overt toxicity and the 

 

1 March 25-28, 2008 Session of the FIFRA Scientific Advisory Panel entitled “Review of the Endocrine Disruptor Screening 

Program (EDSP) Proposed Tier 1 Screening Battery”. 
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potential for chemical interactions with endocrine systems will be presented in at the WoE SAP 

during July 30 through August 2, 2013. 

In 2008, the SAP was also asked to comment on the extent of redundancies in the battery and to 

recommend ways in which the battery could be made more efficient. In addressing these 

questions, the 2008 SAP considered each Tier 1 assay in the context of a conceptual construct 

that used different “modalities” that span multiple levels of biological organization including 

molecular interactions, and cellular, organ and organism responses for each hormonal class (i.e., 

E, A, and T hormonal systems) the battery was expected to assess. The 2008 SAP defined these 

biological levels of organization as consisting of “receptor/target binding, cell responses, enzyme 

activity, organ response, males and female system integrity, development (thyroid) and 

comparative system integrity”. The 2008 SAP examined the “complementarity” and 

“redundancy” in the Tier 1 battery, i.e., “complementarity” describes the concordance of 

endpoints within an assay and “redundancy” describes the concordance of endpoints across the 

assays that are intended to evaluate an interaction with a particular pathway. The extent to which 

different endpoints within and across the assays reflected the test chemicals interaction with E, 

A, and T hormonal system was evaluated in terms of the extent to which the endpoints and 

assays were complementary and redundant. From its analysis, the 2008 SAP concluded that the 

Tier 1 battery is enhanced because the assays provide adequate “replication” (i.e., 

complementary responses within assays) and redundancy across assays. 

The current analysis used the 2008 FIFRA SAP concept as a guide in evaluating battery 

performance by qualitatively describing the degree to which responses within multi-parameter 

assays are “complementary” and the degree to which responses across assays are “redundant”. 

The findings are discussed with consideration to whether there appear to be any unnecessary 

redundancies in assessing chemical interactions with E, A, or T signaling pathways based on the 

diversity of the representative compounds evaluated to date. Because the purpose of the current 

review is to evaluate battery and assay performance in the broader context, this review presents 

qualitative information as to the general presence or absence of responses within assays (i.e.,

complementary responses) and across assays (i.e., redundancy). The directionality or magnitude 

of Tier 1 responses and the process for determining a specific chemical’s potential to interact 

with E, A, or T are not discussed in this evaluation. As discussed previously, the WoE analysis 

of the Tier 1 battery results, with OSRI, will be examined at the WoE SAP during July 30 

through August 2, 2013. 

The range of responses evaluated in the Tier 1 battery using a subset of List 1 chemicals

generally demonstrate the battery contains the necessary complementarity within and redundancy 

across assays to effectively screen for interactions with the E, A, or T hormonal systems. For the 

E and A pathways, there were complementary responses with two or more endpoints responding 

in the pubertal assays and in the Fish Short-Term Reproduction Assay. 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 E and A. The 2008 SAP pointed out that the 
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in vitro assays represent “specificity and sensitivity....and do not provide complementary or 

redundant assessments of androgenic activities”, and in the current evaluation there were also 

cases in which potential specific modes of action were not detected in the in vitro assays; 

however, in vivo responses were obtained in the Hershberger, Pubertals and the Fish Short-Term 

Reproduction Assays. The later observations emphasize the importance of these multiparameter in vivo assays as a component of the battery to evaluate hypothalamic pituitary 

gonadal alterations, as well as potential differences between taxa. In some cases, the results of 

the Estrogen Receptor binding assay and the Uterotrophic assay demonstrated concordance in 

that there was a lack of response in both assays which suggests a lack of estrogenicity among the 

case studies. There was also general agreement among the Hershberger assay, the Male Pubertal 

and/or Fish Short-Term Reproduction Assay as all three generally showed responses in androgen 

dependent tissues. With respect to the thyroid hormone pathway, less complementarity and 

redundancy were established in the design of the battery compared to the E- and A-related 

assays/endpoints, which is consistent with the results in the current analysis. Thus, this current 

analysis reinforces the need of both the Amphibian Metamorphosis Assay and the Rat Pubertal 

assays as essential components of the battery for evaluating the thyroid pathway. 

In general, the current analysis illustrated both complementarity within assays and redundancy 

across assays and demonstrated the battery design provided complementarity and redundancy 

for evaluating potential interactions with the E, A, or T signaling pathways across taxa. 

However, chemicals that elicited a response in the Hershberger also elicited a response in the 

Male Pubertal assay and/or Fish Short-Term Reproduction Assay. Additionally, the 

Uterotrophic assay never showed a response with the subset of chemicals used to evaluate 

battery performance while the multi-parameter female pubertal assay and/or FSTRA did. The 

agency will seek guidance from the 2013 May 21-23 SAP on whether the EDSP Tier 1 battery of 

eleven assays can be optimized and reduced, and more specifically, whether the Hershberger and 

Uterotrophic assays are necessary redundancies in the current battery. 
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I. Introduction

a. Historical background 

In 1996, Congress passed the Food Quality Protection Act (FQPA) and amendments to the 

Safe Drinking Water Act (SDWA). Passage of the FQPA provided for amendments to the Federal 

Food, Drug, and Cosmetic Act [FFDCA, Section 408(p)] which required the US Environmental 

Protection (EPA) to “develop a screening program, using appropriate validated test systems and 

other scientifically relevant information, to determine whether certain substances may have an 

effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other 

such endocrine effect as the Administrator may designate.” 21 U.S.C. 346a(p)(1). In addition, 

amendment to the SDWA (Section 1457) indicated that “the Administrator may provide for 

testing under the screening program ... any other substance that may be found in sources of 

drinking water if the Administrator determines that a substantial population may be exposed to 

such substance.” 42 U.S.C. 300j-17. 

• Expand the number of hormonal pathways by including test systems to detect interactions 

along the androgen and thyroid hormonal pathways, in addition to the estrogen pathway,

• Expand the number of organisms to include wildlife (i.e., amphibian, fish, reptiles, birds, and 

invertebrates), in addition to humans, and,

• Incorporate a two-tiered screening and testing approach.

In general, Tier 1 screening should consist 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 estrogen, androgen, or thyroid (E, A, or T) hormonal pathways and, based on a weight- of -

evidence analysis (WoE), determine whether a chemical is a candidate for Tier 2 testing. Tier 2 

testing would involve more complex in vivo studies to clarify the potential interaction with the 

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

needed for risk assessment.

Based on EDSTAC’s recommendations and pursuant to the Administrator’s discretionary 

authority, the Agency expanded the program to include the androgen and thyroid hormonal 

pathways of the endocrine system and ecological effects within a two-tiered screening and 

testing strategy (EDSTAC, 1998). This framework was generally supported by a joint committee 

of the SAB/SAP (SAB/SAP, 1999) who recommended to the Agency, that after review of the 

initial set of Tier 1 data, the results be subject to external scientific peer review “...with an eye 

towards revising the process and eliminating those methods that don’t work.” 
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b. Tier 1 Screening Assay and Validation Process 

In general, Tier 1 screening inclusive of other scientifically relevant information (OSRI) 

was envisioned to provide data that would be subject to a WoE analysis to determine whether or 

not a chemical has the potential to interact with the E, A, or T hormonal pathways and assess the 

need for Tier 2 testing. Under FFDCA, the EDSP is mandated to use “validated” assays to screen 

for endocrine disrupting chemicals. Validation principles established by the Interagency 

Coordinating Committee Validation of Alternative Methods (ICCVAM, 2000) and Organization 

for Economic Co-Operation and Development (OECD, 2005) were adopted and followed by 

EPA (USEPA, 2007) to develop, standardize, and validate the current EDSP Tier 1 screening 

assays as shown in Table 1. 

Generally, validation has been defined as “the process by which the relevance and 

reliability of a test method is evaluated for a particular use” (OECD, 1996; NIEHS, 1997):

• Relevance describes whether or not a test is meaningful and useful for a particular 

purpose (OECD, 1996). For EDSP Tier 1 screening assays, relevance is generally 

described as the ability of an assay to detect chemicals with the potential to interact with 

the E, A, or T hormonal pathways.

• Reliability is a measure of the variability or reproducibility of assay results within and 

between or among laboratories. 

The validation process of individual EDSP Tier 1 screening assays has largely been a 

collaborative effort coordinated by and funded through the Agency’s EDSP, with other offices 

within EPA, especially the Office of Research and Development (ORD), as well as industry and 

academic institutions providing necessary scientific and technical support. In addition, test 

development, prevalidation, and inter-laboratory validation work for some of the Tier 1 

screening assays were conducted internationally through coordination with the OECD. 

In addition, the EDSP sought guidance under the FACA to provide independent technical 

expertise and recommendations throughout the validation process. Federal advisory committees 

such as the Endocrine Disrupter Methods Validation Sub-committee (EDMVS) and the 

Endocrine Disrupter Methods Validation Advisory Committee (EDMVAC) provided guidance 

from 2001 through 2006. Thereafter, the FIFRA SAP has primarily served the advisory role for 

EDSP. Generally, for each Tier 1 screening assay, the EPA has followed a five-stage validation 

process:

• Test Development, an applied research function using reference chemicals known 

to interact with the endocrine-dependent endpoints culminating in an initial assay 

protocol and establishing relevance
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• Prevalidation consisting of the refinement, optimization, and standardization of 

the protocols. In addition, assessing the transferability and performance of the 

protocols being considered as part of the Tier 1 EDSP assay battery.

• Inter-laboratory Validation in which independent laboratories conducted studies 

with optimized, standardized protocols used to primarily determine interlaboratory variability (i.e., repeatability) and to set or cross-check performance 

criteria.

• Peer Review, an independent scientific review by qualified experts. Peer review 

of individual screening assays was conducted in compliance with the EPA’s Peer 

Review Handbook (EPA, 2006).

• Regulatory Acceptance, adoption for regulatory use by an agency. The inclusion 

of an assay in the proposed Tier 1 battery, review of the proposed battery by the 

SAP (SAP, 2008), and final adoption of the battery by EPA (Federal Register 

Notice issued October 21, 2009, 74 FR 54416) based on SAP recommendations 

constitutes the regulatory acceptance stage of the process. 

c. Tier 1 Screening Battery

Considering the results of assay validation, comments from scientific peer review of 

individual assays, and recommendations from various FACAs, the EPA compiled the current 

EDSP Tier 1 screening battery (Table 1). In accord with EDSTAC’s recommendation (EDSTAC, 

1998), the current combination of assays within the battery was designed to maximize sensitivity 

and reliability for determining the potential of a chemical to interact with the E, A, or T 

hormonal pathways. The basis for selecting individual assays to include in the battery involved 

various factors that generally included the capability of the assays to detect: 

• E, A, and T hormonal signaling in different taxa,

• estrogen- and androgen-mediated receptor binding,

• estrogen-mediated gene transactivation,

• enzyme inhibition involving the reproductive steroidogenesis pathway,

• interactions with gonadal estrogen and androgen production that may alter feedback 

mechanisms involving the hypothalamic-pituitary-gonadal (HPG) axis,

• androgen and estrogen endpoints within and complementary among assays, and 

• interactions with thyroid hormone production that may alter feedback mechanisms involving 

the hypothalamic-pituitary-thyroid (HPT) axis.
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Table 1. Current EDSP Tier 1 Screening Assays.

The current EDSP Tier 1 screening assays encompass key endpoints within a mode of action 

(e.g., receptor binding) as well as along endocrine pathways (e.g., steroidogenesis, effects on 

hypothalamic-pituitary-gonadal and –thyroid axes) through which a chemical has the potential to 

interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal pathways.*

Screening Assay

Test 

Guideline 

Receptor Binding Steroidogenesis HPG 

Axis

HPT 

E Anti-E A Anti-A E A Axis

In vitro

ER Binding

(Rat uterine cytosol)

OCSPP 

890.1250 

■ ■

ERα Transcriptional 

Activation

(Human cell line 

HeLa-9903) 

OCSPP 

890.1300 

OECD 455

■

AR Binding

(Rat prostate cytosol) 

OCSPP 

890.1150 

■ ■

Steroidogenesis

(Human Cell Line 

H295R) 

OCSPP 

890.1550 

■ ■

Aromatase

(Human target tissue 

or cell line 

microsomes)

OCSPP 

890.1200 

■

In vivo

Uterotrophic (Rat) OCSPP 

890.1600 

OECD 440

■

Hershberger (Rat) OCSPP 

890.1400 

OECD 441

■ ■ ■1

Pubertal Male (Rat) OCSPP 

890.1500

■ ■ ■ ■ ■

Pubertal Female 

(Rat)

OCSPP 

890.1450

■ ■ ■ ■ ■

Fish Short-term

Reproduction

OCSPP 

890.1350 

OECD 229

■ ■ ■ ■ ■ ■ ■

Amphibian 

Metamorphosis

(Frog)

OCSPP 

890.1100 

OECD 231

■

* Complementary endpoints across assays are indicated (solid black box) within each column.

1

 5α-reductase inhibition only. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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(i) Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal Pathway 

Seven Tier 1 assays are capable of detecting chemicals with estrogenic and 

antiestrogenic-like activity that may disrupt estrogen hormonal signaling as shown in Table 1.

Details of individual assays are described in respective assays sections within the 

document as well as test guidelines. An introduction and overview of the assays are as follows: 

• ER binding assay, uses rat uterine cytosol and has the potential to detect estrogen receptor 

binding (i.e., ERα or ERβ) of a chemical. Note, cannot distinguish between estrogen agonist 

or antagonist activity.

• ER transcriptional activation assay, uses a human cervical tumor cell line and has the 

potential to detect estrogen agonists through receptor binding (i.e., ERα) and gene 

transcription.

• Aromatase assay, uses human microsomes and has the potential to detect the inhibition of 

aromatase activity (i.e., conversion of androgen to estrogen) 

• Steroidogenesis assay, uses a human cell line and has the potential to detect a change in 

production (increase or decrease) of testosterone and estradiol. 

• Uterotrophic assay, uses adult ovariectomized rat or mouse, or sexually immature intact 

female rats and has the potential to detect estrogen-like activity that may involve receptor 

binding and effect a change (increase) in uterine weight. Note, oral or subcutaneous routes of 

exposure are acceptable where the latter bypasses first-pass liver metabolism. 

• Pubertal female assay, uses intact rats post-weaning and has the potential to detect both 

estrogen and antiestrogen-like activity that may involve receptor binding or inhibition of 

steriodogenesis that may alter hormonal signaling along the HPG axis and affect a change in 

multiple endpoints generally associated with female sexual development (e.g., vaginal 

opening), and gross and histological changes of reproductive organs.

• Fish short-term reproduction assay (FSTRA), uses mature male and female fathead 

minnows and has the potential to detect both estrogen and antiestrogen-like activity that may 

involve receptor binding or inhibition of steroidogenesis that may alter hormonal signaling 

along the HPG axis and affect a change in multiple endpoints generally associated with 

survival, and changes in growth, secondary sex characteristics, histology of gonads, and 

plasma concentrations of vitellogenin.
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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(ii) Assays for Detecting the Effect of Chemicals on the Androgen Hormonal Pathway 

Six Tier 1 assays are capable of detecting chemicals with androgenic and antiandrogenic-like

activity that may disrupt androgen hormonal signaling as shown in Table 1. 

Details of individual assays are described in respective assays sections within the 

document. An introduction and overview of the assays are as follows: 

• AR binding assay, uses rat prostate cytosol and has the potential to detect androgen receptor 

binding; cannot distinguish between androgen agonist or antagonist activity. 

• Aromatase assay, similar to that previously described. 

• Steroidogenesis assay, similar to that previously described. 

• Hershberger assay, uses castrated male peripubertal rats and has the potential to primarily 

detect androgen and antiandrogen-like activity that may involve receptor binding or 

inhibition of steriodogenesis that could effect a change (increase or decrease) on multiple 

endpoints associated with gross changes in organ weights of ventral prostate, seminal 

vesicles, levator ani plus bulbocavernous muscle complex, Cowper’s gland, and glans penis.

• Pubertal male assay, uses intact rats post-weaning and has the potential to detect both 

androgen and antiandrogen-like activity that may involve receptor binding or inhibition of 

steriodogenesis that may alter hormonal signaling along the HPG axis and affect a change in 

multiple endpoints generally associated with male sexual development (e.g., preputial 

separation) and gross and histological changes of reproductive organs.

• The fish short-term reproduction assay, similar to that previously described expect there is 

the potential to detect both androgen and antiandrogen-like activity. 

(iii) Assays for Detecting the Effect of Chemicals on the Thyroid Hormonal Pathway 

Three Tier 1 assays are capable of detecting chemicals that may disrupt the thyroid hormonal 

signaling pathway as shown in Table 1.

Details of individual assays are described in respective assays sections within the 

document. An introduction and overview of the assays are as follows: 

• Pubertal female and males assays, similar to that previously describe except there is the 

potential to detect effects on the thyroid hormonal signaling pathway (i.e., HPT axis) through 

an effect on thyroid gland weight, histology, and serum total thyroxine concentrations. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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• Amphibian metamorphosis assay, uses tadpoles and has the potential to detect effects on the 

thyroid hormonal signaling pathway (i.e., HPT axis) through effects on metamorphosis and 

thyroid gland histology. 

In accord with EDSTAC, “...the value of each individual assay cannot be considered in 

isolation from other assays in the battery, as they have been combined in a manner such that 

limitations of one assay are complemented by the strengths of another” (EDSTAC, 1998). Thus, 

the combination of Tier 1 screening assays and potential for concordance or corroboration of 

results among assays maximizes the sensitivity and reliability for determining the potential of a 

chemical to interact with the E, A, or T hormonal pathways.

d. FIFRA SAP Review of the Tier 1 Screening Battery in 2008 

In 2008, the FIFRA SAP reviewed the combination of EDSP Tier 1 screening assays of 

the current battery based on several charge questions (USEPA, 2008). A couple key questions 

were whether the battery could: 

• Minimize the potential for "false negatives" and "false positives" and

• did not result in unnecessary redundancies for modes of action across the battery.

In addressing these questions, the SAP considered the different modalities within the 

battery for E, A, and T (e.g., receptor binding, cell signaling, organ response) and evaluated the 

degree of redundancy and complementarity of the assays and endpoints across the battery (Table 

1). In response, the SAP indicated the battery provided for both redundancy and 

complementarity, especially in regard to the in vivo assays and recommended to EPA that “...the 

proposed set of Tier 1 assays are appropriate to begin screening for disruptors of the EAT 

axes.” (USEPA, 1998). 

The EPA subsequently published a Federal Register Notice (FRN) in 2009 (USEPA, 

2009c) notifying the public of the current EDSP Tier 1 screening battery (Table 1) and 

availability of harmonized test guidelines (OCSPP Series 890).

e. Test Orders for Tier 1 Screening

The first list of test chemicals for EDSP Tier 1 screening consisted of 67 chemicals, 58 

pesticide active ingredients belonging to several chemical classes and 9 High Production Volume 

(HPV) chemicals used as pesticide inert ingredients (USEPA, 2009d). The criteria for selecting 

chemicals for List 1 were based on the finding of a chemical in at least three of four exposure 

pathways (food, water, residential, or occupational exposures). In October 2009, EPA began 

issuing test orders for Tier 1 screening (USEPA, 2009d). Recipients complied by submitting the 

results of all 11 assays of the battery or, to avoid duplicity in test data according to the FFDCA 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 20 of 160 

statute that directs the EPA to use “... appropriate validated test systems and other scientifically 

relevant information...”, OSRI was submitted and accepted in some instances by the Agency in 

fulfillment of test order requirements for one or more of the Tier 1 assays. In general, 

information that is submitted voluntarily and is applicable to support or clarify an EPA action is 

commonly referred to as “other” scientifically relevant information (USEPA, 2002b). Sources of 

OSRI may include results from EPA or OECD equivalent test guideline studies (e.g., EPA Part 

158 studies) and information from published or publically available peer-reviewed studies. 

f. FIFRA SAP Review of the Tier 1 Screening Assay and Battery Performance in 2013 

It is emphasized that the current 2013 SAP performance review of EDSP Tier 1 screening 

assays and battery is primarily based on the 2008 SAP review of the current battery of assays as 

shown in Table 1. The fundamental difference between then and now is 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, whereas the current review is based the application of 

the entire battery of 11 assays with chemicals not necessarily known to interact with the 

endocrine system. Moreover, the current SAP is consistent with the joint SAB/SAP panel in 

1999 who recommended the EPA “convene a panel of independent scientists to review all the 

screening data for 50-100 compounds, with an eye towards revising the process and eliminating 

those methods that don’t work” (SAB/SAP, 1999) and SAP panel in 2008 who recommended 

“... that the EPA will continue to develop, refine, and review the battery.” 

II. Methods

Section III provides a preliminary analysis of the performance of each EDSP Tier 1 assay 

using an initial subset of chemicals which were issued Test Orders (List 1) in October 2009. The 

purpose of Section III is to:

• Evaluate how well each Tier 1 assay test guideline can be consistently executed based on 

defined performance criteria

• Highlight any issues associated with interpretation of responses within each assay

It is important to note that all 11 Tier 1 assays are validated methods. This analysis is not 

a “revalidation” of the Tier 1 assays. Each of the Tier 1 assays was validated with a known set of 

reference compounds (FR Notice, October 21, 2009) for endocrine screening specific to the 

estrogen, androgen, and thyroid (EAT) pathways through an extensive process of test method 

development, prevalidation and interlaboratory validation which culminated in evaluation by a 

FIFRA SAP in 2008 (USEPA, 2008). 

This review is intended to transparently communicate observations on performance of the 

assays with a set of test chemicals drawn from data submitted in response to the first set of Tier 1 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 21 of 160 

test orders issued for 67 chemicals by EPA on October 29, 20092. Fifteen chemicals were 

voluntarily cancelled or the manufacturer/importer agreed to cease all sales and distribution of 

the chemical in the pesticide market. Tier 1 assay data were submitted on a total of 52 chemicals. 

To facilitate timely feedback, a sub sample consisting of 21 test chemicals that adequately 

represented the chemical space of the 52 pesticide actives were available for consideration. 

These chemicals represent a broad range of physical-chemical properties including water 

solubility, octanol-water partition coefficient (Kow), and volatility. The chemicals also represent 

a range of chemical classes (e.g., organophosphates, imides, azoles) and pesticidal activities 

(e.g., fungicides, herbicides, insecticides). The review of Tier 1 submitted data is an ongoing 

process. Thus, for the purposes of this review the chemicals were selected to be illustrative and 

representative of the 52 chemicals included in the test orders. Given the breadth of physicochemical properties and that many of these chemical are designed to be biologically active (i.e., 

pesticidal modes of action), this group of chemicals represents a robust domain over which to 

evaluate assay and battery performance.

As with other data requirements intended to support regulatory decisions, EPA has 

developed a set of standard evaluation procedures (SEPs) which provide guidance for the review 

and evaluation of environmental and human health effects data. Similar guidance documents 

have been developed for the review of the data submitted in response to the test orders and these 

are outlined in the 890 Guideline Series3

 for the EDSP Tier 1 battery. The SEPs are used in 

conjunction with the appropriate EDSP Series 890 Guideline. Using the guidance within the 

relevant SEP, Agency reviewers develop Data Evaluation Records (DERs) for each of the 

submitted assays using study profile templates4 that have been established for this purpose. 

These DERs capture data to determine how well the assay conforms to the Series 890 EDSP Test 

Guidelines and document basic information on the materials, methods and results of the study. 

The DER provides both registrant conclusions and EPA reviewers’ conclusion.

The Agency currently follows a multi-tiered approach to reviewing EDSP data 

submissions. Primary reviews originate with an Agency staff scientist or contract scientist who 

is responsible for transcribing the materials, methods and results into the appropriate study 

profile template. These reviews generally consist of a reanalysis of study raw data for 

independent verification of study results and the Agency’s interpretation of the study is recorded 

in the DER. Secondary review of the DER is conducted by an Agency scientist who is trained to 

review the test guideline study and is experienced in reviewing data for the specific test 

chemical. A division-level consistency check may then be performed before the DER is elevated 

to an interdisciplinary committee of scientists with specialized knowledge of the Tier 1 assays. 

The committee reviews several data submissions for a given guideline concurrently to ensure 

 

2 Federal Register. 2009.Endocrine Disruptor Screening Program: Tier 1 Screening Order Issuing Announcement. 

http://www.gpo.gov/fdsys/pkg/FR-2009-10-21/html/E9-25352.htm 3 USEPA 2009. OCSPP Harmonized Test Guidelines Series 890. Endocrine Disruptor Screening Program Test Guidelines. 

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series890.htm 4 USEPA. 2011. Study Profile Templates. Endocrine: Series 890 – Endocrine Disruptor Screening Program Test Guidelines. 

http://www.epa.gov/pesticides/regulating/studyprofile_templates/studyprofile_templatelist.htm#series-890 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 22 of 160 

consistency in interpretation. Any changes recommended by the committee or at any previous 

point in the review process are reflected in the draft DER. The Agency anticipates that each 

review will remain preliminary until all Tier 1 data submissions for a given chemical are 

reviewed as a battery along with other scientifically relevant information consistent with the 

approach outlined in the WoE guidance document5

. Given the preliminary nature of the reviews, 

actual chemical names are omitted from this document. 

Up to 21 chemicals were evaluated in this analysis. The number of case study chemicals 

evaluated for each assay type and the pathways evaluated for battery performance differ since 

some of the assays were previously satisfied by OSRI. Table 2 summarizes the number of case 

study chemicals for the assay and battery analysis. This assay/battery performance review 

focuses only Tier 1 test guideline data. All available OSRI will be considered in the future WoE 

determinations for each chemical. 

Table 2. Number of Chemicals Included in the Assay and Battery Performance Analysis 

Assay Performance

Assay Type Number of Case Studies Evaluated

In Vitro

Estrogen Receptor Binding 18

Estrogen Receptor Transcriptional Activation 19

Androgen Receptor 17

Steroidogenesis 18

Aromatase 18

In Vivo

Uterotrophic 17

Hershberger 17

Pubertal Female 17

Pubertal Male 16

Fish Short-Term Reproduction 21

Amphibian Metamorphosis 18

Battery Performance

Interaction with the Estrogen Hormonal Pathway 17

Interaction with the Androgen Pathway 17

Interaction with the HPT Axis 15

Table 3 provides an overview of the product types and chemical classes represented by 

the case study chemicals.

 

5 USEPA. 2011. Weight-of-evidence: Evaluating results of EDSP Tier 1 screening to identify the need for Tier 2 testing. 14 

September 2011. Office of Chemical Safety and Pollution Prevention, Washington, DC. Regulations.gov Document ID: EPAHQ-OPPT-2010-0877-0021
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 23 of 160 

Table 3. Overview of Case Study Chemical Product Types and Chemical Classes.

Number of Case Study Chemicals Product Type Chemical Class

1 Herbicide Phenoxy

2 Herbicide Dinitroaniline

1 Herbicide Chlorinated benzoic

1 Herbicide Benzonitrile

1 Herbicide Chloroacetanilide

1 Herbicide Triazinon

1 Herbicide Triazine

1 Herbicide Fluorinated pyridazinone

1 Insecticide, nematicide N-methyl carbamate

4 Insecticide, nematicide, acaracide Organophosphate

1 Insecticide Pyrethroid

1 Acaricide Organotin

1 Acaricide Organosulphite

1 Fungicide Imide

2 Fungicide Azole

1 Pesticide Inert N/A

In some cases, the Agency’s preliminary review of data submissions identified procedural 

inadequacies or other confounding factors, such as excess overt toxicity or cytotoxicity, which 

may compromise the interpretation of results. The recommendations for successful performance 

of a Tier 1 assays and for gauging overt toxicity are guideline-specific and are addressed in more 

detail in the preceding chapters for each assay

Whether a particular measurement endpoint within a study was deemed affected 

depended on its statistical significance at a particular level of confidence, its biological relevance 

in terms of the nature and magnitude of the effect, and the extent to which the response appeared 

to be concentration dependent. In some cases, the biological relevance in terms of magnitude of 

the effect was used to indicate an effect even in the absence of statistical significance.

Within each assay, a particular pathway (E, A, or T) was scored based on the guidance 

provided by the SAP (USEPA, 2008).
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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III. Evaluation of Results from Individual Assays

This section describes the technical conduct of individual assays and interpretation of 

results in accordance with respective test guidelines and SEPs. Up to 21 chemicals were included 

for this analysis. 

1. In vitro

a. ER Binding

(i) Background 

The purpose of the Estrogen Receptor Binding (Rat Uterine Cytosol) Assay (OCSPP 

980.1250)6 is to provide information on the ability of a compound to interact with estrogen 

receptors (ERs) isolated from the rat uterus. The assay measures the ability of a test chemical to 

interact with ERs as reflected by the displacement of radiolabeled [3

H]-17β-estradiol. As 

conducted, this assay utilizes all cytosolic estrogen receptor subtypes that are expressed in this 

tissue including ERα and ERβ. The ER binding assay only has the ability to determine if a 

chemical can interact and displace the endogenous hormone; it provides no information on 

whether a chemical will act as an agonist or antagonist at the receptor(s) to either activate or 

inactivate an estrogen-dependent response. 

The Estrogen Receptor Binding (Rat Uterine Cytosol) Assay consists of a saturation 

binding experiment and a competitive binding experiment. The purpose of the saturation binding 

experiment is to demonstrate that each batch of rat uterine cytosol contains an adequate number 

of ER with a binding affinity for the native ligand, 17β-estradiol that is consistent with historical 

values. ER saturation binding experiments measure total and non-specific binding of increasing 

concentrations of [3H]-17β-estradiol under conditions of equilibrium. From these measurements, 

specific binding at each concentration can be calculated. A graph of specific [3H]-17β-estradiol 

binding versus radioligand concentration should reach a plateau indicative of saturation of the 

ER with the radioligand. In addition, analysis of the data should document the binding of the 

[

3

H]-17β-estradiol to a single, high-affinity binding site (i.e., Kd = 0.03 to 1.5 nM). The 

competitive binding assay measures the binding of a single concentration of [3H]-17β-estradiol in 

the presence of increasing concentrations (10-10 to 10-3 M) of a test substance. Each test 

concentration is evaluated in triplicate (three concurrent replicates) within one run. Three 

independent (non-concurrent) runs are performed for each chemical tested. 

 

6

Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1250: Estrogen Receptor Binding (Rat Uterine Cytosol). 

Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740-

C-09-005. October 2009.

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0005. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Each run of the competitive binding assay includes concurrent solvent (vehicle), a 17βestradiol reference curve, a weak positive control, and a negative. Dose response curves for the 

negative, weakly positive and reference estrogen are evaluated to demonstrate that the assay is 

conducted properly. The solvent control indicates the maximum specific binding that is possible 

under the conditions of that run. The negative control (octyltriethoxysilane) provides assurance 

that the assay as conducted does not report binding for chemicals that do not bind to the ER. A 

weak positive substance (norethynodrel) is included to demonstrate the sensitivity of each 

experiment and to allow an assessment of variability in the conduct of the assay across time. The 

reference standard (17β-estradiol) is also included to ensure that the run has been properly 

performed, and to allow an assessment of variability in the conduct of the assay across time.

Results from each run of the competitive binding assay are used to classify the test 

chemical as “interacting”, “not interacting”, “equivocal”, or “equivocal up to the limit of the 

concentrations tested”. The classification criteria are summarized below.

Interactive:

A chemical is classified as “interactive” with the ER if the lowest point on the fitted response 

curve within the range of the data is less than 50% (i.e., more than 50% of the radiolabeled 

estradiol has been displaced from the receptor). 

Not Interactive:

A chemical is classified as “not interactive” if the lowest data point on the fitted response curve 

is above 75%. (i.e., less than 25% of the radiolabeled estradiol has been displaced) or if a binding 

curve cannot be fitted and the lowest average percent binding among the concentration groups in 

the data is above 75%. 

Equivocal up to the limit of concentrations tested:

A chemical is classified as “equivocal up to the limit of concentrations tested” if there are no 

data points at or above a test chemical concentration of 10-6 M, and (A) a binding curve can be fit 

but 50% or less of the radiolabeled estradiol is displaced by concentration 10-6 M, or (B) a 

binding curve cannot be fit and the lowest average percent binding among the concentration 

groups in the data is above 50%.

Equivocal: 

A chemical is classified as “equivocal” if it does not meet the criteria of the categories above.
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Chemical Classification: 

After each chemical is classified from each run, the chemical is classified by assigning the 

following values to each run and averaging across runs: 

interactive: 2 

equivocal: 1 

not interactive: 0 

Chemical classification, based on the average of all the runs performed for a chemical: 

interactive: average ≥ 1.5 

equivocal: 0.5 ≤ average < 1.5 

not interactive: average < 0.5 

(ii) Strengths and Limitations of the Assay 

The hormone binding domain (HBD) of the ER is highly conserved across species. 

Environmental chemicals that compete with the endogenous ER have the potential to either 

induce hormone-dependent processes (agonist) or block normal hormone function by preventing 

endogenous hormone from binding to the receptor (antagonist). The assay cannot distinguish 

between ER agonism and ER antagonism. 

(iii) Observations of Test Chemicals

There were 18 chemicals available at the time of analysis to evaluate laboratory 

performance of this assay. Seven different laboratories were involved in conducting the ER 

Binding Assays, with the majority of the assays being performed by two laboratories (Laboratory 

1, 7 chemicals; Laboratory 2, 5 chemicals). Uterine tissue from Sprague-Dawley rats was used 

as the source tissue for cytosolic ER for all compounds. The cytosols were prepared and stored 

as specified in the Guideline. The protein concentrations of the cytosol preparations ranged from 

1.1 to 5.1 mg/mL, and the final protein concentrations used in the assays (where reported) were 

0.341 to 1.191 mg/mL or 40 to 320 μg/assay tube. The purity of the test chemicals used in the 

assays was >89%. Dimethyl sulfoxide (DMSO) or ethanol (EtOH) was used as the solvent 

vehicle for the test chemicals in all assays. Solvent concentration limits were met for all 

compounds. 

Test chemicals were initially evaluated at serial log concentrations from 1 mM to 100 pM 

(10−10 to 10−3 M) in the assay as recommended in the test guideline, with the exceptions of two 

chemicals (Chemicals Q and S) which were tested to a maximum of 10-4 M and 10-5 M, 

respectively, based on preliminary solubility findings. For the remaining 16 compounds, 10 

were soluble up to the limit concentration of 10−3

 M and six were only soluble up 10-4 M. If a 

chemical is not soluble at 10−3 M, the guideline recommends testing the chemical at a 

concentration that is 1⁄2 log lower (e.g. 10-3.5 M). All but one laboratory (Laboratory 1) followed 

this guidance when insolubility was encountered. In some instances when precipitation was 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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observed, Laboratory 1 added additional concentrations at the lower end of the concentration 

range (10−11 or 10−12 M), rather than utilizing mid-log concentrations at the upper end of the 

concentration range (10−4.5 M or 10−5.5 M) which would better characterize the response curve 

and provide more confidence in the interpretation of the results.

Based on the results of the 18 assays, 16 of the test compounds were classified as noninteractive, and two compounds were classified as equivocal based on the classification criteria 

presented above. None of the 18 chemicals was classified as interactive (Table 4). 

Table 4. ER Binding Classification Results for the 18 Chemicals Evaluated. 

Binding Classification Number of Chemicals

Interactive 16

Not interactive 0

Equivocal 2

(iv) Performance Criteria

ER Guideline performance criteria for the saturation binding experiment are provided in 

Table 5. The performance criteria are provided: (i) to ensure that there are adequate numbers of 

ERs (Bmax) in the uterine cytosolic preparations; (ii) to ensure that the ERs have the appropriate 

affinity (Kd) for [

3

H]-17β-estradiol and (iii) to serve as guidance in identifying competitive runs 

that provide parameters in the preferred ranges. 

Table 5. Performance Criteria for Saturation Binding

Parameter Value Range

Bmax 10-150 fmol/100 μg protein

Kd 0.03 – 1.5 nM

Non-Specific Binding (NSB) <50% of specific binding at maximum concentration

Saturation Binding: The saturation binding data were included within the ER binding study 

reports from five laboratories (representing six chemicals). However, saturation binding data 

were not included with the original ER binding study reports that were submitted by two of the 

testing laboratories (Laboratories 1 and 2). Upon request by the Agency, summary saturation 

binding information was subsequently submitted to support the chemicals. The available data 

from the saturation binding assays indicate that binding reached a plateau and that the Scatchard 

plots for each run were generally linear, although several of the runs showed curving of the plots. 

Some laboratories used a single cytosol preparation to test multiple chemicals. In those cases, a 

single saturation binding experiment was conducted for the cytosol batch and the saturation 

binding data (or a summary of the data) for that batch were submitted for more than one 

chemical. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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As shown in Table 6, all three binding runs for Chemicals A and K and one binding run 

for Chemical G were performed using the same cytosol preparation (Cytosol A). Three runs for 

Chemicals B and 2 runs for Chemical G were tested with Cytosol B; Chemicals V and L were 

tested with Cytosol C; Chemicals E, F, I, J and N were tested with Cytosol D; and Chemicals D 

and M were tested using Cytosol E. The remaining five studies submitted saturation binding 

data that were used for only one chemical. 

Table 6. Cytosolic Preparations Used for More than One Chemical.

Laboratory Cytosol ID Chemicals Tested

1 A A, K (all 3 runs); 1 run for G

1 B B (all 3 runs); 2 runs for G

1 C V, L (all runs)

2 D E, F, I, J, N (all runs)

3 E D, M

Figure 1 below represents a typical saturation binding curve. The measured values for 

total binding and non-specific binding as well as the calculated values for specific binding (total 

binding minus non-specific binding) are shown for three independent runs. 

Figure 1. Saturation Binding Curves.

TB= Total binding; NSB= Non-specific binding; SB= Specific binding; E2= Estradiol
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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The saturation binding performance criteria for Bmax and Kd were met by all seven testing 

laboratories for the cytosol preparations used for the 18 chemicals. Non-specific binding (NSB), 

when reported as a value or depicted in graphical form, was less than 24% of specific binding at 

the highest concentration. A summary of the performance criteria for the saturation binding 

experiments is presented in Figure 2. 

Figure 2. Performance Criteria Results for the Saturation Binding for Cytosolic 

Preparations. 

 

Competitive Binding:

Performance Criteria for Competitive Binding

Guideline performance criteria for positive controls for the competitive binding 

experiment are provided in Table 7. The performance criteria are provided to serve as guidance 

in identifying competitive runs that provide parameters in the preferred ranges and to ensure that 

assay is responding properly.

Table 7. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves 

for Positive Control.

Parameter Unit

Estradiol

Test Guideline Criteria

Range of Non-performance 

Lower

Limit Upper Limit

Loge(Syx)

(i.e. Loge(Residual 

Std. Dev.)

- NA 2.35 Values were not reported for 3 chemicals.

All others met the criteria 

Bottom plateau level % binding -4 1 -5 to 1.1 

Top plateau

level % binding 94 111 93- 111

Hill Slope Log10 (M)-1 -1.1 -0.7 -2.0 to -0.63

Note: 19-norethindrone was used as a positive control. None of the testing laboratories used Norethynodrel. 

NA= not applicable

18

18

Bmax 

Kd (nM)

ER Saturation Binding 

Criteria

Met
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Figure 3 below shows typical competitive binding curves for the positive control (19-

norethindrone), strong positive control (17β-estradiol), negative control (octyltriethoxysilane) and 

a test chemical that was classified as not interactive in this assay.

Figure 3. ER Competitive Binding Curves

All competitive binding performance criteria for the positive control, 17β-estradiol, were 

met for 11 of the 18 chemicals. For the remaining chemicals, the laboratory did not meet one or 

more of the 17β-estradiol positive control performance criteria; however, in most cases the 

values were only slightly outside the expected range and did not impact the interpretation of the 

study. The Loge(Syx), with an upper maximum performance criteria of 2.35, was met for 16 of 

the 19 compounds, but was not reported for three studies (Chemicals D, M, P). The top % 

binding (94 to 111%) was generally met for all reviewed studies; the exceptions included only a 

single run for three different chemicals (D, L, V), each only slightly below (93%) the lower 

recommended limit of 94%. The bottom % binding (−4 to 1%) was also slightly below range for 

a single run in two studies (L, V; −5%) and above range for single runs with for two other 

chemicals (Chemicals C and K; 2.1 and 5%, respectively). The Hill slope was slightly below the 

recommended range (−1.1 to −0.7) in one of the five runs in one study (Chemical P; −1.126), and 

the Hill slope performance criteria were not met in any run for two studies from the same 

laboratory (Chemicals M, D; −2 to −1.7) The Hill slope was greater than -1.1 in one or two runs 

in two other studies: 2 of 3 runs in the study in Chemical K (−1.4 and −1.7), and in a single run 

in the study for Chemical A (−1.6). A summary of the performance for the 17β-estradiol

positive control is presented in Figure 4.
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 31 of 160 

Figure 4. Performance Criteria Results for the Strong Positive Control. 

Although the Guideline recommends the use of norethynodrel as the weak positive 

control ligand, it allows the use of 19-norethindrone as a replacement if norethynodrel is 

unavailable. All 18 of the reviewed studies used 19-norethindrone in place of norethynodrel. 

The Guideline does not contain performance criteria for 19-norethindrone. 

The single performance criterion for the negative control, octyltriethoxysilane, requires 

that the displacement of radioligand from ER be <25%. This criterion was met in all but three 

cases (Chemicals L and V, which shared control data; and Chemical Q) that were all conducted 

by the same laboratory (Laboratory 1). For these three chemicals, the negative control criterion 

was met in one run and was not met in the two remaining runs, with displacement values ranging 

from 51.8 to 58.5%. NSB (no criteria provided for NSB in the Guideline) was generally less 

than 10%. 

(v) Issues or Problems with Laboratory Performance 

One of the issues identified in the laboratory performance of the ER Binding Assay was 

where two sets of negative control runs by Laboratory 1 did not meet the negative control 

performance criterion in two out of three assay runs (data were used for three separate study 

compounds). Two of the compounds, Chemical L and T, shared the same control data, and 

Chemical Q had different control data. The assays with Chemicals L and T used the same 

cytosol preparation and the Chemical Q used a cytosol prepared on a different date; however, the 

other compounds assayed with either of these cytosol preparations did not show a similar issue 

with the negative control. Although the study report for Chemical T stated that precipitation was 

observed at an octyltriethoxysilane concentration of 10−3 M in all three competitive binding runs 

(resulting in exclusion of this concentration), the study reports for Chemical L (which shares 

control data with Chemical T) and Chemical Q stated that precipitate was not observed at any 

14

16

15

16

2 

3 

4 

3 

3 

Hill Slope

Top plateau level

Bottom plateau level

Loge(Syx) (i.e. Loge(Residual Std. Dev.)

17β-Estradiol Performance Criteria

Met (all runs) Did not meet in one run

Did not meet in >1 run Not reported
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concentration of octyltriethoxysilane. Although an explanation for the >25% displacement is 

proposed in the two latter compound study reports, a positive response with the negative control 

and/or precipitation at a concentration that has been validated by the EPA may indicate an issue 

with the laboratory performance for those assay runs. Otherwise, only minor deficiencies were 

noted in the 18 studies, and few of these impacted study interpretation. In general, the data were 

considered reliable. 

(vi) Overall conclusion

The assay performance was generally consistent across all 18 test compounds. Although 

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

laboratories performance of the ER binding assays was generally acceptable. 

b. ERα Transcriptional Activation

(i) Background 

The Estrogen Receptor Transcriptional Activation Assay (OSCPP 890.1300)7

 is used to 

determine if a chemical interacts with the endocrine system by functioning as an ERα ligand and 

activating transcription of an estrogen-dependent reporter gene. This protocol is validated for the 

detection of an agonistic response. The assay exclusively uses the stably-transfected hERαHeLa-9903 cell line derived from human cervical tumor cells (Japanese Collection of Research 

Bioresources [JCRB] Cell Bank). The cells were transfected with an expression construct 

encoding human ERα and a reporter construct containing an estrogen response element and 

encoding firefly luciferase; the bioluminescence product is evaluated to determine the agonist 

response. The cells are required to be grown for more than one passage from frozen stock, but 

not more than 40 passages. The stability of the cells is monitored with four reference chemicals: 

17β-estradiol (strong agonist), 17α-estradiol (weak agonist), 17α-methyltestosterone (very weak 

agonist), and corticosterone (negative control). A group of ten compounds should be used to 

evaluate the proficiency of the cells prior to initiation of the assay runs. The proficiency 

compounds include positive [diethylstilbestrol, 17-ethynyl estradiol, hexestrol, genistein, estrone, 

butyl paraben, and 1,3,5-tris(4-hydroxyphenyl)benzene] and negative [dibutyl phthalate, 

atrazine, and corticosterone] chemicals. The proficiency test should be performed in duplicate 

on two different days (and include the three positive reference chemicals).

The assay is performed under cell culture conditions in 96-well plates as described in 

Guideline 890.1300. After allowing the cells to attach for 3 hours, cells are exposed to multiple 

concentrations of the test chemical (in triplicate wells) for 20-24 hours. Medium is then 

removed; luciferin substrate is added; and the luciferase activity is measured using a 

 

7

 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1300: Estrogen Receptor Transcriptional Activation 

(Human Cell Line (HeLa-9903). Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and 

Toxic Substances (7101). EPA 740-C-09-006. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2009-0576-0006
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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luminometer. The amount of light emitted is proportional to the potency and/or concentration of 

the estrogen (or estrogenic compound). A test chemical is determined to be positive if the 

maximum response induced by the test chemical (RPCMax) is equal to or exceeds 10% of the 

response of the positive control in at least two of two or two of three runs. 

(ii) Strengths and Limitations of the Assay

The use of an immortalized cell line in this assay eliminates the use of live animals for 

this purpose. The assay can detect chemicals that bind ERα and activate transcription of an ERαdependent gene. The activity of the test compound can be quantified and compared to the activity 

of the well characterized ERα ligand, 17β-estradiol. 

This assay was validated for ER agonist activity only (does not identify ER antagonists). 

The construct used in this cell line identifies only chemicals that activate ERα (does not detect 

activation of ERβ or membrane-associated ERs). Also, luminescence signals in excess of signals 

produced by the known endogenous ligands have been reported at phytoestrogen and synthetic 

estrogen concentrations in the micromolar range. The guideline includes an additional test cotreating cells with test chemical and a known ERα antagonist, to be used when a false positive is 

suspected. Finally, the HeLa-9903 cell line exhibits limited metabolism so the assay may not 

adequately evaluate metabolites for potential estrogenic activity.

(iii) Observations of Test Chemicals

Nineteen compounds were used to evaluate the performance of the estrogen receptor 

transcriptional activation assay (ERTA). Test chemical purity was >89%. All of the test facilities 

used the recommended hERα-HeLa-9903 cell line from JCRB, and the cell passages used for the 

test runs were maintained between 1 < n < 40. All test materials were dissolved in either DMSO 

or EtOH and final vehicle concentrations in the assays did not exceed 0.1% meeting Guideline 

requirements. Only a single chemical (Chemical N) was tested up to the test limits of 10-3 M; all 

other chemicals were limited by solubility and/or cytotoxicity. Selected test concentrations 

ranged from 10-3.3 and10-3.5 to 10-13 M. Most test facilities spaced the test chemical 

concentrations by orders of whole logs; however, one chemical was tested with half log 

separations (Chemical L) after solubility and cytotoxicity issues were observed in the first assay 

run. 

All test facilities, with the exception of Laboratory 1 included the appropriate figures 

and/or tables for calculation of the positive control (PC, 1 nM 17β-estradiol) values (PC10) 

relative to the vehicle control (VC). Laboratory 1 did not report if PC10 values were greater than 

1 plus 2 standard deviations (SD) of the concurrent VC; however, PC10 and 1 plus2 SD values

could be determined from the reported data and did conform to the Guideline. 
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Proficiency Test

The Guideline-required proficiency testing of the cell passage(s) to be used for assay runs 

was conducted, but reported differently, by each testing facility. A few laboratories (3/8) 

provided proficiency data and classification per guideline requirements. However several

laboratories (4/8) provided some data or assessment that did not completely meet guideline 

requirements. Also, Laboratory #1 did not provide proficiency testing information initially (see 

below). 

• None of the reports issued by Laboratory 1 (Chemicals: A, B, G, K, L, P, Q, S and V) 

contain any information regarding proficiency testing, other than a statement claiming 

that all study personnel had demonstrated proficiency in performing the assay. 

Subsequently, on request, a summary proficiency data report was submitted to the 

Agency. This report included the methodology and qualitative results, but no data or 

figures, from replicate tests for the ten proficiency chemicals (over three assay runs with 

three different passages of cells). Several criteria for the reference chemicals included 

with each proficiency run were not met. 

• Reports issued by Laboratory #2 (Chemicals: E, F, I, J, N and O) stated that proficiency 

testing was performed and that the results demonstrated proficiency; however, the data 

and results were included in a separate, unreferenced report. 

• Reports issued by Laboratory #3 (Chemicals D and M) included summary and 

classification data from two proficiency test runs, but did not include the individual data

While nine of the ten proficiency chemicals were classified accurately, corticosterone 

was positive in the first run and negative (correct) in the second run. 

• Laboratory #4 (Chemical C) did not include any proficiency data or a summary, but 

stated that a separate study was conducted and the results met guideline requirements. 

• Laboratory #5 (Chemical H) provided curve fit, RPCmax data and classifications for 

proficiency chemicals (2 to 4 runs), as an appendix to the main study report.

Transcriptional Activation Test 

Of the 19 chemicals tested, the RPCmax values were <10% for 14 chemicals that were 

classified as negative (except for one, Chemical Q, that was considered inconclusive). Of the 

five chemicals that had RPCmax values >10% in at least two runs, Chemical E (19.1%),), 

Chemical F (18.0%), Chemical I (25.4%) and Chemical L (29.7%), and Chemical F (18.0%) 

were considered positive agonists. Chemical B (RPCmax = 115%) was suspected to be a false 

positive because the ERα antagonist ICI 182,780 did not inhibit the response. A PC50 value was 

determined only for Chemical B, and PC10 values were reported for three of the chemicals 

(Chemicals E, F, and I) with a positive response. Neither PC50 nor PC10 were reported for 

Chemical L. LogEC50 and/or Hill Slope estimates were determined for all positive chemicals

except Chemical L, as well as for Chemical M (negative response). A summary of the results is 

shown in Table 8. 
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Table 8. Classification of the 19 Chemicals Evaluated in the ERTA. 

Assay Classification Number of Chemicals

Positive 5*

Negative 13

Inconclusive 1

*Positive includes one suspected false positive (Chemical B).

(iv) Performance Criteria

The performance criteria for the four reference compounds were determined in 

conjunction with each test chemical. The ranges for each reference compound met the Guideline 

required ranges; Laboratory 1 included an additional lower concentration (beyond the lowest 

Guideline-recommended concentration) for each reference compound. This additional low 

concentration had no impact on the study. 

Few issues were observed for any testing facility regarding the negative reference 

compound, corticosterone, namely, cytotoxicity at the high concentration (10-4 M) observed by 

Laboratory #1 during five test chemical runs (Chemicals: B, G, K, and L). Corticosterone 

showed a slight positive response in 1 of 3 runs for Chemical C by Laboratory #4. 

All response curve parameters for the strong estrogen agonist (17β-estradiol) were met 

for 10 of the 19 chemicals. Nevertheless, out-of-range values were very close to the test 

guideline ranges except the LogEC50 for Chemical O and Chemical F as noted below: 

• LogEC50 (-9.4 versus the criteria range of -11.3 to -10.1) and Hill Slope (0.3 versus the 

criteria range of 0.7 to 1.5) estimates were out of range for two of four runs for Chemical 

O and one of the four runs for Chemical F tested by Laboratory #2 that shared the same 

reference compound data. 

• Hill Slope estimates were out of range (0.4 versus the criteria range of 0.7 to 1.5) for one 

out of three runs for three chemicals (Chemicals I, J, N) tested by Laboratory #1 that 

shared the same reference compound data. 

• LogEC50 (-9.7), Hill Slope (0.66), and logPC10 (-10.9 versus the criteria range of -11.4 to 

-10.1) estimates were out of range for one out of three runs for Chemical C tested by 

Laboratory #4 

• The LogPC50 estimate (-11.5) was out of range for one out of two runs for Chemical A 

tested by Laboratory #1. 
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• LogEC50 (-11.5 and -11.4) and LogPC50 (-11.7 and -11.5) estimates were out of range for 

Chemicals G and L in two out of two valid runs tested by Laboratory #1 that shared the 

same reference compound data; Hill Slope (1.6) was out of range for one run only. 

Response curve parameters for the weak estrogen agonist (17α-estradiol) were met for 

only 5 of the 19 chemicals. Nevertheless, out-of-range values were very close to the guideline 

ranges except for Chemicals M, D and Q as noted below:

• LogPC50 (-9.9 versus validated range of -9.6 to -8.1), LogPC10 (-11.0 versus criteria range 

of -10.7 to -9.3), and LogEC50 (-10.0 versus criteria range of -9.6 to -8.4) estimates were 

out of range for Chemicals G and L, in one out of two valid runs tested by Laboratory #1 

that shared the same reference compound data.

• LogPC50 (-10.1), LogPC10 (-10.9), and Hill Slope (0.5 versus criteria range of 0.9 to 2.0) 

estimates were out of range for three out of four runs, for Chemicals O and F tested by 

Laboratory #2 that shared the same reference compound data. 

• LogPC10 estimate (-11.9) was out of range for one run and Hill Slope estimates (0.3 and 

0.8 versus 0.9 to 2.0) were out of range for both two runs, for Chemicals D and M that 

were tested by Laboratory #1 that shared the same reference compound data. 

• Hill Slope estimates (0.6) were out of range for one out of three runs for Chemicals I, J

and N tested by Laboratory #2 that shared the same reference compound data, as well as 

one out of three runs (0.7) for Chemical E (Laboratory #2), and one out of two runs (2.5) 

for Chemicals Q and P (Laboratory 1). 

• LogEC50 estimates (-8.2) were out of range for two of three runs for Chemical C that was 

tested by Laboratory #4. 

All of the test facilities had difficulties meeting the Guideline ranges for the very weak 

estrogen agonist, 17α-methyltestosterone. Often, the logPC10 and logPC50 estimates were less 

than the lower limits described in the Guideline, indicating increased sensitivity of the assay 

system to weak agonists during tests for 11 of the 19 chemicals. Alternatively, eight test (from a 

total of three laboratories) had logPC10 and/or logPC50 values for methyltestosterone that were 

above the Guideline range upper limit (Chemicals P and S, Laboratory 1; Chemical C, 

Laboratory 4; Chemical H, Laboratory 5) or were not calculable due to cytotoxicity (Chemicals 

B, K, Q and V; Laboratory #1). 

(v) Issues or Problems with Laboratory Performance

Majority of the deviations are considered minor. However, cytotoxicity observed with 

corticosterone in Laboratory 1, and cytotoxicity and high logPC10 and/or logPC50 estimates 

observed with 17α-methyltestosterone in Laboratories #1, 4 and 5 impact interpretations for eight 

chemicals.
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(vi) Overall conclusion

While performance criteria were generally not met for the majority of the chemical, outof-range values were often close to the Guideline ranges. All but one chemical were classified 

as either negative or positive in the assay, however inability to fulfill the performance criteria 

may affect the interpretation of at least 8 of 19 chemicals.

c. AR Binding

(i) Background 

The Androgen Receptor Binding (Rat Prostate Cytosol) (OSCPP 890.1150)8 Assay is 

used to determine if a chemical competes with the reference [3

H] androgen ([

3

H]-R1881) for 

binding of the cytosolic androgen receptor (AR) from rat ventral prostate tissue homogenate. 

The assay measures the ability of radiolabeled [3

H]-R1881 to interact with the AR in the 

presence of increasing concentrations of a test chemical. Rat prostate cytosol containing AR is 

incubated in test tubes with increasing concentrations of the test substance and an aliquot of 

radiolabeled [3

H]-R1881. If the test substance interacts with the hormone binding domain of the 

AR, less radiolabeled [3

H]-R1881 can bind to the AR producing a descending concentrationresponse curve.

The Androgen Receptor Binding Assay consists of a saturation binding experiment and a 

competitive binding experiment. The purpose of the saturation binding experiment is to ensure 

that the AR in the cytosolic preparation is present in reasonable numbers and is functioning with 

the appropriate affinity for the radiolabeled reference ligand. AR saturation binding experiments 

measure total and non-specific binding of increasing concentrations of [3

H]-R1881 under 

conditions of equilibrium. From these measurements, specific binding at each concentration can 

be calculated. A graph of specific [

3

H]-R1881 binding versus radioligand concentration should 

reach a plateau indicative of saturation of the AR with the radioligand. In addition, analysis of 

the data should document the binding of the [3

H]-R1881 to a single, high-affinity binding site 

(i.e., Kd = 0.685 to 1.57 nM and a linear Scatchard plot). The competitive binding assay 

measures the binding of a single concentration of [3H]-R1881 in the presence of increasing 

concentrations (10-10 to 10-3 M) of a test substance. Each test concentration is evaluated in 

triplicate (three concurrent replicates) within one run. Three independent (non-concurrent) runs 

are performed for each chemical tested. 

Each run also includes concurrent solvent (vehicle), and weak and strong positive 

controls. The solvent control indicates the total binding that is possible under the conditions of 

that run. A weak positive substance (dexamethasone) is included to demonstrate the sensitivity 

 

8 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1150: Androgen Receptor Binding (Rat Prostate 

Cytosol). Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). 

EPA 640-C-09-003. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0003. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 38 of 160 

of each experiment and to allow an assessment of variability in the conduct of the assay across 

time. The reference standard (unlabeled R1881) is also included to ensure that the run has been 

properly performed, and to allow an assessment of variability in the conduct of the assay across 

time.

Although the classification terminology used in the AR binding Test Guideline differs 

from the ER binding assay, the term “binder” in the AR assay and “interacting” in the ER assay 

are considered equivalent for the purposes of chemical classification. 

Binder Classification

Results from each run of the competitive binding assay are used to classify the test 

chemical as a “binder”, “non-binder”, “equivocal”, or “untestable”. The classification criteria 

are summarized below.

If the data fit a 4-parameter nonlinear regression model, the test chemical would typically be 

classified as:

Binder: 

A chemical is classified as a “binder” if the average curve for the test chemical across runs 

displaces 50% or more of the radioligand.

Equivocal: 

A chemical is classified as “equivocal” if the average lowest portion of the curves across runs is 

between 50% and 75% activity, or if the slope of the curve falls outside the range for the weak 

positive control (-0.6 to -1.4). 

Non-Binder: 

A chemical is classified as a “non-binder” if the average lowest portion of the curves across runs 

is greater than 75% activity, or the data do not fit the model. 

Untestable: 

A chemical is classified as “untestable” if the test compound is not soluble above 10-6 M and the 

binding curve does not cross 50%. 

(ii) Strengths and Limitations of the Assay 

Environmental chemicals that compete with the endogenous AR have the potential to 

either induce hormone-dependent processes (agonist) or block normal hormone function by 

preventing endogenous hormone from binding to the receptor (antagonist). There is a high 

degree of DNA sequence conservation in the AR across mammalian phylogenetic lines (Kelce et 

al, 1998; Betney and McEwan, 2003). Therefore substances that bind the AR from rats are 

presumed to be capable of binding the AR in humans and other species. The assay cannot 

distinguish between AR agonism and AR antagonism. 
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(iii) Observations of Test Chemicals

Seventeen compounds were evaluated in the AR Binding Assay. Six different 

laboratories were involved in conducting the 17 AR Binding assays, with the majority of the 

assays being conducted by Laboratory 1 (7 tests) or Laboratory 2 (4 tests). Each assay utilized 

ventral prostate tissue from castrated, male Sprague-Dawley rats as the source of cytosolic AR. 

The cytosol was prepared and stored as specified in the Guideline. The protein concentrations of 

the cytosolic preparations ranged from 4.3 to 8.8 mg protein/mL, and the final protein 

concentrations used in the assays were 1.6 - 6.57 mg/mL, or 0.48-1.97 mg protein/assay tube. 

The purity of the test chemicals used in the assays was >89%. DMSO and EtOH were used as 

the solvent vehicles for the test chemicals in all assays. Solvent concentration limits were met 

for all compounds (max: 3%). Test chemicals were initially tested at serial log concentrations 

from 10-10 to 10-3 M (as recommended in the Test Guideline), with the exception of one chemical 

(Chemical S) which was tested at a maximum of 10-5 M based on preliminary solubility findings. 

No solubility issues were noted for nine of the compounds tested at up to 10-3 M. However, 

seven of the test chemicals were insoluble in the assay at a concentration of 10-3 M, and two of 

these compounds were also not soluble at 10-4 or 10-5 M (Chemical G and C, respectively). 

Similar to what was observed in the ER Binding Assay, when precipitation was observed, 

Laboratory 1 added additional concentrations at the lower end of the concentration range (10−11

or 10−12 M), rather than utilizing mid-log concentrations at the upper end of the concentration 

range (10−4.5 M or 10−5.5 M) which would better characterize the response curve and provide 

more confidence in the interpretation of the results those runs. Based on the results of the 17 

assays, 10 of the test compounds were classified as non-binders, and three compounds 

(Chemicals A, H and K) were classified as “binders”, with Log IC50 values of -5.0 to -3.8 M. 

Another three compounds were classified as equivocal, based on minimum 3H-ligand binding 

between 50 and 75% (Chemicals I and P) or because of an excessively steep Hill Slope 

(Chemical J). One compound (Chemical C) was classified as untestable as it was not soluble 

>10-6 M and binding was >75% at all lower concentrations. A summary of the classification of 

the chemicals is shown in Table 9. 

Table 9. AR Binding Classification Results for the 17 Chemicals Evaluated 

Binding Classification Number of Chemicals

Binder 3

Non-binder 10

Equivocal 3 

Untestable 1 

 
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(iv) Performance Criteria:

Saturation Binding

The AR Binding Guideline performance criteria for the saturation binding experiment are 

provided in Table 10. The performance criteria are provided to serve as guidance in identifying 

competitive runs that provide parameters in the preferred ranges and to ensure that there are 

adequate numbers of ARs in the prostate cytosolic preparations and that the ARs have the 

appropriate affinity for [

3

H]-R1881. 

Table 10. Performance Criteria for Saturation Binding 

Parameter Value Range

Kd 0.685 – 1.57 nM 

Non-Specific Binding (NSB) <20% of specific binding at 

maximum concentration

Saturation binding data were not always included with the original AR binding study 

reports; however, saturation binding information was subsequently submitted to the Agency 

upon request. Saturation binding data were often submitted as summary data in graph and 

tabular form without the accompanying raw data. Some laboratories used a single prostate 

cytosol preparation to test multiple chemicals. In those cases, a single saturation binding 

experiment was conducted for the cytosol batch and the saturation binding data (or summary 

data) for that batch were submitted for more than one chemical. As shown in Table 11, 

Chemicals E, I, J, and N were tested using the same cytosol preparation (Cytosol A); Chemicals 

B, G, K, U and V were tested with Cytosol B; and Chemicals D and, M, were tested using 

Cytosol C. For Chemicals A and L, one competitive binding run was conducted with Cytosol B, 

with Cytosol D and a third run was conducted using a different cytosol prep (Cytosol E). The 

remaining four studies (Chemicals C, H, P and S) submitted saturation binding data used for only 

one chemical. 
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Table 11. Prostate Cytosol Preparations Used for More Than One Chemical.

Laboratory Cytosol ID Chemicals Tested

2 Cytosol A E, I, J, N (all 3 runs)

1 Cytosol B B, G, K, U, V (all 3 

runs)

3 Cytosol C D, M (all 3 runs)

1 Cytosols

B, D and E A, L (1 run per cytosol)

The available data from the saturation binding experiments indicated that binding reached 

a plateau. The saturation binding performance criteria for Kd (0.685 to 1.57 nM) was not met for 

the cytosol prepared by Laboratory 2 (Cytosol A) (Kd: 0.464 nM), which was utilized in assays 

for 4 chemicals. The Kd saturation binding performance criteria were also not met for two 

cytosols prepared by Laboratory 1, Cytosol preparation D (0.614 nM) and Cytosol E (0.613 nM). 

A third batch of cytosol prepared by Laboratory 1, Cytosol B, met the criteria for Kd (1.080 nM). 

Additionally, the saturation binding performance criteria for Kd was not met by Laboratory 5 for 

the cytosol preparation used for Chemical H (0.65 nM). The saturation binding performance 

criteria were met for the rat prostate cytosol prepared by Laboratory 3 (2 studies), and the 

individual studies conducted by the two remaining laboratories. A summary of the performance 

for the saturation binding experiments is presented in Figure 5. This figure represents the 

saturation binding performance for 6 different testing laboratories and 8 different cytosol 

preparations. The non-specific binding criterion was met for all cytosol preparations. 

Figure 5. Performance Criteria Results for the Saturation Binding for Cytosolic 

Preparations. 

 

18

9 

0 

9 

0% 20% 40% 60% 80% 100%

Non-Specific Binding (NSB)

Kd (nM)

AR Saturation Bindng

Met Did not meet
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Figure 6 below represents an AR saturation binding curve where the performance criteria 

were met. The measured values for total binding and non-specific binding as well as the 

calculated values for specific binding (total binding minus non-specific binding) are shown for 

one independent run. 

Figure 6. Saturation Binding Curves.

TB= Total binding; NSB= Non-specific binding; SB= Specific binding

(v) Performance Criteria:

Competitive Binding

Guideline performance criteria for positive controls for the competitive binding 

experiment are provided in Table 12. The performance criteria are provided to serve as guidance 

in identifying competitive runs that provide parameters in the preferred ranges and to ensure that 

assay is responding properly.

Table 12. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves 

for Strong and Weak Positive Controls. 

Parameter Unit R1881 Dexamethasone

Lower Limit Upper Limit Lower Limit Upper Limit

Bottom plateau level % binding -2.0 2.0 -12 12

Top plateau level % binding 82 114 87 106

Hill Slope Log10 (M)-1 -1.2 -0.8 -1.4 -0.6
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Figure 7 below shows representative competitive binding curves for the positive control 

(dexamethasone), strong positive control (R1881) and a test chemical that was classified as a 

binder in this assay. 

Figure 7. Competitive Binding Curves

The performance criteria for the Competitive Binding Assays were generally met with 

only a few exceptions. For the positive control, R1881, the top % binding (82 to 114%) was met 

for all reviewed studies, and bottom % binding (-2.0 to 2.0%) was met except in Laboratory 2 

studies (Chemical E and I) and the Laboratory 5 study (Chemical H), which had values slightly 

below the bottom range (-2.6 to -2.1%). Except for one run (-1.588) in the study conducted for 

Chemical P, the Hill Slope performance criteria for R1881 (-1.2 to 0.8) were met. The Log IC50

for the reference androgen ranged from -10.1 to -8.6 Log M in runs across all the assays, but was 

typically -9.9 to -9.0 log M. 

For the weak positive control, dexamethasone, the performance criteria for top % binding 

(87 to 106%) and bottom % binding (-12 to 12%) were met in all 17 studies, and the Hill Slopes 

for dexamethasone (-1.2 to -0.6) also met the performance criteria (-1.4 to 0.6). The Log IC50 for 

the weak positive control were relative consistent across all the assays, ranging from -4.6 to -4.0 

log M. The % relative binding affinity (%RBA) for dexamethasone compared to R1881 was 

0.0013-0.0071%, with the range typically being 0.0021-0.0036%. 

A summary of how well the positive controls met the performance criteria is shown in 

Figure 8 and Figure 9. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 44 of 160 

Figure 8. Performance Criteria Results for Strong (R1881) Positive Control. 

Figure 9. Performance Criteria Results for Weak (Dexamethasone) Positive Control. 

(vi) Issues or Problems with Laboratory Performance

The performance criteria for the saturation binding experiments were not consistently 

met by two of testing laboratories. In addition, for compounds with limited solubility at 10-4

to 10-3 M, the addition of 1⁄2 log concentrations at the upper end of the test concentration 

range would provide more useful information than including testing concentrations at the 

lower end (<10-10 M). Otherwise, only minor deficiencies were noted in the 17 studies, and 

none of these had an impact on study interpretation.

17

18

15

1 

3 

Hill Slope

Top plateau level (%)

Bottom plateau level (%)

Competive Binding - R1881 

Met Did not meet

18

18

18

Hill Slope

Top plateau level (%)

Bottom plateau level (%)

Competitive BindingDexamethasone 

Met Did not meet
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 45 of 160 

(vii) Overall conclusion

The laboratory performance of the assay was generally consistent across all 17 test 

compounds. Although several laboratories generated rat prostate cytosolic preparations that did 

not meet the performance criteria for the saturation binding experiment, when these same 

preparations were used in the competitive binding assays, the preparations were able to identify 

test chemicals that bound to the AR (binders) and the control chemicals responded appropriately 

as indicated by meeting (or only slightly exceeding) the competitive binding performance criteria 

for R1881 and dexamethasone. Overall, the laboratories performance of the AR binding assays 

was generally acceptable and the data were considered reliable.

d. Steroidogenesis 

(i) Background 

The Steroidogenesis Assay (OCSPP Guideline 890.15509

) is an in vitro screen intended to 

identify xenobiotics 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 [follicle-stimulating hormone receptor (FSHR) and luteinizing 

hormone receptor (LHR)] are activated. The steroidogenesis assay is not intended to identify 

substances that affect steroidogenesis due to effects on the hypothalamus or pituitary gland (i.e. 

HPG axis). The assay utilizes a human H295R adreno-carcinoma cell line (NCI-H295R) which 

expresses genes that encode all the key enzymes needed for steroidogenesis as shown in Figure 

10. The assay is intended to identify chemicals that have the potential to induce or inhibit the 

production of testosterone and/or estradiol. 

 

9 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1550: Steroidogenesis (Human Cell Line - H295R). 

Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101).

EPA 740-C-09-004. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0004. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 46 of 160 

Figure 10. Steroidogenic Pathway in H295R cellsa. 

a

Taken from OECD (2011), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD 

Publishing. Gray background indicates corticosteroid pathways/products.

Sex steroid pathway/products are circled. CYP= cytochrome P450; HSD = hydroxysteroid hydrogenase;

DHEA= dehydroepiandrosterone. 

The assay is typically conducted using 24-well culture plates. H295R cells are plated 

under standard culture conditions and allowed to acclimate for 24 hours. The cells are then 

exposed for 48 hour to seven concentrations (plus solvent control) of the test chemical (10-4 - 10-

10 M) in triplicate. A separate 24-well plate containing solvent control and positive controls for 

steroid hormone inhibition (prochloraz) and induction (forskolin) are included as a quality 

control (QC) plate for each run. A minimum of three runs is recommended. At the end of the 

exposure period, the culture medium is removed, cell viability is evaluated and the 

concentrations of estradiol and testosterone in the medium are measured. Laboratories may use 

the hormone measurement system of their choice to analyze production of testosterone and 

estradiol. Data are expressed as fold change relative to the control. The Guideline defines a 

chemical to be positive in this assay if the fold induction is statistically significantly different 

from the solvent control at doses that fall within the increasing or decreasing portion of the 

concentration-response curve. Statistically significant (p≤ 0.05) differences at concentrations 

that do not follow a concentration-response curve may be due to random effects; such results are 

considered equivocal. Concentrations exceeding the limits of solubility or at cytotoxic (<80% 

cell viability) concentrations are not included in the data analyses.
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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(ii) Strengths and Limitations of the Assay

The assay uses a cell line available from the American Type Culture Collection (ATCC). 

It can respond with both increases and decreases in the production of both testosterone and 

estradiol and allows direct assessment of cell viability within the assay to distinguish effects on 

steroidogenesis due to chemical interaction with hormone pathways from effects due to 

cytotoxicity. (OECD Guideline). The inclusion of the H295R Steroidogenesis Assay in the 

battery provides some redundancy with the aromatase assay and it also addresses multiple targets 

in the steroidogenic pathway (SAP, 2008). The total metabolic capacity of the cell line is 

unknown. H295R cells possess enzymes that are capable of catalyzing the production of 

glucocorticoids and mineralocorticoids so chemicals that alter production of these hormones 

could influence the levels of testosterone and estradiol production. 

(iii) Observations of Test Chemicals

Eighteen compounds were used to evaluate the test laboratories performance of the 

Steroidogenesis Assay. The purity of the test chemicals used in the assays was >89%. Five

different laboratories were involved in conducting the 18 steroidogenesis assays, with the 

majority of the assays being conducted by Laboratory 1 (eight chemicals) or Laboratory 2 (six 

chemicals). All assays were performed with H295R cells (ATCC CLR-2128) grown in DMEMHam’s F-12 medium in a 24-well plate setup as specified in the Guideline. Due to changes in 

hormone production in H295R cells with increasing cell passages, specific guidance is provided 

in the test guideline to ensure that the cells are responsive in this assay. 

In the 18 studies evaluated, the cells were all frozen at passage 5 and cultured at least an 

additional four passages before use in the assays. The total number of passages was ≤10 in all 

but two assays. Passage 11 cells were used in two assays (Chemicals E, F), but it was stated that 

since the QC plate met the criteria for hormone production, induction, and inhibition, the assay 

results were acceptable. All assays used forskolin as a positive control for hormone induction (1 

and 10 μM) and prochloraz as a positive control for hormone inhibition (0.1 and 1 μM). DMSO 

was used as the solvent in all but one assay. EtOH was used as the solvent for the remaining 

assay (Chemical S). The concentration of solvents was acceptable for all studies.

One laboratory (Laboratory 3) provided details of the laboratory proficiency test for two 

chemicals and Laboratory 4 provided details of an abbreviated proficiency test (single high 

concentrations of forskolin and prochloraz) for Chemical C; the results indicated that the ED50

values were within ranges specified by the guidelines. All other performing laboratories either 

only stated that a laboratory proficiency test was conducted or made no reference to one. All 

assays provided dosing schematics for the test chemicals, and QC plates indicated that the cells 

were seeded at an appropriate concentration (2-3 × 105 cells/mL), and stated that the cells were 

cultured under the appropriate conditions (5% CO2 at 37°C for 24 h) prior to exposure. 

Cytotoxicity was determined by one of three assays: the MTT assay; the modified MTT assay; 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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or the XTT assay. Cytotoxicity was observed in 4 of the 18 steroidogenesis assays, at the highest 

test compound concentrations (1-100 μM).

Hormone concentrations were determined by high pressure liquid chromatography 

coupled with tandem mass spectroscopy (HPLC/MS/MS) in 14 assays; enzyme linked 

immunosorbent assays (ELISAs) were used in the remaining four assays (Chemicals C, D, H, 

M). The limit of quantitation for testosterone (Guideline requirement: 100 pg/mL) ranged from 

25-100 pg/mL for HPLC/MS/MS and from 5-95.7 pg/mL for ELISA (the single exception was a 

MDL [minimum detection level] of 200 pg/mL for Chemical C). The limit of quantitation for 

estrogen/estradiol (Guideline requirement: 10 pg/mL) was 10 pg/mL for HPLC/MS/MS and 5.7-

8.7 pg/mL for ELISA (the single exception was a MDL of 25 pg/mL for Chemical C). 

Acceptable recovery of testosterone and estrogen from spiked samples was demonstrated in all 

but three assays. Hormone cross-reactivity in the ELISAs was checked in two of the four studies 

that used ELISA for determining hormone concentrations. Hormones were extracted from the 

culture medium with either methylene chloride or methyl tert-butyl ether in the HPLC/MS/MS 

studies. 

Test chemicals were tested at concentrations from 100 to 0.0001 μM, with the exceptions 

of Chemicals C and S. Chemical C was tested at concentrations from 37.5 to 0.01 μM in Run 2 

and 20 to 0.01 μM in Run 3, based on results from the previous runs. Chemical S was tested at 

concentrations of 0.1 to 0.00001 μM, as cytotoxicity was observed at 1 μM. No solubility issues 

were noted for 16 of the compounds tested at up to 100 μM; however, precipitation was noted at 

100 μM in one run for Chemical A, and at 10 μM in one run and at 100 μM in all three runs for 

Chemical B.

All eight steroidogenesis assays that were conducted by Laboratory 1 included 10 μM 

22R-hydroxycholesterol (Guideline recommendation is 20 to 40 μM) to increase the basal 

production of estradiol. Background wells (that did not include 22R-hydroxycholesterol) were 

included on the QC plates; the background values were subtracted from the forskolin values to 

correct for the influence of 22R-hydroxycholesterol on forskolin induction. 

(iv) Performance Criteria

Guideline performance criteria for QC plates in the steroidogenesis assay are provided in 

Table 13. The performance criteria are provided to serve as guidance in identifying runs that 

provide parameters in the preferred ranges and to ensure that the cell line is responding properly 

to known inducers and inhibitors. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Table 13. Performance Criteria to be Met on Each QC Plate

Parameter Testosterone Estradiol

Minimum Basal Production 500 pg/mL 40 pg/mL

Basal Production ≥5-times MDL ≥2.5-times MDL

Induction (10 μM forskolin) ≥1.5-times SCa ≥7.5-times SC

Inhibition (1 μM prochloraz) ≤0.5-times SC ≤0.5-times SC

a

This value was presented as ≥2-times SC in the Test Guideline (OCSPP 890.1550). Abbreviations: 

MDL= minimum detectable limit; SC= solvent control

QC Plate Results

All of the QC plates were considered to have had acceptable hormone production for 

basal levels, as well as for induction (forskolin) and inhibition (prochloraz). Basal testosterone 

production met or exceeded the minimal basal level of 500 pg/mL for 13 of the 18 assays (range 

719-10897 pg/mL). For the five plates that did not (Chemicals E, K, N, J, F; all from the same 

laboratory), the lowest concentration range was 433-470 pg/mL. All plates met the guideline 

requirement of having a basal testosterone production ≥5 times the MDL. Two assays by 

Laboratory 1 had initial induction levels with 10 μM forskolin that were below the minimum 

(1.5 times the solvent control [SC]), but all values were ≥2 after subtracting the background 

values. The remaining 16 chemicals had induction levels with 10 μM forskolin that exceeded 

1.5 times the SC. Two assays had inhibition levels with 1 μM prochloraz that were above the 

minimum (≤0.5 times the SC), ranging from 0.52 times to 0.53 times the SC. Basal estradiol 

production met or exceeded the minimal basal level of 40 pg/mL for 13 of the 18 assays (range 

41-370 pg/mL). For the five plates that did not, the lowest concentration range was 26.7-35.5 

pg/mL. All plates met the guideline requirement of having a basal estrogen production ≥2.5 

times the minimum detection level (MDL). All assays had induction levels with 10 μM 

forskolin that were ≥7.5 times the solvent control. Seven assays had inhibition levels with 1 μM 

prochloraz that were above the minimum (0.5 times the SC), ranging from 0.53 times to 0.60 

times the SC. All intraplate coefficients of variation (CVs) were within acceptable limits 

(≤30%), but interplate CVs for the A, D, M, N, Q and SQC plates were ≥30%. Overall, when the 

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

data do not indicate that the assay was not responding appropriately. A summary of the QC plate 

performance data is shown in Figure 11.
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Page 50 of 160 

Figure 11. Quality Control Plate Performance for the 18 Steroidogenesis Studies. 

Test Compound Results

Table 14 summarizes the results of changes in estrogen and testosterone levels. Six test 

compounds increased estrogen levels, with no effect on testosterone production. Estrogen levels 

were significantly (p<0.05) increased by Chemicals, P, D, and F (10 μM and 100 μM). 

Chemical B increased estrogen levels (p<0.05) in one run at 1 μM and in two runs at 10 μM 

(precipitation was noted in one run at 10 μM). Estrogen levels were significantly (p<0.05) 

increased at 100 μM by Chemicals V and O. Testosterone levels were decreased by Chemical A 

(p<0.05), with no effect on estradiol production. Two test compounds showed decreased 

testosterone levels and increased estrogen levels. Chemical I decreased (p<0.05) testosterone 

levels at 10 and 100 μM and increased (p<0.05) estrogen levels at 10 and 100 μM. Chemical C 

decreased (p<0.05) testosterone levels at 5 (2 runs), 10 (3 runs), and 20 μM (3 runs) and 

increased (p<0.05) estrogen levels at 5 (2 runs), 10 (3 runs), and 20 μM (3 runs). Chemical J 

decreased (p<0.05) both testosterone (100 μM) and estrogen levels (0.1 μM, 2 runs; 1 μM, 3 

runs; 10 μM, 3 runs; and 100 μM, 3 runs). Two test compounds (Chemicals M and H) were 

considered to have equivocal effects on testosterone and estrogen levels due to variable findings 

between runs, and the remaining six test compounds (Chemicals E, G, K, N, S, U) were 

considered to have negative results for effects on testosterone and estrogen concentration. A 

summary of the results is shown in Table 15. 

11

18

18

13

16

18

13

18

7 

5 

2 

5 

Estradiol Inhibition (1 μM prochloraz) 

Estradiol Induction (10 μM forskolin) 

Basal Production Estradiol

Minimum Basal Production Estradiol

Testosterone Inhibition (1 μM prochloraz) 

Testosterone Induction (10 μM forskolin) 

Basal Production Testosterone

Minimum Basal Production Testosterone

Performance Criteria for QC Plates

Met Did not meet
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Table 14. Summary of Fold-Change in Estradiol and Testosterone Levels

Chemical Concentration 

(μM)

Significant Change in

Estradiola 

Significant Change in

Testosteronea 

P 

10 ↑ --b

100 ↑ --

A 

10 -- ↓ 

100 -- NAc

D 

10 ↑d --

100 ↑d --

F 

10 ↑ --

100 ↑ --

B 

1 ↑ (1 run) --

10 ↑ (2 runs)e --

V 100 ↑ --

O 100 ↑ --

I 

10 ↑ ↓ 

100 ↑ ↓

Cg 

5 ↑ (2 runs) ↓ (2 runs)

10 ↑ ↓

20 ↑ ↓

J 

0.1 ↓ (2 runs) --

1 ↓ --

10 ↓ --

100 ↓ ↓ 

M 0.0001 -100 equivocal equivocal

H 0.0001 -100 equivocal equivocal

a

 = statistically significant fold-change relative to solvent (p<0.05) in all 3 runs, unless otherwise noted.

b No significant changes noted.

c

NA = not analyzed due to precipitation. Precipitation noted in all 3 runs 

d Statistical significant only for the overall mean of 3 runs per concentration. 

e

1 run not analyzed due to precipitation. Precipitation noted in all 3 runs at 100 μM.

f

not tested. g

Extra concentrations beyond protocol requirements tested. Precipitation or cytotoxicity noted at ≥ 37.5 

μM. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Table 15. Results from the 18 Steroidogenesis Studies.

Assay Results Number of Chemicals

Negative 6

Induced Estradiol 6

Inhibited Testosterone 1 

Induced Estradiol and Inhibited Testosterone 2 

Inhibited Estradiol and Testosterone 1 

Equivocal 2 

All intraplate CVs were within acceptable limits (≤30%), but interplate CVs for the 

Chemicals A, H, M and S test plates were slightly ≥30%. MRIDs submitted by Laboratories 2 

and 3 reported if CV criteria were met or not met, but did not include individual values.

(v) Issues or Problems with Laboratory Performance 

Four of the five performing laboratories (16 of 18 compounds) did not provide data and 

results for the laboratory proficiency test which is recommended to demonstrate laboratory 

proficiency with running the assay.

(vi) Overall conclusion

The laboratories performance of the assay was generally consistent across all 18 test 

compounds, and the performance criteria were generally met for all compounds. In most cases 

where the performance criteria were not met, the values only slightly exceeded the expected 

values and did not impact the interpretation or reliability of the study. The results demonstrate 

that the Steroidogenesis Assay as performed by the testing laboratories can distinguish between 

chemicals that alter or do not alter testosterone and/or estrogen levels in vitro. 
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e. Aromatase (Human Recombinant) Assay

(i) Background 

The Aromatase Assay (OCSPP Guideline 890.120010) is used to determine 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. The assay utilizes 

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

and measures the release of 3H2O during the conversion of 3H-androstenedione to estrone. 

Competitive inhibition of aromatase activity by a test chemical is detected by serial reaction 

tubes containing increasing concentrations of the chemical of interest. A depiction of the 

reaction is presented below:

Following a 15 minute incubation, the reaction is terminated using methylene chloride. 

The aqueous fraction is extracted and the amount of 3H2O is quantified for each assay tube by 

liquid scintillation spectrometry. Each mole of 3H2O generated represents the conversion of one 

mole of substrate to estrone. Each run is an independent experiment and contains replicate tubes 

of full activity control, background activity control, positive control and test chemical. The 

known aromatase inhibitor 4-hydroxyandrostendione (4-OH ASDN) is used as the positive 

control. Eight concentrations of the positive control (10-10 to 10-5 M) and eight concentrations of 

test chemical (10-10 to 10-3 M) are evaluated in each run. A minimum of three runs is 

recommended. After the completion of the first run, the Test Guideline recommends adjusting 

the concentration of the test chemical in the second and third runs if evidence of insolubility is 

observed at the highest concentration (10-3 M). When insolubility is observed, it is 

recommended that mid-log concentrations (i.e. 10-3.5 M) be tested to define the lower portion of 

the concentration response curve. 

 

10 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1200: Aromatase Assay (Human Recombinant). Office 

of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 

740-C-09-004. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0004. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 54 of 160 

The data are analyzed by first converting raw data to aromatase activity (mmol/mg 

protein/min) and calculating percent of control values. The percent of control values are plotted 

versus logarithm of the test chemical concentration. Chemicals that reduce enzyme activity 

levels by 50% or more and fit a four parameter, one-site competitive binding inhibition model 

are considered to be inhibitors of aromatase. Chemicals that fit the inhibition curve, but allow 

50-75% activity, i.e. reduce activity by 25-50%, are considered equivocal. Chemicals that do not 

fit the model or that fit the model, but do not reduce activity by 25%, are considered to be noninhibitors of aromatase. Typically, an inhibition curve will fall from 90% to 10% over 2 log 

units with a slope near -1. Unusually steep curves may indicate solubility issues or protein 

denaturation. If the slope of the curve is steeper than -2.0, the classification is considered 

equivocal. A typical positive control response curve is shown in Figure 12 below:

Figure 12. Concentration Response Curve for Positive Control 4-OH ASDN.

(ii) Strengths and Limitations of the Assay

The assay utilizes commercially available recombinant microsomes and does not require 

culturing cell lines or animal testing. The assay is considered an important assay in the battery 

since it might indirectly inform on the potential developmental effects mediated by disruption of 

testosterone to estradiol conversion. The assay detects chemicals that inhibit aromatase activity, 

and cannot detect chemicals that may induce aromatase activity through an upregulation of 

aromatase gene expression. The assay provides limited/no ability to metabolize xenobiotics. 

(iii) Observations from Initial Screening of Test Chemicals

Eighteen compounds were used to evaluate the performance of the Aromatase Assay. 

Seven different laboratories were involved in conducting the Aromatase Assays, with the 

majority of the assays (13) being conducted by two labs. Although any source of human 

recombinant microsomes containing CYP19 + reductase may be used in the assay, all 

laboratories utilized recombinant microsomes purchased from one source (BD Biosciences). The 

assays were generally conducted in accordance with the recommendations in Guideline 

890.1200. The purity of the test chemicals used in the assays was >89%. Dimethyl sulfoxide 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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(DMSO) or ethanol was used as the solvent vehicle in all studies, each at ≤1% of assay volume. 

Each assay included data from three successful runs, although a total four or seven test runs were 

conducted in five studies. 

In general, the test chemicals were initially tested at serial log concentrations from 10-10

to 10-3 M as recommended in the Test Guideline. There was one exception (Chemical S) where 

a solubility test was performed prior to conducting the study using DMSO and ethanol as 

solvents. The test chemical was soluble at 10-5 M in ethanol and that concentration was used as 

the high test concentration in the initial run. Additional mid-log concentrations of 10-5.5 and 

10-7.5 M were included in the runs for that chemical. Of the remaining 17 chemicals, no 

solubility issues were noted for eight of the compounds tested at up to 10-3 M. However, 10 of 

the test chemicals were not soluble in the assay at a concentration of 10-3 M, and five of these 

compounds were also not soluble at 10-3.5 M. When solubility issues were noted, additional midlog concentrations (10-3.5, 10-4.5 or 10-4.5 M) were added at the upper end of the concentration 

range for six compounds, in order to better define the lower portion of the inhibition curve. 

However, for three chemicals with solubility issues, additional mid-log concentrations were not 

added to the assay. These values are summarized in Table 16. 

Table 16. Summary of the High Test Concentration for the 18 Aromatase Assays. 

Number of Test 

Chemicals

Highest Soluble 

Concentration 

Tested

Mid-log 

Concentrations 

Tested

Comments

8 10-3 M N/A Soluble up to 10-3 M

3 10-4 M Yes Insoluble at 10-3.5 M

3 10-4 M No Insoluble at 10-3 M

2 10-4.5 M Yes Insoluble at 10-4 M

1 10-5 M Yes Insoluble at 10-4.5 M

1 10-5 M Yes Solubility pre-test performed.

N/A= not applicable

Two of the studies included data demonstrating the laboratory’s proficiency in running 

the assay by testing either the four Guideline-recommended proficiency chemicals (econazole, 

fenarimol, nitrofen and atrazine) or a variation of these chemicals (prochloraz, fenarimol, 

nitrofen and ronidazole), and another five study reports stated that proficiency testing was 

successfully conducted, but results were not included in the study reports. The remaining 12 

studies provided no information regarding proficiency testing.

Results 

Of the 18 chemicals tested, 11 were classified as non-inhibitors (Table 17). However, 

two of these compounds had precipitation at 10-3 M and did not include mid-log concentrations at 

the higher concentrations to better define the inhibition curve, consequently there is less 

confidence in their classification. Five of the chemicals were classified as equivocal due to 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 56 of 160 

having either steep Hill slopes (Chemicals M, P) or minimum inhibition between 50-75% at the 

highest tested concentration (Chemicals A, D, and S). However, there is less confidence in the 

classification for one (Chemical M) of these compounds as it did not include mid-log 

concentrations in the upper range. The remaining 2 compounds were classified as inhibitors 

(Chemicals J and K).

Table 17. Classification of the 18 Chemicals Evaluated in the Aromatase Assay. 

Assay Results Number of Chemicals

Inhibitor 2

Non-inhibitor 11

Equivocal 5 

(iv) Performance criteria 

Guideline performance criteria for the aromatase assay are provided in Table 18. The 

performance criteria are provided to serve as guidance in identifying runs that provide 

parameters in the preferred ranges. 

Table 18. Performance Criteria for Aromatase Assay 

Parameter Recommended Values

Minimum Aromatase Activity 0.1 nmol/mg-protein/min

Mean Background Control Activity ≤ 10% of Full Activity 

Coefficient of Variation (CV) for replicates within 

each sample type and concentration of 4-OH ASDN <15%

Criteria for Positive Control Response Curves

Parameter Lower Limit Upper Limit

Slope -1.2 -0.8 

Top (%) 90 110 

Bottom (%) -5 +6

Log IC50 -7.0 -7.3 

Background and Full Activity Controls 

Full enzyme activity control samples contain substrate, NADPH, propylene glycol, 

vehicle and microsomes. Aromatase activity in the full enzyme activity controls was above the 

recommended minimum activity level (0.1 nmol/mg protein/min), except in two studies, which 

were slightly below the recommended levels with minimum activities of 0.090 and 0.098 

nmol/mg protein/min in selected runs. In all the assays, the average enzyme activity across all 

three runs ranged from 0.114 to 0.616 nmol/mg protein/min. Background activity controls 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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(samples that contain all components of the full aromatase activity controls minus NADPH) were

≤11.5% of the full activity in all assays and was typically ≤3.5% of full activity. The 

recommended mean background control activity is ≤ 15% of full activity control. The range of 

values for the full activity controls (as % of average full activity) were within the recommended 

90-110% range in 8 of the 18 studies, and the remaining 10 studies had only a few full activity 

values slightly outside the range (84-115%). Quality control-assay drift is monitored by 

measuring the aromatase activity of the full enzyme activity controls and background activity 

controls at the beginning and end of each assay run. No assay drift was noted in 10 of the 18 

studies, and slight assay drift was occasionally noted in the other eight studies. When drift was 

noted, the changes were minor and did not adversely impact the study results.

Positive Control 

All studies utilized 4-hydroxy-androstenedione (4-OH ASDN) as the positive control 

compound for inhibition of aromatase activity. The performance criteria for the 4-OH ASDN 

were generally met with a few exceptions. The top of the mean inhibition curve for the positive 

control was within the recommended range (90-110%) in 12 of the studies and was out of range 

in the remaining 6 studies (77.5-117%). The bottom of the mean inhibition curve for the positive 

control was also within the recommended range (-5 to 6%) in 17 studies, and was only slightly 

out of range in the remaining study (-5.2%). The Hill Slope values for 4-OH ASDN were 

acceptable (-1.2 to -0.8), with the exception of one run in one study (-0.76). The Log IC50 values 

for 4-OH ASDN were within the recommended range (-7.3 to -7.0) in all runs for 10 of the 18 

studies. Of eight studies with Log IC50 values outside the recommended range, six studies had 

runs that were lower values (-7.79 to -7.35 log M) and two tests had slightly higher values (-6.90 

to -6.74 log M). However, the average log IC50 values (across runs for a chemical) were 

generally within range, with the exception of the two studies, which had average log IC50 values 

of -7.54 and -7.38 log M, respectively. The variability (%CV) within % inhibition values for 4-

OH ASDN at each concentration tested were generally acceptable (<15%), although 10 of the 

studies had %CV in excess of 15% at one or more concentrations. The higher %CVs typically 

occurred at the higher concentrations of 4-OH ASDN, which had high levels of inhibition 

(>95%). At these low activity levels, small changes in the standard deviations resulted in large 

changes in the %CV. A summary of how well the positive controls met the performance criteria 

is shown in Figure 13. 
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Page 58 of 160 

Figure 13. Aromatase Performance Criteria Results.

 

(v) Issues or Problems with Laboratory Performance

The issues were noted in the conduct of the Aromatase Assays relating to laboratory 

proficiency testing and the selection of test chemical concentrations. Although the assay 

generally performed as expected in each study, actual laboratory proficiency data utilizing four 

reference compounds was provided in only two study reports. The other challenge, which was 

noted in 4 of the 18 studies, was that the activity of the test compounds was not adequately 

characterized at higher concentrations. For three chemicals with solubility issues at 10-3 M, 

additional mid-log concentrations, such as 10-3.5 or 10-4.5 M, were not included in the assay to 

better characterize the response of the test chemical and provide a complete inhibition curve.

(vi) Overall conclusion

The assay performance was generally consistent across all 18 test compounds, and the 

performance criteria for the assay were generally met in each study. Aside from the lack of midlog concentrations for some studies, the deviations noted in the performance criteria were often 

minor, slightly exceeding the ranges for the performance criteria and did not adversely impact 

study interpretation. Overall the assay as performed by the testing laboratories is able to 

distinguish between inhibitors and non-inhibitors of aromatase activity.

17

10

17

12

17

8 

18

16

1 

8 

1 

6 

1 

10

2 

Mean Positive Control (mean of all runs)- Log 

IC50

Positive Control (all runs)- Log IC50 

Positive Control Response Curve- Bottom (%)

Postive Control Response Curve- Top (%) 

Positive Control Slope

Coefficient of Variation (CV) for replicates of 4-

OH ASDN

Mean Background Control Activity

Minimum Aromatase Activity

Aromatase Performance Criteria

Met Did not meet
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2. In vivo

a. Uterotrophic

(i) Background 

The Uterotrophic Assay (OCSPP 890.1600)11 is intended as an in vivo screening assay, 

providing data about a single endocrine mechanism (i.e., the estrogenicity of a test compound). 

The Uterotrophic assay employs an animal test system in which there are very low levels of or 

no endogenous estrogen and concomitant low baseline uterine weights with minimal variation. 

This enhances the ability to evaluate the uterine response to exogenous 17β-estradiol and 

potential estrogen agonists. Two animal models have been validated for this assay: young adult 

females (mouse or rat) after ovariectomy with adequate time for uterine tissue regression; or 

immature female rats after weaning and prior to puberty. The Guideline states a preference for 

the adult ovariectomized rat. The Uterotrophic Assay evaluates the ability of test chemical(s) to 

elicit agonism of estrogenicity as determined by a statistically significant increase in uterine 

weight.

(ii) Strengths and Limitations of the Assay 

The Uterotrophic Assay is a relatively rapid screen which incorporates metabolism. The 

test guideline states a preference for subcutaneous injection as the route of administration to 

provide absorption, distribution, metabolism, and excretion (ADME) insight and contrast with 

the rat pubertal assays with gavage administration. However, oral administration is acceptable 

and is the most frequent route of administration in uterotrophic studies reviewed to date. For 

humans, oral administration will model a primary exposure route and incorporates ADME of the 

test chemical. A positive finding in this assay can be corroborated by information such as ER 

binding or transcriptional activation. 

(iii) Observations of Test Chemicals

Of the 17 chemicals evaluated, 10 assays utilized mature, ovariectomized female rats at 

ages of 56-75 days, and the other seven assays (Chemicals D, E, F, J, K, M, Q) utilized sexually 

immature females at an age of 19 days. All compounds were evaluated for estrogenic activity, 

and none of the tests included the optional testing for anti-estrogenic activity. The route of 

exposure for the test compounds was via oral gavage, with the exception of one chemical (N) 

which was administered subcutaneously (s.c.). The dietary concentrations of phytoestrogens 

(total isoflavones or genistein equivalents) were reported for all but one assay (Chemical C). 

Dietary phytoestrogen concentrations were reported within two distinct ranges: 6.2-43 μg/g diet, 

reflecting the actual measured concentrations, or <300-350 μg/g diet, meeting Guideline 

specified limits. The Guideline recommends that the highest dose level be at or just below the 

 

11 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1600: Uterotrophic Assay. Office of Safety and 

Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740-C-09-0010. October 

2009. 
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MTD but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the agency also 

considers the toxicity profile of the chemical (i.e., ≥10% decrease in body weight, cholinesterase 

inhibition, target organ toxicity, etc.) in dose selection. As a screen, large intervals, e.g., one half 

log units (corresponding to a dose progression of 3.2) or even one log units, between dosages 

were acceptable.

As shown in Table 19, of the 17 chemicals tested in this assay, dose levels were selected 

based on the results of range-finding studies (11 chemicals), Part 158 standard toxicology studies 

(3 chemicals), or the Limit Dose (3 chemicals). Seven chemicals had two dose levels (A, B, D, 

G, K, Q, and S) tested, and 10 chemicals had three doses tested. No overt toxicity was seen in 16 

of 17 chemicals; overt toxicity was seen for Chemical A as describe below. The doses tested 

were determined to be adequate for all chemicals.

Table 19. Summary of Overt Toxicity Observed in the Uterotrophic Assay

Chemical

Code

Highest Dose Tested

(mg/kg/day)

Body weight

(>10%)

Clinical 

Signs

Methods Used to 

Select Dose Levels

A 250 X X Part 158 studies

B 1000 Limit Dose

C 1000 Limit Dose

D 120 Range finding study

E 200 Range finding study

F 225 Range finding study

G 400 Range finding study

H 180 Range finding study

I 4.0 Range finding study

J 450 Range finding study

K 20 Part 158 studies

M 20 Range finding study

N 50 Range finding study

P 1000 Limit Dose

Q 400 Range finding study

S 75 Range finding study

V 0.3 Part 158 studies

X= Toxicity which was observed only at the highest dose tested

Chemical - A: Doses were selected based on the results of Part 158 standard toxicology studies 

where diarrhea, rust colored fur, and tremors were observed in female rats within 9 hrs following 

a single oral dose at 300 mg/kg. Based on these studies, the highest dose selected for the main 

study was 250 mg/kg/day. In the main study, rats at the highest dose tested (HDT) exhibited the 

following adverse clinical effects: 2 rats displayed nasal discharge, thinness, and fur 

discoloration on Day 2; on Day 3, those same 2 rats plus 3 additional rats (total of five) displayed 

head and scapula fur discoloration, lethargy and hunched posture; these same 5 rats displayed 

head and scapula fur discoloration prior to euthanasia. There was a significant decrease (15%) in 

mean body weight at HDT.
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Chemical - D: In the range finding study, when tested at doses of at doses of 0, 5, 20, 50, 80 or 

150 mg/kg/day, clinical signs and body weight changes were seen at 150 mg/kg/day. Based on 

these results, the highest dose selected for the main study was 120 mg/kg/day.

Chemical - E: In the range finding study, when tested at 250, 500, 750 and 1000 mg/kg/day by 

Day 15, females weighed 12.2%, 9.0%, 13.5% and 12.9% less than controls, respectively. Based 

on these results, the highest dose selected for the main study was 200 mg/kg/day.

Chemical - F: In the range finding study, when tested at doses of 0, 50, 200, 400, or 600 

mg/kg/day for 15 days, absolute and relative liver weights were increased at doses ≥ 200 

mg/kg/day. Based on these results, the highest dose selected for the main study was 225 

mg/kg/day.

Chemical - G: Two range finding studies were performed. In the first study, no clinical signs of 

toxicity or change in body weights were observed in doses up to 150 mg/kg/day. In the second 

study mortality was observed in ≥600 mg/kg dose groups. Based on these results, the highest 

dose selected for the main study was 400 mg/kg/day.

Chemical - H: In the range finding study, when tested at doses of 0, 20, 60, or 180 mg/kg/day 

for 3 consecutive days, no adverse effects were seen at any dose including the high dose. The

highest dose tested in the main study was 180 mg/kg/day where mean body weights were 

decreased (p<0.05) by 8 and 9% on Days 3 and 4, respectively, at this dose. 

Chemical - I: In the range finding study, when tested at doses of 0, 1, 2, 4, or 8 mg/kg/day for 15 

days, decreased body weight gains were observed in the 4 and 8 mg/kg/day dose groups. Red 

blood cell cholinesterase (ChE) activity was inhibited by 75-81% at 1 mg/kg/day and ≥ 95% at 

doses greater than 2 mg/kg/day; brain ChE was inhibited at doses ≥2 mg/kg/day. Based on these 

results, the highest dose selected for the main study was 4 mg/kg/day. 

Chemical - J: In the range finding study, when tested at doses of 0, 25, 100, 300, or 450 

mg/kg/day for 15 days, no adverse effects were seen at any dose. Based on these results, the 

highest dose selected for the main study was 450 mg/kg/day. 

Chemical - K: Dose levels were selected based on data available from the standard Part 158 

studies. No mortality was observed in the repeated-dose dietary studies conducted at doses of up 

to 27 mg/kg/day, or in repeated-dose gavage studies with doses up to 15 mg/kg/day. Based on 

these data, the highest dose selected for the main study was 20 mg/kg/day. 

Chemical - M: In the range finding study, when tested at doses of 20 or 40 mg/kg/day for 10 

days, mortality, severe clinical signs and reduced body weight gain were seen at 40 mg/kg/day. 

Based on these results, the highest dose selected for the main study was 20 mg/kg/day. 
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Chemical - N: In a range-finding study, rats were given subcutaneous (s.c) injections at 0, 25, 75 

or 250 mg/kg/day. Rats given 75 mg/kg/day, struggled and vocalized during dosing, suggesting 

irritation due to s.c administration; therefore, the 250 mg/kg/day dose was not administered. 

Based on body weight loss and the presence of a small focus of hemorrhage at the injection site, 

the dose of 75 mg/kg/day was determined to exceed MTD. Based on these results, the highest 

dose selected for the main study was 50 mg/kg/day. 

Chemical - Q: In the range finding study, when tested at dose levels of 0, 100, 300, 500, 750 and 

1000 mg/kg for 14 consecutive days, mortality occurred at doses >500 mg/kg/day. At 300 

mg/kg/day, transient uncoordinated movement was seen in all rats. Based on these results, the 

highest dose selected for the main study was 400 mg/kg/day. 

Chemical - S: In the range finding study, when tested at doses of 0, 10, 50, or 100 mg/kg/day for 

28 days, mortality, clinical signs, and decreases in body weight were seen at 100 mg/kg/day. 

Based on these results, the highest dose selected for the main study was 75 mg/kg/day. 

Chemical - V: In Part 158 standard toxicology studies, brain ChE activity was significantly 

decreased by 50-59% in adult rats at 0.3 mg/kg/day. Based on these results, the highest dose 

selected for the main study was 0.3 mg/kg/day. In the main study, the cholinesterase phase of 

the study was conducted separately; significant inhibition of red blood cell (RBC) and whole 

brain ChE activity was seen at the high dose (0.3 mg/kg/day) on the first and last day of dose 

administration. All animals survived until scheduled termination and no clinical signs of toxicity 

were observed. 

(vi) Results

None of the 17 test compounds evaluated showed a response of estrogenic activity under 

the conditions of this assay. Except for Chemical A, doses tested in all studies were deemed 

adequate based on the absence of overt systemic toxicity at the doses tested, attainment of an 

adequate dose and/or inclusion of the Limit Dose. 

Positive Control: 17α-ethynyl estradiol (EE) is recommended as the positive control for this 

assay. In the examined studies, EE was administered by subcutaneous (s.c.) injection at doses 

ranging from 0.27 to 0.5 μg/kg/day in three studies, at 3 μg/kg/day in two studies, and at 300 

μg/kg/day in two studies. Additionally, EE was administered by oral gavage at doses of 1.0 

μg/kg/day in three studies, 10 μg/kg/day in four studies, and 100 μg/kg/day in three studies.
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The expected effect of the positive control, EE, in the agonist assay is increased uterine 

weight. Following s.c. administration, EE increased absolute wet uterine weights by 1.9- to 3.9-

fold at doses of 0.27-0.5 μg/kg/day, 8.7- to 9.3-fold at doses of 3 μg/kg/day, and 6.6- to 10.4-fold 

at doses of 300 μg/kg/day. The concomitant increases in blotted uterine weights were less 

varied, with increases ranging from 1.92 - to 2.8-fold at 0.27 - 0.5 μg/kg/day, 3.3 - 3.5 at 3 

μg/kg/day and 3.3- 6.0 at 300 μg/kg/day. Following administration by oral gavage, EE increased 

absolute wet uterine weights by 2.1- to 2.9 -fold at 1 μg/kg/day, 6.3- to 9.9-fold at 10μg/kg/day, 

and 2.6- to 5.1-fold at 100 μg/kg/day. As with the s.c. dosing, the increases in blotted uterine 

weights following oral dosing were less varied, ranging from 2.1- to 3.0-fold at 1 μg/kg/day, 5.0- 

to 6.2-fold at 10 μg/kg/day, and 2.3- to 3.3-fold at 100 μg/kg/day. EE doses were high enough 

for the positive control to show the appropriate response in terms of statistically (p≤ 0.05) 

increased uterine weights in all 17 assays. However, for three assays, the EE doses used were 

above the maximum dose (10 μg) validated by OECD for this assay (Kanno et al., 2001); such 

high doses are often beyond the maximal response plateau for EE and may lead to nonmonotonic positive control results.

(iv) Performance Criteria: The performance criteria for the Uterotrophic assay are:

• Blotted uterine weights after dosing of the vehicle control are expected to be 0.09% 

or 0.04% of final body weight in immature or ovariectomized female rats,

respectively.

• A baseline positive control study with ≥ 4 doses of EE to show dose response vs. 

uterine weight is expected to have been conducted, and the dose of EE administered 

in the assay should be 70-80% of the effective dose determined from the baseline 

study.

The first criterion related to relative uterine weight was met in all studies; however, the 

second criterion was not met consistently. Issues regarding the second performance criterion are: 

• Baseline control studies were not reported for 2 test chemicals. 

• One laboratory (8) conducted a baseline control study, but selected the reference EE dose 

based on other study results. 

• Data were not routinely reported to assess the dose response of the positive control (EE) 

vs. effective dose from the control baseline study.

(v) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of 

the assay were identified. 
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(vi) Overall Conclusion

The assay performance was consistent across all 17 test compounds. Vehicle control uterine 

weights were ≤ 0.09% or 0.04% of final body weight in immature or ovariectomized female rats, 

respectively, and positive control animals showed the appropriate response of statistically 

(p≤0.05) increased uterine weights. Although a wide range of doses were utilized for the EE 

positive control, the resulting maximal increases in wet and blotted uterine weights consistently 

reached at doses ≥10μg and demonstrated that the assay was performing as expected.

b. Hershberger 

(i) Background 

The Tier 1 Hershberger Bioassay (OCSPP 890.1400)12 is a short-term in vivo screening 

test that is designed to detect chemicals that act as androgen agonists (androgenic) or androgen 

antagonists (anti-androgenic) or chemicals that inhibit 5α-reductase. Androgens are essential for 

the initial development of the reproductive system in utero, the maturation of the reproductive 

system and accessory sex organs, development of secondary and tertiary sex characteristics at 

puberty, and maintenance of the reproductive system and accessory sex organs structures and 

functions until death. Androgens are needed for feedback regulation of the hypothalamuspituitary-gonadal (HPG) axis and spermatogenesis.

In this assay, castrated pubertal rats are dosed with the test chemical by oral gavage or 

subcutaneous (s.c) injection for 10 consecutive days. Critical measurements include the weights 

of five androgen-dependent sex accessory tissues (ventral prostate [VP], seminal vesicle [SV] 

with fluids and coagulating glands, levator ani-bulbocavernosus [LABC] muscle, paired 

Cowper’s glands [CG] and glans penis [GP]). These five tissues all respond to androgens with an 

increase in absolute weight. When testing for anti-androgenicity, these tissues are stimulated to 

increase in weight by s.c. administration of a potent reference androgen (testosterone propionate 

[TP]) and the five tissues measured respond to anti-androgens with a decrease in absolute 

weight. Other endpoints include clinical observations and body weight changes. For androgen 

agonism, a statistically significant (p≤0.05) increase in two or more of the five androgen 

responsive tissues is considered a positive result. For androgen antagonism, a statistically 

significant (p≤0.05) reduction in two or more of the five androgen responsive tissues is 

considered a positive result.

 

12 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1400: Hershberger Assay. Office of Safety and 

Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740-C-09-008. October 

2009. 
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Page 65 of 160 

(ii) Strengths and Limitations of the Assay

The Hershberger Assay is a relatively rapid screen that is quite specific to androgenic 

effects. The assay can effectively detect (and distinguish) androgen agonists, antagonists, and 

inhibitors of 5α-reductase. The assay incorporates metabolism and can detect chemicals that 

require metabolic activation. Oral administration models a primary exposure route and 

incorporates ADME. Compounds other than androgenic agonists can alter the weight of certain 

tissues; however, the growth response of several tissues concomitantly substantiates a more 

androgen-specific mechanism.

(iii) Observations of Test Chemicals

Of the 17 available Hershberger Bioassays, 16 used Sprague-Dawley rats as the test 

species, and one (Chemical S) utilized Wistar rats. All of the assays utilized male rats, surgically

castrated on post-natal day (PND) 42-48. The rats were at PND 51-60 and weighed 194-314 g at 

the initiation of the 10-day treatment period. All test compounds were evaluated for both 

androgenic and anti-androgenic activity. The route of exposure for all of the test compounds was 

oral gavage. The Guideline recommends that the highest dose level be at or just below the MTD 

but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the agency also 

considers the toxicity profile of the chemical (i.e., ≥10% decrease in body weight, cholinesterase 

inhibition, target organ toxicity, etc.) in dose selection. In addition to the guideline 

recommended endpoints, blood and brain cholinesterase activity were measured in 2 studies, and

serum testosterone levels were measured in three studies. 

(iv) Results

As shown in Table 20, of the 17 chemicals tested in this assay, dose levels were selected 

based on the results of range-finding studies (11 chemicals), Part 158 standard toxicology studies 

(5 chemicals), or the Limit Dose (1 chemical). For the androgen phase (A) of the assay, 9 

chemicals had two dose levels tested and 7 chemicals had three doses (E, F, I, J, N, P and V) 

tested. For the anti-androgen phase (aA), three dose levels were used for all chemicals. 

No overt toxicity was seen for 14 of 17chemicals and the dose levels tested were 

determined to be adequate for these chemicals. Overt toxicity was seen for the remaining 3 

chemicals, (B, E and K). No overall androgenic response was seen for any chemical. In contrast, 

anti-androgenic response was seen for 7 chemicals (A, B, E, G, H, K and S). For 3 of the 7 (B, E 

and K), effects were only seen in the presence of overt toxicity (e.g., ≥10 % decreases in body 

weight and/or clinical signs). Anti-androgenic responses were seen in the absence of overt 

toxicity for the remaining 4 Chemicals (Table 20). 
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Table 20. Summary of Overt Toxicity Observed in the Hershberger Assay. 

Chemical

Code

Highest Doses 

Tested

(mg/kg/day)

Body 

Weight 

(> 10%)

Food 

Consumption

Clinical 

Signs

Methods Used to Select 

Dose Levels

A 150 Part 158 studies

B 1000 x x Range finding study

C 1000 Limit dose

D 80 Range finding study

E 750 x x Range finding study

F 450 Range finding study

G 150 Part 158 studies

H 180 Range finding study

I 12 Range finding study

J 500 Range finding study

K 25 x Part 158 studies

L 100 Range finding study

M 20 Range finding study

N 800 Range finding study

P 500 Part 158 studies study

S 75 x Range finding study

V 0.3 Part 158 studies

X = Toxicity seen only at the highest dose tested 

Shaded rows indicate a response in the assay

Chemical - B: Based on the results of a range finding study, the highest dose tested (HDT) was 

1000 mg/kg/day for both the androgen agonist and anti-androgen phase of the assay. In 

androgen phase of the Hershberger assay, at 1000 mg/kg/day, mean body weights were 

decreased (p≤0.05) on Days 3 (↓13%), 4 (↓15%), and 5 thru 9 (↓11%). Food consumption was 

affected. In the anti-androgen phase, at the HDT, mean body weights were (p≤0.05) decreased on 

Days 3 (12%), 4 (18%), 5 and 6 (↓21%), 7 (↓22%), 8 (↓24%), 9 (↓23%), 10 (↓22%) and 11 

(↓22%), and food consumption was decreased (p<0.05) by 31% on Days 1-11. Effects on 

androgen sensitive tissues were seen only at the HDT, but not at the lower doses [300 mg/kg/day 

and 100 mg/kg/day] where no overt toxicity was seen. 

Chemical - E: Based on the results of a range finding study, the high dose selected was 750 

mg/kg/day. In the androgen phase of the Hershberger assay, at 750 mg/kg/day, mean body 

weights were decreased (p≤0.05) on Days 4 (↓11%), 7 (↓7%) and 11 (↓7%, not significant). 

Food consumption was decreased (p≤0.05) in the mid (250 mg/kg/day; ↓12%) and high (750 

mg/kg/day; ↓44%) dose groups during Days 1-4. There were no effects on food consumption 

during Days 4-7 or 7-11 in any of the dose groups. In the anti-androgen phase at the HDT, mean 

body weights were significantly (p<0.05) decreased on Days 4 (↓14%), 7 (↓10%) and 11 (↓12%). 

Food consumption was decreased (p≤0.05) in the mid (250 mg/kg/day; ↓23%) and high (750 

mg/kg/day; ↓50%) dose groups during Days 1-4, and only in the high dose (750 mg/kg/day; 
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↓13%) group during Days 4 through 11. Effects on androgen sensitive tissues were seen only at 

the HDT but not at the lower doses, 250 mg/kg/day and 50 mg/kg/day.

Chemical - K: Based on the results of a range finding study, the highest dose selected was 25 

mg/kg/day. In the androgen phase of the Hershberger assay at the HDT, tremors (Days 3-9) and 

uncoordinated movement (Days 4 and 5) were seen in one rat, and tremors were noted in six 

other rats on Days 3-9. There were no effects on body weight at any dose level. In the antiandrogen assay, tremors (Days 3 and 7) and uncoordinated movement (Day 3) were seen in one 

rat at the HDT. Another rat at the HDT exhibited tremors on Days 3-9 and uncoordinated 

movement on Day 5; six other rats at this dose also exhibited tremors between Days 4 and 8. At 

24 hours after dosing, no clinical signs were observed in any dose group. Mean body weights 

were decreased (↓12%; p≤0.05) on Day 11 only at the high dose. Effects on androgen sensitive 

tissues were seen only at the HDT but not at the lower doses, 15 mg/kg/day and 5 mg/kg/day.

Chemical – S: Based on the results of a range finding study, the highest dose selected was 75 

mg/kg/day. In the Hershberger assay, clinical signs of toxicity were limited to salivation in 3/6 

and 6/6 rats at the 10 and 75 mg/kg/day groups, respectively, in the androgen agonist phase, and 

in 4/6 and 6/6 rats in the 40 and 75 mg/kg/day groups in the anti-androgen study.

Positive Controls: The Guideline recommended reference androgen agonist is TP administered 

at 0.2 or 0.4 mg/kg/day by s.c. injection. In the examined studies, TP was administered by s.c. 

injection at 0.2 mg/kg/day in four studies and at 0.4 mg/kg/day in 13 studies. The Guideline 

recommended reference androgen antagonist is flutamide (FT) administered at a dose of 3 

mg/kg/day by oral gavage, and co-administered with TP. FT was administered at the 

recommended dose and by the recommended route in all 17 assays. 

The expected effect of the reference androgen agonist, TP, in the androgenic assay is 

increased weight of the androgen-dependent sex accessory tissues. Below are the ranges of 

reference androgen responses across the 17 assays evaluated. Following administration by s.c. 

injection at 0.2 mg/kg/day, TP increased tissue weights in all 5 sex accessory organs examined 

compared to solvent control as follows: ( i) SV by 327-381%;( ii) VP by 381-676%; (iii) LABC 

by 132-188%; (iv) CG by 322-579%; and (v) GP by 48-72%. Following administration by s.c. 

injection at 0.4 mg/kg/day, TP increased sex accessory tissue weights compared to the control as 

follows: i) SV by 530-1926%; ii) VP by 493-2200%; iii) LABC by 96-287%; iv) CG by 

285-794%; and v) GP by 49-112%. 

The expected effect of the reference androgen antagonist, FT, when co-administered with 

TP in the anti-androgenic assay is decreased weight of the androgen-dependent sex accessory 

tissues compared to the TP controls. Below are the ranges of reference androgen agonist 

responses across the 17 assays evaluated Following administration by oral gavage at 3 

mg/kg/day with TP co-administered by s.c. injection at 0.2 mg/kg/day, FT decreased sex 
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accessory tissue weights compared to the solvent control as follows: (i) SV by 70-74%; (ii) VP 

by 73-75%; (iii) LABC by 48-58%; (iv) CG by 67-75%; and (v) GP by 18-33%. Following 

administration by oral gavage at 3 mg/kg/day with TP co-administered by s.c. injection at 0.4 

mg/kg/day for the 17 assays, FT decreased sex accessory tissue weights compared to the control 

as follows: (i) SV by 62-86%; (ii) VP by 59-81%; (iii) LABC by 36-58%; (iv) CG by 51-69%; 

and (v) GP by 14-35%. 

In the androgenic assay, the reference androgen agonist (TP) showed the appropriate 

response in terms of significantly (p≤0.05) increased sex accessory tissue weights in all 17 

assays. In the anti-androgenic assay, the reference androgen antagonist (FT), co-administered 

with the reference androgen agonist, showed the appropriate response in terms of significantly

(p≤0.05) decreased sex accessory tissue weights in all 17 assays.

(v) Performance Criteria: The performance criteria for the Hershberger Assay are 

presented in Table 21. 

Table 21. Maximum recommended % CVs for the Mandatory Sex Accessory Tissues.

Tissue Anti-androgenic effects Androgenic effects

Seminal vesicles 40% 40% 

Ventral prostate 40% 45%

LABC 20% 30% 

Cowper's glands 35% 55%

Glans penis 17% 22% 

In the androgenic assays, the CVs [expressed as a percent (%CVs)] for the sex accessory 

tissues from the control and high-dose animals were less than the specified maxima for all five 

tissues in 10 of the 17 assays. For the control animals, the maximum recommended %CVs were 

exceeded in one assay for the SV (49%, Chemical D), four assays for the VP (48-94%; 

Chemicals D, E, F, and G), one assay for the CG (71%; Chemical G), and one assay for the GP 

(38%; Chemical G). For the high-dose animals, the maximum recommended %CVs were 

exceeded in one assay for the SV (49%; Chemical D), three assays for the VP (49-78%; 

Chemicals D, N, P), one assay for the CG (83%; Chemical G), and three assays for the GP (25-

36%; Chemicals C, D, P). Although %CVs above the recommended maxima were noted in 7 of 

the assays, only 2 (Chemicals D and G) of the 17 assays had %CVs that exceeded maximum 

performance criteria for multiple tissues from both the control and high-dose animals. For the 

positive control (TP), the %CVs were less than the recommended maxima for all five androgen 

sensitive tissues in all 17 evaluated assays.

In the anti-androgenic assays, the %CVs for the sex accessory tissues from the control 

and high-dose animals were less than the specified maximums for all five tissues in 13 of the 17 

assays. 
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• For the control animals, the %CVs were less than the recommended maxima for the SV, 

VP, LABC, and CG tissues in all 17 assays, and the maximum %CV for the GP was 

exceeded only slightly in one assay (22%; Chemical B).

• For the high-dose animals, the %CVs were less than the recommended maxima for the 

SV, GC, and GP in all 17 assays, and the %CV maxima were exceeded in one assay for 

the VP (49%; Chemical J) and three assays for the LABC (24-33%; Chemicals A, B, K). 

Only one of the anti-androgenic assays (Chemical B) had %CVs above the recommended 

maxima in multiple tissues from both the control and high-dose animals.

• For the positive control (FT+TP) animals, the %CVs were less than the recommended 

maxima for all five androgen sensitive tissues in 8 of the 17 evaluated assays, and another 

7 of the assays had %CVs above the recommended maxima in only one tissue. The 

maximum recommend %CVs for the positive control were exceeded in one assay for the 

SV (44%), two assays for the VP (41% and 57%), four assays for the LABC (22%-30%), 

one assay for the CG (37%), and two assays for the GP (19% and 20%). The maximum 

%CV was exceeded by ≥10% in only one tissue (SV or VP) in two different assays. 

(vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of 

the assay were identified. The %CVs in excess of the performance criteria were noted in 14 of 

the 17 studies; none of these had an impact on study interpretation. Two of the studies 

(Chemicals D and G) had %CVs in excess of the recommended maxima in at least three separate 

tissues in the androgenic assay for the control and/or high-dose animals, and both these studies 

were negative for androgenic activity. One Chemical (B) had high %CVs in the anti-androgenic 

assay in at least three separate tissues for the control and/or high-dose animals; Chemical B 

showed a response for androgen antagonist activity in this assay. 

(vii) Overall Conclusion

Overall, the assays were conducted in accordance with the test guidelines, including 

proper dose selection, methodology, study design, and statistical analyses. The assay 

performance was relatively consistent across all 17 test compounds. With regards to changes in 

sex accessory tissue weights, the reference androgen agonist and antagonist both performed as 

expected. Although the maximum %CVs were occasionally exceeded for all tissues across all 17 

assays in the anti-androgen assay, the overall responses were relatively consistent and 

demonstrated that the screening assay can detect the androgenic and anti-androgenic potential of 

chemicals.
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c. Pubertal Female

(i) Background 

The purpose of the female pubertal assay (OCSPP 890.1450)13 is to identify chemicals 

that have the potential to interact with the endocrine system, by identifying effects on pubertal 

development and thyroid function in the intact juvenile/peripubertal female rat. This assay is 

also capable of detecting not only anti-thyroid, estrogenic, or anti-estrogenic chemicals, but also 

agents that alter pubertal development through mechanisms that induce changes in luteinizing 

hormone, follicle stimulating hormone, prolactin, or growth hormone levels or via alterations in 

hypothalamic function.

Endpoints assessed in the Female Pubertal Assay include body weights, age and weight at 

vaginal opening, organ weights (thyroid, liver, kidneys, pituitary, and adrenals), histology 

(uterus, ovary, thyroid, and kidney), hormones [thyroxine (T4) and thyroid-stimulating hormone 

(TSH)], estrous cyclicity (age at first vaginal estrus after vaginal opening, length of cycle, 

percent cycling, and percent cycling regularly), and standard blood panel (including creatinine 

and blood urea nitrogen). 

(ii) Strengths and Limitations of the Assay 

The Female Pubertal Assay is an apical assay performed in the intact animal that has 

redundant confirmatory endpoints, involves the pubertal period of development, and has a wellestablished base of knowledge of the relationship of the endpoints to endocrine activity. 

Collectively, all of these factors contribute to the strength of this assay to detect potential 

interactions with the E or T pathways. Moreover, they also contribute to an evaluation of a 

chemical’s potential to affect the HPG axis. Having redundant confirmatory endpoints is helpful 

in an assay such as this where variability in proficiency in measuring specific endpoints can be a 

factor in the ability of the assay to detect a response.

Metabolism takes place in this in vivo assay, which is advantageous over in vitro assays. 

For chemicals whose metabolites are the most active agents, in vitro systems may not identify 

the parent compound as having the potential to interact with the endocrine system. Thus, the use 

of an in vivo system reduces the likelihood of false negative or false positive results. In addition, 

the integrated nature of the endocrine system in the developing organism and the relationship of 

the endocrine toxicity to other systemic effects cannot be simulated in vitro. Also, the use of a 

 

13 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1450: Pubertal Development and Thyroid Function in

Intact Juvenile/Peripubertal Female Rats. Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides 

and Toxic Substances (7101). EPA 740-C-09-009. October 2009. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 71 of 160 

mammalian model usually gives rise to greater confidence that results are relevant to humans 

than if a phylogenetically more removed model is used.

The Female Pubertal Assay focuses on a period of development when the endpoints

selected for the assay are particularly sensitive to endocrine modulation. Therefore, sensitivity is

greater than for other life stages which are practically feasible to include in a screen for

endocrine activity. One of the strengths particular to the female pubertal protocol lies in the fact 

that the measures included for the identification of endocrine effects are based on a solid 

knowledge of how the reproductive and thyroid axis mature in the rat. Thus, the extensive basic 

literature in this area, reviewed in the detailed review paper, provides the background for the 

underlying assumptions in the assay endpoints and assists in the interpretation of results. The 

assay offers certain advantages over the proposed in vitro tests for determining alterations in AR 

and ER binding or steroidogenesis in that the in vitro assays cannot account for metabolic 

activation of xenobiotics which would result in “false negative” responses.

The Female Pubertal Assay does have limitations. One limitation is the cyclical 

variability of the female reproductive organ weights after sexual maturity has been reached. The 

female pubertal assay utilizes ovary-intact animals. Thus, once puberty is attained, the 

fluctuations associated with the normal ovarian cycle could be viewed as problematic, as 

evidenced by the high coefficients of variation. Although identification of mechanism of action 

is not necessary for identification of interaction with the endocrine system, the inability of this 

assay to identify mechanisms of action is a limitation. However, the pubertal assays are not 

stand alone assays, and the results are to be interpreted within the context of the Tier 1 battery. 

Thus, effects observed with the pubertal assay are used to complement any data in the other 

battery assays that may be more informative as far as mechanisms are concerned.

(iii) Observations of Test Chemicals

Of the 17 Female Pubertal Assays, Sprague-Dawley rats were used as the test strain for 

all but one chemical (Chemical S), which utilized Wistar rats. Dose groups in each assay 

consisted of 15-16 juvenile female rats. Six different laboratories were involved in testing. The 

route of exposure for all of the test compounds was oral gavage, using corn oil, aqueous 0.4-

1.0% methylcellulose, or 1% gum tragacanth as the dosing vehicle. The Guideline recommends 

that the highest dose level be at or just below the MTD but need not exceed the Limit Dose 

(1000 mg/kg/day); however, typically, the Agency also considers the toxicity profile of the 

chemical (i.e., ≥10% decrease in body weight, cholinesterase inhibition, target organ toxicity, 

etc.) in dose selection. The two or three dose levels used for the test compounds were considered 

to be adequate in all assays. In all 17 tests, juvenile female rats were dosed daily from PND 22 

to PND 42. 
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The dietary concentrations of phytoestrogens (total isoflavones or genistein-equivalents; 

aglycone forms of genistein plus daidzein) in the study diet were reported for all assays. The 

most common diets provided were manufactured by Teklad (14-18% protein), except for two 

studies conducted by Laboratory 2, which used A04C-10 diet. Reported dietary phytoestrogen 

concentrations fell into two distinct ranges; either 6.2 to 29 μg/g diet, reflecting the actual 

measured concentrations, or ≤350 μg/g diet, indicating the concentrations were within the 

Guideline specified limits. Two or three dose groups of the test compounds plus a vehicle 

control group were included in the studies. The dose levels used were considered to be adequate 

in all assays.

The Guideline recommends that females be sacrificed on PND 42 beginning 2 hours after 

the final dose, and that sacrifices are to be completed by 1300 hours due to normal diurnal 

fluctuation in thyroid hormone levels. The Guideline recommendation is sacrifice by injectable 

anesthetic, followed immediately by decapitation. Decapitation without anesthesia is an 

acceptable, although less preferred method. Only one laboratory (for Chemical S) used an 

injectable anesthetic (sodium thiopentone); eight laboratories used decapitation without 

anesthetic, and eight laboratories used decapitation after isoflurane inhalation.

In all 17 assays, the animals were observed for clinical signs throughout the assay. At 

sacrifice, the appropriate reproductive organs were collected, along with the liver, pituitary, 

adrenals, kidney and thyroid. All samples were weighed and selected organs were examined 

histologically. Blood samples were also collected for analyses of clinical chemistry parameters

(including creatinine and blood urea nitrogen) and serum T4 and TSH levels. Of the four 

cholinesterase inhibiting compounds that were tested, two of the tests included analysis of RBC 

and brain cholinesterase (ChE) activity. The statistical analyses were deemed appropriate in all 

17 assays.

(iv) Results

As shown in Table 21, of the 17 chemicals evaluated in this assay, dose levels were 

selected based on the results of range-finding studies (11 chemicals), Part 158 standard 

toxicology studies (5 chemicals), or the Limit Dose (1 chemical). Eleven chemicals had two 

doses tested and 6 chemicals had three doses (E, F, I, J, N, and V) tested.

No overt toxicity was seen for 12 of 17 chemicals, and the dose levels tested were 

determined to be adequate for these chemicals. For the remaining 5 chemicals (D, G, K, M and 

S), effects on estrogen-dependent and/or thyroid-related endpoints were seen only at doses that 

elicited overt toxicity (e.g., ≥10% decreases in body weight, food consumption, and clinical 

signs) as shown in Table 22. 
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Table 22. Summary of Overt Toxicity Observed in the Pubertal Female Assay

Chemical

Code

Highest 

Dose Tested 

(mg/kg/day)

Mortality

Body 

weight

(>10%)

Food 

Consumption

Clinical 

Signs

Methods Used to 

Select Dose 

Levels

A 100 Part 158 studies

B 500 Range finding study 

C 1000 Limit Dose

D 120 x Range finding study 

E 300 Range finding study 

F 250 Part 158 studies

G 125 x x x Range finding study 

H 60 Range finding study 

I 2.0 Range finding study 

J 400 Range finding study 

K 20 x xx Part 158 studies

M 20 x Range finding study 

N 800 Range finding study 

P 600 Part 158 studies

Q 300 Range finding study 

S 75 x x x Range finding study 

V 0.3 Part 158 studies

X = Toxicity seen only at the highest dose tested 

XX = Toxicity seen at both doses tested

 

Chemical - D: Based on the results of a range finding study, the highest dose selected for the 

main study was 120 mg/kg/day. In the female pubertal assay, increased incidences of salivation 

were seen in 6/15 rats at the low dose (60 mg/kg/day) and in 7/15 rats at the high dose (120 

mg/kg/day) when compared to controls. No treatment-related effects were seen in estrogendependent endpoints at any dose level. Treatment-related changes in thyroid-related end points 

were seen only at the HDT, but not at the lower dose, 60 mg/kg/day.

Chemical - G: Based on the results of a range finding study, the highest dose selected for the 

main study was 125 mg/kg/day. In the pubertal assay at the HDT, piloerection, diarrhea 

thinness, hunched posture and/or a distended abdomen was seen in one rat on Days 10 to 15 

which was sacrificed on Day 15 (not attributed to dosing error). Another rat died on Day 19. 

Also, at the HDT, there was a significant (p< 0.01) decrease (↓14%) in final body weights. 

Treatment-related effect on estrogen-dependent and thyroid-related endpoints were seen only at 

the HDT, but not at the lower dose (62.5 mg/kg/day). 

 

Chemical - K: Based on the data available from the standard Part 158 toxicology studies, the 

highest dose selected for the main study was 20 mg/kg/day. In the pubertal assay, tremors were 
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seen at various times post-dose in up to 5 of 16 rats at the low dose (10 mg/kg/day) on 10 days, 

and up to 16 rats at the high dose (20 mg/kg/day) on 18 days. At the high dose, mean body 

weights were significantly (p<0.01) decreased on Days 27 (↓8%), 19 (↓10%) and 36 (↓9%). 

Treatment-related effects were seen in estrogen-dependent endpoints only at HDT whereas 

thyroid-related endpoints were seen both at the HDT and at the lower dose (10 mg/kg/day). 

Overt toxicity was seen at both dose levels. 

Chemical - M: Based on the results of a range finding study, the highest dose selected for the 

main study was 20 mg/kg/day. In the pubertal assay, increased incidence of salivation was seen 

in 5 of 15 rats at HDT. Treatment-related effects on estrogen-dependent endpoints were seen 

only at the HDT, but not at the lower dose (10 mg/kg/day). No treatment-related effects were 

seen in thyroid-related endpoints at any dose level.

Chemical - S: Based on the results of a range finding study, the highest dose levels selected for 

the main study was 75 mg/kg/day. In the pubertal assay, the following findings were noted at 

HDT: (i) decreases (p<0.01) in mean body weights on Days 24 (↓8%), 28 (↓14%), 36 (↓20%) 

and 42 (↓13%); (ii) decreases (p<0.01) in food consumption during PND 22-29 (↓37%) and 

during PND 29-36 (↓27%); and (iii) increased incidences of slight to moderate salivation (postdose) in 15/15 rats compared to controls (0/15). Treatment-related effects were seen in estrogen 

endpoints at both low and high dose groups; however, overt toxicity was seen only at the HDT. 

Effects on thyroid-related endpoints were seen only at the HDT, but not at the lower dose (10 

mg/kg/day). 

Of the 17 chemicals evaluated, 6 showed responses in at least one estrogen-dependent 

endpoint (E, G, K, M, Q, and S). The remaining 11 chemicals did not show any estrogendependent responses. Estrogen-dependent responses were defined as: (i) an advance or delay in 

VO (>2 days); (ii) advance or delayed first estrous; (iii) estrous cyclicity disruption; and/or (iv) 

uterine and ovarian weight changes. 

For 4 of 6 chemicals that showed estrogen-dependent responses, effects occurred at doses 

that elicited overt toxicity (Chemicals G, K, M and S).

• For Chemical G, estrogen-dependent effects seen at the HDT in the presence of overt 

toxicity (mortality, decreased body weight and clinical signs) included delayed VO and 

changes in ovarian and uterine weighs. 

• For Chemical K, estrogen-dependent effects seen at the HDT in the presence of overt 

toxicity (decreased body weight and tremors) were changes in ovarian histopathology. 

• For Chemical M, estrogen-dependent effects seen at the HDT in the presence of overt 

toxicity (salivation) were delayed VO and first estrus. 
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• For Chemical S, estrogen-depended effects seen at the HDT in the presence of overt 

toxicity (decreases in body weight and food consumption, salivation) included delayed 

VO and changes in ovarian and uterine weights. 

For the remaining two (Chemicals E and Q), estrogen-dependent responses were seen in the 

absence of overt toxicity.

Of the 17 chemicals evaluated, 11 showed an alteration for at least one thyroid-related 

endpoint (Chemicals A, B, C, D, E, F, G, K, P, Q and S). The remaining six chemicals (H, I, J, 

M, N and V) did not show a response in any thyroid related endpoints. Thyroid-related 

responses were defined as: (i) changes in serum thyroxine (T4); (ii) changes in serum TSH; (iii) 

alterations in thyroid weights, and/or (iv) thyroid histopathology. 

For 4 of 11 chemicals that showed a thyroid-related response, effects occurred at doses 

that elicited overt toxicity (Chemicals D, G, K and S). 

• For Chemical D, changes in thyroid weight alterations in serum T4 levels were seen at the 

HDT in the presence of increased incidence of salivation.

• For Chemical G at the HDT, alterations in serum T4 levels and thyroid histopathological 

changes were seen in the presence of mortality, decreased body weight and clinical signs. 

• For Chemical K, alterations in serum T4 level and histopathological changes (increase in 

the follicular cell height) were seen at the HDT.

• For Chemical S, changes in thyroid weight were the sole finding at HDT, which was seen 

in the presence of decreases in body weight and food consumption and salivation.

• For the remaining 7 chemicals, thyroid-related responses were seen in the absence of 

overt toxicity.
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(v) Performance Criteria

The Guideline includes performance criteria for the vehicle-treated control animals 

(Sprague-Dawley for the Female Pubertal assay (OCSPP Guideline 890.1500, Section Q

[reproduced as Table 23)]. The table lists eleven performance criteria, including the average 

value and coefficient of variation (CV) in percentage for critical organ weights; age and body 

weight at vaginal opening (VO); final body weight; and serum concentration of T4. The “mean”, 

“2 SDs”, “CV”, and “1.5 CV” columns in Table 19 provide the mean, two standard deviations, 

coefficient of variation, and 1.5 times the coefficient of variation for that endpoint in historical 

controls. Mean values and CVs for the vehicle control group must fall within the acceptable 

range of each to be considered fully acceptable. The Guideline states that similar results have 

been produced with Wistar and Long-Evans strains.

The performance criteria for blotted uterus, ovaries, thyroid, T4, and pituitary weights 

were met for all 16 chemicals tested in SD rats. The most common exception to the performance 

criteria not fully met was mean adrenal weight, which was below the acceptable range (38.34-

48.84 mg) for 11 chemicals (range was 31.6-37.4 mg). Although adrenal weight is a relevant 

secondary endpoint, it is not the most sensitive endpoint for this assay. The performance criteria 

for the remaining endpoints were met in all 16 assays performed in SD rats, with the following 

exceptions. 

• For age at VO, the mean was above the acceptable range for two chemicals (Chemical C, 

PND 36.1; and Chemical F, PND 36.4), and the CV was slightly above the acceptable 

range for four chemicals (Chemical A, 8.6%; Chemical P, 9.39%; Chemical D, 7.5%; 

Chemical G, 11.7%).

• For body weight at VO, the mean was above the acceptable range for two chemicals 

(Chemical C, 137.1 g; Chemical A, 135.7 g) and the CV was above the acceptable range 

for three chemicals (Chemical A, 14.2%; Chemical G, 18.3%; Chemical F 15.58%).

• For final body weight, the CV was above the acceptable range for three chemicals 

(Chemical E, 11.91%; Chemical B, 9.57%; Chemical G, 10.1%).

• For kidney weight, the CV was above the acceptable range for three chemicals (Chemical 

B, 12.75%; Chemical K, 11.4%; Chemical G, 12.7%).

• For liver weight, the CV was above the acceptable range for five chemicals (Chemical E, 

14.71%; Chemical N, 13.29%; Chemical P, 13.36%; Chemical B, 15.26%; Chemical G, 

14.76%). 

 

For the one test chemical that utilized the Wistar rat, the CV exceeded the acceptable 

range for kidney weight and total serum T4 concentration, based on performance criteria for the 

SD strain. 
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Table 23. Performance Criteria for Controls (Sprague-Dawley Strain). 

Endpoint Mean 2 SDs Acceptable range CV 1.5 CV 

Top of 

Acceptable 

Rangea

Uterus, blotted (milligrams)

298.89 111.49 187.40 to 410.38 28.26 9.48 37.73 

Ovaries (milligrams)

75.65 39.11 36.54 to 114.77 17.37 5.83 23.20 

T4(total, μg/dl)

4.03 1.34 2.69 to 5.38 21.38 8.01 29.39 

Thyroid weight (milligrams)

14.20 8.00 6.20 to 22.20 23.97 14.61 38.58 

Age at VO (postnatal day, where day of birth = PND 0)

33.15 2.47 30.67 to 35.62 4.69 1.82 6.52 

Weight at VO (grams)

116.58 14.86 101.71 to 131.44 9.81 4.16 13.97 

Final body weight (grams)

154.70 49.85 104.86 to 204.55 6.69 2.25 8.93 

Adrenals (milligrams)

43.59 5.25 38.34 to 48.84 16.02 6.95 22.97 

Kidneys (grams)

1.57 0.63 0.95 to 2.20 8.42 2.34 10.76 

Liver (grams)

8.05 3.73 4.32 to 11.78 9.58 3.55 13.13 

Pituitary (milligrams)

8.97 3.11 5.86 to 12.08 15.76 11.22 26.97 

a= Bottom of the acceptable range for coefficient of variation is zero.

(vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of 

the assay were identified. Although not a technical deficiency in the conduct of the assay, 

prolonged delays in VO precluded an evaluation of cyclicity for some assays. Minor 

deficiencies, including mean body or organ weights or %CV outside the limits of the 

performance criteria, were noted in 16 of the 17 studies (Chemical Q had no deficiencies), and 

none of these minor deficiencies had an adverse impact on study interpretation. One study 

(Chemical G) had six exceptions to the performance criteria with one exception, % CV for age at 

VO that was higher than acceptable criterion (11.7% treated vs. 6.52% control), indicating 

variability of the data. All other studies had three or fewer exceptions. Eleven of the 16 studies 

used the body weight on PND 21 instead of PND 22 as the covariate for statistical analyses, but 

this was not considered to have any meaningful effect on study interpretation.
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Several assays utilized radioimmunoassay for T4 and TSH measurements. Details on the 

methodology were not provided in the study report for three assays. 

(vii) Overall Conclusion

Overall, the assays were conducted in accordance with the test guidelines, including 

proper dose selection, methodology, study design, and statistical analyses. The assay 

performance was generally consistent across all 17 test compounds. Although the %CV and 

mean body or organ weights were occasionally outside the limits of the performance criteria, the 

overall responses were relatively consistent and demonstrated that the screening assay can detect 

potential effects of chemicals on pubertal development and thyroid function in the intact 

juvenile/peripubertal female rat.

 

d. Pubertal Male 

(i) Background 

The Male Pubertal Assay (OCSPP 890.1500)14 is designed to determine if a chemical 

interacts with the endocrine system by identifying effects on pubertal development and thyroid 

function in the intact juvenile/peripubertal male rat. This assay is capable of detecting chemicals 

with anti-thyroid, androgenic, or anti-androgenic androgen receptor (AR) or steroid-enzymemediated activity. This assay may also detect agents which alter pubertal development via

changes in gonadotropins, prolactin, or hypothalamic function. Therefore, this screening assay

can be useful in detecting chemicals that act on androgens or through the hypothalamuspituitary-gonadal (HPG) axis that controls the estrogen and androgen hormone systems, as well 

as chemicals that may interfere with the thyroid system.

Endpoints assessed in the Male Pubertal Assay include age and weight at preputial 

separation, body weights, organ weights (seminal vesicles with coagulating glands, ventral and 

dorsolateral prostate, levator ani/bulbocarvernosus (LABC) muscle complex, epididymides, 

testes, thyroid, liver, kidneys, pituitary, and adrenals), histology (epididymis, testis, thyroid, and 

kidney), hormones (testosterone, thyroxine [T4], and thyroid stimulating hormone [TSH]), and 

standard blood panel including creatinine and blood urea nitrogen.

 

14 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1500: Pubertal Development and Thyroid Function in 

Intact Juvenile/Peripubertal Male Rats. Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides 

and Toxic Substances (7101). EPA 740-C-09-012. October 2009. 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 79 of 160 

(ii) Strengths and Limitations of the Assay 

The Male Pubertal Assay is an apical assay performed in the intact animal that has 

redundant confirmatory endpoints, involves the pubertal period of development, and has a wellestablished base of knowledge of the relationship of the endpoints to endocrine activity. 

Collectively, all of these factors contribute to the strength of this assay to detect potential 

interactions with the A or T pathways. Moreover, they also contribute to an evaluation of a 

chemical’s potential to affect the HPG axis. Having redundant, confirmatory endpoints is helpful 

in an assay such as this where variability in proficiency in measuring specific endpoints can be a 

factor in the ability of the assay to detect a response.

Metabolism takes place in this in vivo assay, which is advantageous over in vitro assays. 

For chemicals whose metabolites are the most active agents, in vitro systems may not identify 

the parent compound as having the potential to interact with the endocrine system. In addition, 

the integrated nature of the endocrine system in the developing organism and the relationship of 

the endocrine toxicity to other systemic effects cannot be simulated in vitro. Also, the use of a 

mammalian model usually gives rise to greater confidence that results are relevant to humans 

than if a phylogenetically more removed model is used.

As is the case with any study design, the male pubertal assay also has limitations. One 

potential limitation is the variability of the testosterone measurements. The hormone assays 

themselves are not difficult, as they are available commercially and include their own quality 

control samples. However, sample collection methodology and the changing levels of 

testosterone over time in the juvenile male rat present possible sources of increased variability in 

the assay. Collecting the sample in a non-stressed animal will reduce the variability. However, 

the pubertal assay are not stand alone assays nor is testosterone measurement the only endpoint 

that might be associated with endocrine disruption in the male, and the results are to be 

interpreted within the context of the Tier 1 battery. Thus, effects observed with the pubertal 

assay are used to complement any data in the other battery assays that may be more definitive as 

far as mechanisms are concerned.

(iii) Observations of Test Chemicals

Of the 16 Pubertal Male Assays, Sprague-Dawley rats were used as the test strain for 15 

assays, and the remaining assay utilized Wistar rats. Dose groups in each assay consisted of 15 

or 16 juvenile male rats. Six different laboratories were involved in the testing. For each test 

compound, the route of exposure was oral gavage, using either corn oil, aqueous 0.5-1.0% 

carboxymethyl cellulose, or 1% gum tragacanth as the dosing vehicle. The Guideline 

recommends that the highest dose level be at or just below the MTD but need not exceed the 

Limit Dose (1000 mg/kg/day); however, typically, the Agency also considers the toxicity profile 

of the chemical (i.e., ≥ 10% decrease in body weight, cholinesterase inhibition, target organ 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 80 of 160 

toxicity, etc.) in dose selection. The two or three dose levels used for the test compounds were 

considered to be adequate in all assays. In all 16 assays, juvenile male rats were dosed daily 

from PND 23 to PND 53 or 54. 

The dietary concentration of phytoestrogens (total isoflavones or genistein-equivalents; 

aglycone forms of genistein plus daidzein) in the study diet is recommended to be ≤ 300 μg/g 

diet. The most common diets were Teklad diets (14-18% protein), except for studies conducted 

by Laboratory 3 (Diet A04C-10). Reported dietary phytoestrogen concentrations fell into two 

distinct ranges, either 6.2 to 29 μg/g diet or ≤300-350 μg/g diet.

In each assay, animals were sacrificed by 1300 hours on the final day of dosing, 2 hours 

after the last dose. With regards to anesthesia, the Guideline recommends sacrifice by 

decapitation without anesthesia in order to minimize any effect on testosterone. The use of CO2

inhalation is not considered acceptable as an anesthetic, but if used must be administered for ≤60 

seconds. In the 16 tests, six laboratories used decapitation without anesthetic, eight laboratories 

used decapitation after isoflurane inhalation, and one laboratory used decapitation after brief CO2

inhalation (<60 sec).

In all 16 assays, the animals were observed for clinical signs throughout the assay. At 

sacrifice, the appropriate reproductive organs were collected, along with the liver, pituitary, 

adrenals, and thyroid. All samples were weighed and selected organs were examined 

histologically. Blood samples were also collected for clinical chemistry analyses and analysis of 

serum T4, TSH, and testosterone; none of the assays tested for serum levels of 3,3’5-

triiodothyronine (T3). Of the four cholinesterase (ChE) inhibiting compounds that were tested, 

two of the assays included analysis of red blood cell (RBC) and brain ChE activity. The 

statistical analyses were deemed appropriate in all 16 assays.

(vi) Results

As shown in Table 24, of the 16 chemicals evaluated in this assay, dose levels were selected 

based on the results of range-finding studies (12 chemicals), Part 158 standard toxicology studies 

(3 chemicals), or at the Limit Dose (1 chemical). Ten chemicals had two dose levels tested and 6 

chemicals had three doses (E, F, I, J, N, and V) tested.

No overt toxicity was seen for 13 of 16 chemicals and the dose levels tested were 

determined to be adequate for these chemicals. For the three chemicals (D, G and S) effects on 

androgen-dependent and/or thyroid-related endpoints were seen only at doses that elicited overt 

toxicity (e.g., as < 10% decreases in body weight, food consumption, and clinical signs) as 

shown in Table 24. 
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Table 24. Summary of Overt Toxicity Observed in the Pubertal Male Assay. 

Chemical

Code 

Highest Dose 

Tested

(mg/kg/day)

Body weight

(>10%) 

Food 

Consumption

Clinical Signs Methods Used to Select 

Dose Levels

A 100 Part 158 studies

B 375 Range finding study

C 1000 Limit Dose

D 120 xx Range finding study

E 400 Range finding study

F 250 Range finding study

G 125 x Range finding study

H 60 Range finding study

I 2.0 Range finding study

J 400 Range finding study

K 20 Part 158 studies

M 20 Range finding study

N 800 Range finding study

P 800 Range finding study

S 75 x x x Range finding study

V 0.3 Part 158 studies

X = Toxicity seen only at the highest dose tested 

XX = Toxicity was seen at both doses tested

Chemical - D: Based on the results of a range finding study, the highest dose selected for the 

main study was 120 mg/kg/day. In the male pubertal assay, increased incidences of salivation 

were seen in 5/15 rats at the low dose (60 mg/kg/day) and in all rats at the high dose (120 

mg/kg/day) groups. No treatment-related effects were seen in androgen-dependent endpoints at 

any dose level. Treatment-related changes in thyroid-related end points were seen at both dose 

levels.

Chemical - G: Based on the results of a range finding study, the highest dose selected for the 

main study was 125 mg/kg/day. In the male pubertal assay at the HDT, final body weights were 

decreased (23%; p <0.01). Treatment-related effects in androgen-dependent and thyroid-related 

endpoints were seen in the absence of any toxicity at the low dose (62.5 mg/kg/day) but in the 

presence of overt toxicity at HDT.

Chemical - S: Based on the results of a range finding study, the highest dose selected for the 

main study was 75 mg/kg/day. At the HDT, mean body weights were significantly (p<0.01) 

decreased on PND 27 (↓10%), PND 41 (↓22%) and PND 53 (↓20%). Food consumption was 

significantly (p<0.01) decreased (10-28%) during the measured intervals from PND 23-53. 

Increased incidence of slight to moderate salivation was seen in 15/15 rats compared to 0/15 in 

the controls. Treatment-related effects in androgen-dependent endpoints were seen only at HDT.
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Of the 16 chemicals evaluated, 9 showed responses in at least one androgen-dependent 

endpoint (B, C, E, F, G, J, N, P and S). The remaining 7 chemicals did not show any androgendependent responses. Androgen-dependent responses were defined as: (i) a delay preputial 

separation (ii) changes in testosterone levels; and/or (iii) androgen-sensitive organ weights 

changes. 

For 2 of 9 chemicals that showed androgen-mediated responses, effects occurred at doses 

that elicited overt toxicity (Chemicals G and S). 

• For Chemical G, at the HDT, androgen-dependent effects, seen in the presence of 

decreased body weight, included increased age at PPS; alterations in testosterone levels; 

and changes in androgen-sensitive organ weights. 

• For Chemical S, at the HDT high dose, androgen-dependent effects were increased age of 

PPS, alterations in testosterone levels, and changes in androgen-sensitive organ weights. 

These changes were seen in the presence of increased salivation and decreases in body 

weight and food consumption. 

For the remaining 7 chemicals, androgen-dependent responses were seen in the absence of overt 

toxicity. 

Of the 16 chemicals evaluated, 7 showed a response for at least one thyroid-related 

endpoint (Chemicals C, D, E, F, G, J, and P). The remaining 9 chemicals did not show a 

response in any thyroid-related endpoints. Thyroid-related responses were defined as: (i) 

changes in serum thyroxine (T4); (ii) changes in serum TSH; (iii) alterations in thyroid weights, 

and/or (iv) thyroid histopathology. 

For 2 of 7 chemicals that showed thyroid-related responses, effects occurred at doses that 

elicited overt toxicity (Chemicals D and G). 

• For Chemical D, alterations in serum T4 levels, changes in thyroid weight, and enlarged 

thyroid were seen at the HDT in the presence of overt toxicity (salivation). 

• For Chemical G, alterations in serum T4 levels, changes in thyroid weight and thyroid 

histopathological changes were seen in the presence of decreased body weight.

(v) Performance Criteria

The Guideline includes the performance criteria for the vehicle-treated control animals 

(Sprague-Dawley and Wistar strains) for the Male Pubertal assay (OCSPP Guideline 890.1500 

[reproduced as Table 25]). The table lists the performance criteria (average value and CV in 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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percentage) for critical organ weights; age at PPS; and serum concentrations of TSH, 

testosterone, and total T4. The “mean”, “2 SDs”, “CV”, and “1.5 CV” columns in Table 20 

provide the mean, two standard deviations, coefficient of variation, and 1.5 times the coefficient 

of variation for that endpoint in historical controls. Mean values and CVs for the vehicle control 

group must fall within the acceptable range of each criterion to be considered fully acceptable. 

Performance criteria are not included for LABC and thyroid weight and TSH concentration for 

Wistar rats, for the testis weight for Sprague-Dawley rats, or for the dorsolateral prostate (DP) of 

either strain. For Sprague-Dawley rats, there are currently 16 parameters having performance 

criteria for the average value (weight, age, or concentration) and the %CV, totaling 32 

performance criteria values. As stated in the Guideline, more emphasis is placed on meeting 

performance criteria for the CV than for the endpoint control means since the CVs relate directly 

to sensitivity of the endpoints.

The performance criteria were generally met for each assay, although all tests had 2 or 

more values that were outside the acceptable range. For the 15 assays that utilized SprgueDawley rats, each assay had 2 to 11 values out of range. However, the majority of these out-ofrange values were only marginally outside the performance criteria. If these marginal values are 

excluded, then five of the assays met all the performance criteria; nine of the assays had only 1-3 

values out of range; and one assay had only 4-6 values out of range. For the one assay that 

utilized Wistar rats, 16 of the 28 performance criteria values exceeded the normal range. 

The individual performance criteria for each parameter (organ weight, body weights, age, 

or hormone concentration) across the 15 assays that utilize Sprague-Dawley rats are as follows. 

• For the weight of the seminal vesicle plus coagulating gland (with fluid), only 1 

assay had an average weight above the performance criteria (Chemical C), and 8 

assays had %CVs in excess of the performance criteria maximum value (Chemicals 

B, C, E, F, I, J, K, and N). 

• For the ventral prostate weight, the average weight met the performance criteria in 

all 15 assays, and 4 assays (Chemicals A, B, K, and P) had %CVs in excess of the 

recommended maximum. The %CVs were substantially out of range in only 2 

assays (Chemicals A and B).

• For the LABC weight, only 1 assay (Chemical N) had an average weight below the 

performance criteria; otherwise, the performance criteria for LABC were met in all 

15 assays. 

• For the weight of the epididymis (combined left and right), the average weight met 

the performance criteria in all 15 assays, and only 3 assays (Chemicals D, K, and S) 

had %CVs in excess of the recommended maximum. The %CV was substantially 

out of range in only 1 assay (Chemical K).

• The T4 concentration was low in 1 assay (Chemical M); otherwise, the performance 

criteria for T4 were met in all 15 assays. 
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• For TSH concentration, 5 of the assays (Chemicals A, D, G, K, and M) had 

concentrations below the performance criteria, and 7 assays (Chemicals D, F, G, K,

M, N, and S) had %CVs above the maximum. The %CVs were substantially out of 

range in only 3 assays (Chemicals F, M, and S).

• The performance criteria for testosterone concentration were met in all 15 assays.

• For thyroid weight, the average weights were below the performance criteria in 7 

assays (Chemicals E, G, H, I, J, N, and P), and 3 assays (Chemicals B, K, and N) had 

%CV above the recommended maximum. The %CV was substantially out of range 

in only 1 assay (Chemical B).

• For adrenal weight, the maximum %CV was slightly exceeded in only 1 assay 

(Chemical P); otherwise, the performance criteria for adrenal weight were met in all 

15 assays. 

• For kidneys, average weights were below the performance criteria in 12 of the 15 

assays, but the maximum %CV was only slightly exceeded in 1 assay (Chemical B).

• For liver, the average weight met the performance criterion in all 15 assays, and the 

maximum %CV was exceeded in only 2 assays (Chemicals B and I).

• For the pituitary gland, the average weight met the performance criteria in all 15 

assays, and recommended maximum %CV were exceeded in 5 assays (Chemicals G,

N, P, S, and V). The %CVs were substantially out of range in only 3 assays 

(Chemicals N, P, and S).

• For the initial body weight, the average weight was above the performance criteria in 

6 assays (Chemicals A, C, D, G, K, and P), but the maximum %CVs was only 

slightly exceeded in 3 assays (Chemicals B, F, and P).

• For the final body weight, the average weight met the performance criteria in all 15 

assays. The maximum %CVs was exceeded in 12 assays; however, the values were 

substantially out of range in only 3 assays (Chemicals B, I, and P). 

• For the body weight at PPS, the average weight was slightly high in 1 assay

(Chemical D) of the 15 assays. The maximum %CVs was exceeded in 12 assays, 

and the values were substantially out of range in 6 assays (Chemicals A, B, G, J, K,

and I). 

• The average age at PPS met the performance criteria in all 15 assays, but the 

maximum %CV was slightly exceeded in 5 of the assays (Chemicals B, C, D, M,

and P).

Although the Guideline for this assay does not currently specify performance criteria 

(weight ranges and CVs) for the testes or the dorsolateral prostate (DP) of Sprague-Dawley rats, 

the data from the 15 assays that utilized Sprague Dawley rats indicate that testis weights (and 

variances) for control animals were consistent across the assays. For each assay, the left testis 

weights averaged 1.343-1.502 g with CVs of 5.2-27.7% (typically 6.7-9.2%). Right testis 

weights averaged 1.352-1.543 g with CVs of 5.7-22.2% (typically 6.7-9.2%). Across the 15 

assays, the overall average weights %(CV) were 1.428 g (3.0%) for the left testis and 1.449 g 

(3.3%) for the right testis. For the dorsolateral prostate (DP), average study weights ranged from 

90.4 to 194.6 mg with CVs of 13.9-31.1% (typically 18.4-27.6%). Across the 15 assays, the 

overall average weights (%CV) for the DP were 140.0 mg (19.0%). 
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Table 25. Performance Criteria for Controls (Sprague-Dawley and Wistar Strains). 

Endpoint Rat

Strain Mean 2 SDs Acceptable Range CV 1.5 CV

Top of 

Acceptable 

Rangea 

Ventral 

prostate wt 

(grams)

Wistar 0.223 0.072 0.151 to 0.295

16.67 5.65 22.32 Sprague-Dawley 0.246 0.086 0.160 to 0.332

LABC wt 

(grams)

Wistar NA NA NA 15.77 11.33 27.100 Sprague-Dawley 0.651 0.204 0.447 to 0.855

Epididymis wt 

(g)

Wistar 0.474 0.124 0.350 to 0.598 10.94 5.45 16.39 Sprague-Dawley 0.446 0.082 0.364 to 0.528

Seminal 

vesicle w/ 

coagulating 

gland wt. (g)

Wistar 0.576 0.234 0.342 to 0.810 

20.61 0.45 21.06 

Sprague-Dawley 0.507 0.212 0.295 to 0.719 

Testis wt 

(grams)

Wistar 1.341 0.250 1.091 to 1.591 9.27 8.35 17.62 Sprague-Dawley NA NA NA

Thyroid wt 

(milligrams)

Wistar NA NA NA 15.39 8.24 23.63 Sprague-Dawley 20 6 14 to 26

Adrenals wt 

(mg)

Wistar 54.597 13.768 40.829 to 68.365 15.42 7.34 22.77 Sprague-Dawley 46.478 14.636 31.842 to 61.114

Kidneys wt (g) Wistar 2.516 0.550 1.966 to 3.066 9.56 5.20 14.76 Sprague-Dawley 2.646 0.404 2.242 to 3.050

Liver wt (g) Wistar 14.070 2.874 11.196 to 16.944 10.24 4.69 14.93 Sprague-Dawley 12.670 2.680 9.990 to 15.350

Pituitary wt 

(mg)

Wistar 8.051 1.934 6.117 to 9.985 12.14 3.83 15.98 Sprague-Dawley 10.354 2.544 7.810 to 12.898

T4 concen. 

(μg/dl)

Wistar 5.478 2.164 3.314 to 7.642 18.27 9.20 27.46 Sprague-Dawley 5.716 1.660 4.056 to 7.376

TSH concen. 

(ng/mL)

Wistar NA NA NA 34.04 24.26 58.29 Sprague-Dawley 14.162 9.950 4.212 to 24.112

Testosterone 

concen. 

(ng/mL)

Wistar 2.118 2.540 0 to 4.658

58.82 30.88 89.70 Sprague-Dawley 2.110 1.850 0.260 to 3.960

Weaning wt (g) Wistar 58.238 11.058 47.180 to 69.296

8.04 2.21 10.25 

Sprague-Dawley 52.642 7.170 45.472 to 59.812

Age at PPS 

(PND, PND 0 

= day of birth)

Wistar 43.124 2.948 40.176 to 46.072

3.64 2.03 5.67 

Sprague-Dawley 43.147 3.366 39.781 to 46.513

Weight at PPS 

(grams) Wistar 209.142 31.850 177.292 to 240.992 7.54 0.03 7.57 

Sprague-Dawley 222.223 33.946 188.277 to 

256.169 

Final body 

weight (grams) Wistar 291.818 41.578 250.24 to 333.396 6.62 0.85 7.47 

Sprague-Dawley 295.647 36.412 259.235 to 

332.059 a Bottom of the acceptable range for coefficient of variation is zero
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(vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of 

the assay were identified. Five of the 16 studies used the body weight on PND 23 (instead of 

PND 21) as the covariate for statistical analyses, but this was not considered to have any 

meaningful effect on study interpretation.

(viii) Overall Conclusion

Overall, the assays were conducted in accordance with the OPPTS guidelines, including 

proper dose selection, methodology, study design, and statistical analyses. Assay performance 

was generally consistent across all 16 test compounds. Although the average values and %CVs 

were occasionally outside the limits of the performance criteria, the overall responses were 

relatively consistent and demonstrated that the screening assay can detect potential effects of 

chemicals on pubertal development and thyroid function in the intact juvenile/peripubertal male 

rat.

e. Fish short-term reproduction

(i) Background 

The fish short-term reproduction assay (FSTRA; OCSPP Guideline 890.135015) is 

intended to evaluate reproductive fitness as an integrated measure of toxicant effects. The test 

includes a suite of biochemical and histological endpoints that reflect effects associated with 

interactions to the hypothalamic-pituitary-gonadal (HPG) endocrine axis and its downstream 

hormonal pathways. The assay uses sexually mature male and female fathead minnow fish 

(Pimephales promelas). During a 21-day chemical exposure, survival, reproductive behavior, 

secondary sex characteristics, fecundity, and fertility are monitored. At the end of the test, 

additional measurements that are meant to be directly reflective of the status of the reproductive 

endocrine system such as secondary sex characteristics, gonadal histopathology, gonado-somatic 

index (GSI), and plasma concentrations of vitellogenin (VTG) are collected. The FSTRA is 

intended to serve in a screening capacity to provide an indication of potential endocrine 

interactions, not necessarily to confirm any specific mechanism or mode of action. Specifically, 

the FSTRA is intended to provide information on potential estrogen agonists or antagonists, 

androgen agonists or antagonists, and/or chemicals that act on steriodogenesis or at a higher level 

along the HPG axis. The specifics on the test methodology are presented in the OCSPP 890.1350 

Fish Short-term Reproduction Assay guideline (USEPA 2009). A summary of the assay is 

presented below.

 

15 USEPA 2009. Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1350: Fish Short-term Reproduction 

Assay. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances 

(7101). EPA 740-C-09-007. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0007 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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Prior to initiating the chemical exposure, breeding groups (consisting of two males and 

four females) of mature fish, typically 4-6 months old, are monitored for a minimum of 14 days 

for survival, behavior, fecundity and fertility. This is to ensure adequate spawning performance 

and to allow for a systematic (typically a randomized block) distribution of the breeding groups 

to individual treatment group replicates. Each treatment group (including the dilution water-only 

negative control) has four replicate tanks. There are typically three chemical treatment 

concentrations and a negative control group. If a solvent carrier is required to facilitate dosing, 

then a solvent control group is also included.

The exposure route for the test chemical is by way of tank water using an appropriate 

flow-through delivery system. Breeding groups are exposed for 21-days during which mortality, 

reproductive behavior, secondary sex characteristics, fecundity and fertility are monitored daily. 

At test termination, additional measurements are collected, which include nuptial tubercle score, 

gonadal histopathology, GSI, and plasma concentrations of VTG. Additionally, plasma sex 

steroids (17β-estradiol and testosterone) may be measured at test termination. Collectively, these 

measurement endpoints are meant to be indicative of the status of the reproductive endocrine 

system.

According to the test guideline, “statistically significant effect in one or more of the core 

endpoints (fecundity, secondary sex characteristics, VTG, GSI, and histopathology) may be 

indicative of the test material to potentially disturb the HPG axis of fishes.” (pg 20). Effects on 

fertility should also be considered when evaluating potential disruptions. Throughout this 

chapter, unless otherwise specified, “HPG axis” is used generically and should be considered 

inclusive of its downstream pathways. As the statistical methods used to analyze study data tend 

to favor detection of monotonic response, any statistically significant effect on one of these 

endpoints may be considered indicative of a “positive” response. Also, the test guideline 

suggests that results which are indicative of a trend but which are not necessarily statistically 

significant at the 95% confidence level may be considered biologically relevant. However, if a 

given treatment results in substantial mortality (> 2 mortalities in any replicate per the guideline) 

or other overt signs of toxicity, responses in the core endpoints may be due to general toxicity 

and not necessarily mediated primarily via interaction with the HPG axis. In such a case, the 

guideline recommends that the lower treatment level(s) be examined for potentially HPG-related 

effects outside of the range of general toxicity.

It is recommended that the suite of responses across the FSTRA and the overall weight of 

evidence across the EDSP Tier I battery be examined collectively to provide a comprehensive 

assessment of any potential to interact with the HPG axis in fish. Furthermore, fish are intended 

to serve as surrogates for other species as HPG pathways are conserved across vertebrate species. 

Data obtained through the FSTRA are considered applicable as a line of evidence in evaluating 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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the potential for a chemical to impact endocrine- mediated processes beyond fish, although the 

degree of applicability for other vertebrates is partially dependent upon other factors such as 

anticipated route of exposure and the pharmacodynamics of a given chemical.

Evaluation of the assay performance was conducted using data submitted in response to 

Tier 1 EDSP test orders. Strengths and previously identified limitations of the assay are 

acknowledged. However, as acknowledged by the 2008 FIFRA SAP, the previously identified 

limitations do not affect the general appropriateness of the experimental design to screen for 

potential endocrine interacting chemicals. The performance of the assay is evaluated using the 

test validity criteria in the guideline as a benchmark for comparison between studies. Finally, 

issues identified during the preliminary review of the submitted studies are discussed. These 

issues do not suggest critical flaws of the FSTRA experimental design, but they may indicate that 

additional, targeted guidance may be warranted to improve the laboratory execution of the assay. 

(ii) Strengths and Limitations of the Assay

The FSTRA is intended for detection of effects potentially associated with disturbance to 

the HPG axis using the suite of measurement endpoints identified in the guideline. The assay is 

conducted using whole fish reflecting an intact HPG axis and its complex feedback mechanisms, 

which may be relevant to other taxa. Since the assay is conducted on mature fish, it reflects 

relevant pharmacokinetics and pharmacodynamics that may impact the potential exposure in 

terms of absorption, distribution, metabolism, and excretion of the test chemical and alterations 

at various levels of biological organization, i.e., biochemical (VTG, plasma steroids), tissue 

(gonad histology), organ (gonado-somatic index), and the whole animal (phenotype, survival). 

The test species, fathead minnow is a routinely-used species for which husbandry conditions are 

relatively well defined thus enabling environmental conditions in the laboratory to be optimized 

to reduce confounding effects on reproduction that might be attributed to test conditions. The 

test design utilizes standard aquatic toxicology methodology where measurement endpoints are 

well defined and accepted internationally as measures of reproductive fitness (OECD 2012). The 

results of this assay have also been reproduced across several laboratories and have yielded 

relatively consistent results for reference chemicals (USEPA 2007a). 

In 2008, a FIFRA SAP charged with evaluating the proposed Tier 1 EDSP battery 

(USEPA 2008) emphasized the importance of the FSTRA because of its inclusion of apical 

endpoints. The inclusion of the apical endpoints may allow for detection of potential endocrine 

active chemicals that may operate by mechanisms not yet specified. The assay also has utility as 

a screen for perturbations of the VTG and secondary sex characteristic endpoints in intact 

animals.

While there are many strengths of the FSTRA, some limitations have been previously 

identified in other summary reports for the FSTRA. The technical document for the FIFRA 
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 89 of 160 

Scientific Advisory Panel in 2008 on the EDSP Tier 1 screening battery (USEPA 200816) 

identified the inherent difficulties of testing poorly soluble test materials and the specialized 

technical expertise that would be required for analysis of the sex steroids. However, difficulties 

in testing poorly soluble materials are generally addressed in Organization for Economic 

Cooperation and Development (OECD) Guidance Document 23: Guidance Document on 

Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000) as well as the 

OCSPP Guideline 850.1000 (draft) (USEPA 199617). 

In an earlier Integrated Summary Report (USEPA 2007), other limitations were discussed 

and included: 

1) VTG analysis requires additional calibration in the individual laboratory; 

2) fecundity can be influenced by both chemical and non-chemical factors [potentially nonendocrine specific toxicity] and requires a pre-exposure period to establish adequate 

spawning; 

3) histology requires additional time and resources; and, 

4) the sensitivity and specificity of secondary sex characteristics are not applicable to all 

modes of action; Secondary sex characteristics are not as amenable to quantitative 

analysis as other endpoints; and the test duration may not be adequate to observe some 

physiological alterations of secondary sex characteristics.

Upon reviewing submitted Tier 1 test order data, it is recognized that for the limitations 

discussed above that involve additional time and/or resources (VTG, plasma sex steroids, 

gonadal histology), the inclusions of these endpoints when blood/tissue samples are available are 

extremely useful measurement endpoints when considered collectively in determining whether a 

chemical is potentially interacting with the endocrine system. The pre-exposure phase to

evaluate fecundity is critical for this assay to establish relatively uniform spawning performance 

and allow for a systematic distribution of breeding groups to replicate treatment tanks. With 

respect to the potential lack of endocrine-mediated specificity of the fecundity endpoint, the 2008 

SAP noted that the fecundity observations were nonetheless “an essential part of apical analysis 

of HPG activity” (USEPA 2008, pg 13). Similarly, although secondary sex characteristics are 

generally evaluated qualitatively or only semi-quantitatively and may vary in their response to 

different modes of action, the 2008 SAP concluded that the presence of the male secondary sex 

characteristic of nuptial tubercles in females was a particularly “robust endpoint to identify 

androgenic compounds” (USEPA 2008, pg 15). Additionally, although the 2008 SAP and prior 

supporting documents noted certain limitations and potential challenges in the performance and 

 

16 USEPA. 2008. Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held on March 25-26, 2008 

to review and consider the Endocrine Disrupter Screening Program (EDSP) Proposed Tier 1 Screening Battery. FIFRA Scientific 

Advisory Panel. Office of Science Coordination and Policy. June 11, 2008. 

17 USEPA. 1996. Ecological Effects Test Guidelines OPPTS 850.1000 Special Considerations for Conducting Aquatic 

Laboratory Studies. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic 

Substances (7101). EPA 7120C096-113. April 1996. 

http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850-1000.pdf
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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conduct of the FSTRA (as with any assay), these limitations did not detract from the overall SAP 

conclusion that the FSTRA was a critical component of the proposed battery (USEPA 2008). 

(iii) Observations from Initial Screening of Chemicals

Currently, of the FSTRAs that tested chemicals of unknown potential for endocrine 

interaction and which were submitted by Test Order Recipients, twenty-one were selected by the 

Agency as case studies to evaluate assay performance, consistency in experimental conduct, and 

interpretation of the results (see Section II). 

Generally, the Agency documents its evaluation of pesticide ecotoxicity or mammalian 

toxicity data in a Data Evaluation Record (DER). Each DER typically reflects a single data 

submission (study); the DER outlines the study conditions, provides a comparison of the reported 

test methods to those outlined in the test guideline (e.g., OCSPP 890.1350), and summarizes and 

discusses the Agency’s interpretation of results based on its own independent analysis of the 

data. In the case of the 21 case study chemicals, the Agency’s reviews of these studies remain 

preliminary. The current review of FSTRA performance is based on these preliminary reviews 

as captured in the associated draft DERs. 

Quality Control 

In the OCSPP 890.1350 guideline, there are six separate test validity elements identified 

(Table 26). These validity criteria provide an indication of the general performance of the 

FSTRA under test conditions. When met, the validity criteria are used to support the scientific 

soundness of a given study and the degree of confidence in its results. An additional test 

performance criterion is that the water quality characteristics described in the test guideline 

should remain within the reported tolerance limits. These characteristics include temperature 

and dissolved oxygen as well as limits on residual chlorine, unionized ammonia, total organic 

carbon and alkalinity. This additional performance criterion is meant to provide information on 

general water characteristics that are typically adequate to promote reproduction and general 

health of the fish. The validity criteria for temperature, control survival, fecundity and fertility 

are also referred to as performance criteria in the guideline (pg 19). In this evaluation, the 

validity criteria were used to evaluate the scientific soundness and level of confidence in the 

results. However, a deviation from a defined test validity criterion does not necessarily indicate 

that the study cannot provide useful information. Rather, as with any of the identified criterion, 

it is anticipated that slight deviations from a criterion will occur and that decisions regarding 

acceptability of a study will be influenced by factors such as the observed variability in the 

endpoint and the magnitude and duration of departure from a criterion. Overall, the conduct and 

results of each study are evaluated as a whole to determine the degree of confidence in the ability 

to discern the potential for endocrine interactions. Each of the identified criteria in Table 26 is 

discussed in more detail below. 
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Table 26. Test Validity Elements for the FSTRA

Criteria Reference Value(s)

Measured Test Concentration Coefficient of 

Variance (CV)

≤20% for all replicates

Control Fertility ≥95% (of eggs produced)

Control Fecundity ≥15 eggs/female/day/replicate or spawning every 4 days 

(per replicate)

Control Survival ≥90%

Dissolved Oxygen ≥60% saturation (at 25°C)

Mean Water Temperature 25 ± 1°C

Evaluations of these six criteria were conducted using the information provided in the 21 

submitted studies. The number of studies that met each of the identified test validity criteria is 

presented in Figure 14. 

Figure 14. Summary of Number of Studies Achieving Each Individual Test Validity 

Criteria

 

Four of the 21 studies met all of the identified test validity criteria (Figure 15). Eleven 

studies met all but one of the listed criteria (Table 27), indicating that 71% of the evaluated 

studies either met all or only missed one of the identified test validity criteria. The six other 

studies had two or more deficiencies. A CV of greater than 20% for test concentration was the 

most common deficiency noted and was observed in multiple laboratories and in both studies 

that did and did not utilize solvent carriers. The increase in CV above the recommended value 

was observed in three studies that also reported decreased dissolved oxygen levels or control 

fecundity rates.

21

15

20

15

17

12

6 

1 

6 

4 

9 

0 5 10 15 20

Mean Temperature 

Dissolved Oxygen 

Control Survival 

Control Fecundity 

Control Fertility 

Test Concentration CV

# of Studies

met

didn't meet
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Page 92 of 160 

Figure 15. Summary of the Number of Individual Test Validity Criteria Achieved by Each 

Study

Table 27. Identification of the Only Test Validity Criteria Not Achieved in 11 Studies 

Test Validity Criteria Number of Studies

Measured Test Concentration Coefficient of Variance (CV) 4

Control Fecundity 3

Dissolved Oxygen 2

Control Survival 1

Control Fertility 1

Water Quality Parameters

Environmental test conditions are described in OCSPP 890.1350 guideline (Table 1 in 

guideline, pg 3) and include dilution water characteristics, appropriate water temperature ranges, 

and minimum dissolved oxygen levels. The guideline test validity criteria for dissolved oxygen 

and water temperature are dissolved oxygen ≥ 60% of saturation (4.9 mg/L at 25°C), and that the 

mean water temperature 25 ± 1°C. Additional water quality characteristics specified in the 

guideline (Table 1 in guideline) include limits on total alkalinity, total organic carbon, unionized 

ammonia and residual chlorine. 

Based on an analysis of the data, the performing laboratories were able to maintain the 

specified mean water temperature of 25 ± 1°C throughout the duration of the test. As depicted in 

Figure 14, all 21 studies reported an overall mean temperature of 25 ± 1°C. However, three of 

the studies were not able to keep the temperature within 1°C between tanks at any one time, and 

in seven other studies there was insufficient temperature data reported to evaluate this. 

all 5 of 6 4 of 6 3 of 6 2 of 6

0 

2 

4 

6 

8 

10

12

# of Met Criteria

# of Studies
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 93 of 160 

Additionally, in two studies, the minimum temperature was reported to have dropped to 23°C. 

However, these types of deviations were not of sufficient duration and/or frequency to affect the 

overall mean temperature and its standard deviation. Short durations of this magnitude of 

temperature fluctuations did not appear to negatively impact the performance of the fish as 

reproductive performance in the affected tanks was within the test validity criteria. 

Dissolved oxygen (DO) levels were also generally maintained above the required 

minimum of 60% saturation. In 15 of the study reports, the individual DO measurements were 

equal to or greater than 60% of saturation. In the other six study reports, at least one 

measurement of DO was below 60% saturation (minimums of 45-58% reported). As DO was 

not measured daily nor in every replicate when measured, the duration of the decline for all the 

studies is unknown. However, in these six studies, once a decrease in DO was measured, efforts 

to raise the DO was initiated, and aeration was generally added to the system sometimes as early 

as at test initiation to Day 2 of the test. In one instance, a drop in DO was measured after 

aeration was initiated; however based on the data provided it appears that this may have occurred 

in two other instances as well. In one case, the temporary drop in DO was reported not to have 

adversely affected the interpretation of the study; the other five studies did not speculate on the 

potential impact. 

In addition to the specified test validity criteria for temperature and DO in Table 1 of the 

guideline, measured limits on several dilution water characteristics were also listed. These 

included total alkalinity, total organic carbon (TOC), unionized ammonia and residual chlorine. 

All of the study reports measured total alkalinity from the control and treatment group test 

solutions, and the reported values were greater than the minimum level specified in Table 1 of 

the guideline. Seventeen of the 21 studies reported measuring TOC, but values were only 

reported for 10 of the studies which were all below the maximum level specified in the guideline. 

Measurements of residual chlorine levels were reported in eight of the studies. However, only 

two of the studies had reported residual chlorine levels below the guideline maximum level 

whereas for the other six, the limit of detection was greater than the maximum recommended 

level. Unionized ammonia levels were recorded in one study which was below the guideline 

maximum level; six other studies reported the nitrogen levels (as either ammonia (as N) or 

ammonium (as N)) in the dilution source water. In these 21 studies, the reported levels of total 

organic carbon, unionized ammonia and residual chlorine were from periodic samples collected 

from the dilution water house source and not from the dilution water collected directly from the 

study chambers. The laboratories that performed these 21 FSTRAs routinely perform other 

ecotoxicological studies in support of pesticide registrations. As such, historical data are 

available on the quality of the dilution water source used in these studies. In general and based 

on the historical data from specific laboratories, the quality of dilution water sources used in 

these studies has been adequate to promote good health and reproduction in fathead minnow.
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Control Survival 

The test validity criterion in the guideline for control survival is 90%. This criterion of 

90% survival applies to the negative control and, if used, the solvent control as well. In all but 

one study, overall control survival was equal to or greater than 90%. In the one study, the 

control survival was 83%. While not used as test validity criteria, the survival of each gender 

between the controls and treatment groups was evaluated to determine if mortality may have 

been gender specific. While no evidence of gender-specific mortality was observed, except for 

in one study, tracking the gender-specific mortality is useful when trying to interpret the results 

for other endpoints. This is especially true for the gender-specific endpoints as a slight decrease 

in the sample size for males or females may have an impact on the ability to reliably interpret or 

discern differences in sublethal endpoints. In regards to mortality, the guideline specifies that if 

all the treatment groups exhibit mortality, then the assay should be repeated at lower test 

concentrations to be better enable reliable inferences about possible endocrine interactions. 

Observations of non-lethal effects such as morbidity and other clinical signs of toxicity (e.g., loss 

of equilibrium and decreased feeding) should also be considered when evaluating whether a test 

should be repeated and also when evaluating for potential endocrine interactions. Overt toxicity 

is described in the guideline as “>2 mortalities in any replicate that can only be explained by 

toxicity rather than technical error” (pg 18). Other signs of overt toxicity identified in the 

guideline include “hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and 

any other clinical signs of disease”. 

Control Fecundity

The guideline indicates that for a successful study, the fish in the control(s) should be 

actively spawning. This can be indicated by the fish spawning at least every 4 days or 

reproducing at a rate of 15 eggs/female/reproductive day/replicate (abbreviated: E/F/D/R) 

during the exposure phase of the assay. While not a test validity criteria, the guideline states that 

in the pre-exposure phase, the fish should spawn at least two times in the 7 days immediately 

preceding the exposure phase and that egg production should exceed 15 eggs per female per 

reproductive day per replicate. In terms of the spawning groups in the pre-exposure phase that 

were carried over into the actual exposure, the majority (20/21) of the evaluated studies met the 

pre-exposure recommended spawning frequency and/or fecundity rate. In the one remaining 

study, the pre-exposure production rate was <15 E/F/D/R for all but two of the tanks used in the 

exposure phase (range approximately 6-16 E/F/D/R); there were insufficient data reported to 

determine whether fish in these two tanks had spawned two times in the 7 days prior to exposure. 

Spawning performance of controls during the exposure phase for each of the 21 studies 

was evaluated. In 17 of the 21 studies, the overall control (includes negative and solvent) mean 

fecundity was >15 E/F/D/R and generally was >20 E/F/D/R. The negative control mean 

fecundity for the other four studies ranged from 8.8-12 E/F/D/R; two of the studies also had a 

solvent control with fecundity values of 6.5 and 13 E/F/D/R. Based on the wording in the 
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guideline, adequate spawning is to be demonstrated in all replicates of the control. Therefore, 

the stated criteria were evaluated against each replicate in the negative control, and solvent 

control if present. On an individual replicate basis, 15 of the studies met the fecundity 

performance criterion. In the other six studies, for at least one replicate in the negative control, 

and solvent control if present, the fecundity was <15 E/F/D and the spawning interval was 

greater than 4 days. It should be noted that in10 studies for at least one control replicate the 

reported fecundity was >15 E/F/D, but spawning still did not occur every 4 days; however, the 

number of eggs produced allowed for demonstration of adequate spawning. It has been reported 

that on average, spawning will occur every 3 - 4 days; however, longer intervals of as much as 

15 days have also been observed (Jensen et al., 2001 in USEPA 2007(ISR report). 

In reviewing the submitted data, it was observed that laboratories are calculating 

fecundity in two slightly different ways. Some of the labs are calculating a daily fecundity rate 

in each replicate expressed as ‘number of eggs/female’ and then taking the mean of the 21 daily 

fecundity estimates for an overall mean ‘number of eggs/female/reproductive day’. Other labs 

are summing the total number of eggs produced and the total number of female reproduction 

days over the 21 days and dividing the two which is then expressed as ‘number of eggs/female 

reproduction day’. Both methods take into account female mortality that occurs during the test. 

If there is no female mortality in the tank, then the overall mean value is the same regardless of 

which method is used. However, if there is mortality, then there may be differences in the 

overall fecundity value for that replicate. Both methods of calculating fecundity have been 

presented in previously published documents on the development of this assay. As long as the 

method is consistent within a study, either methodology should be acceptable. 

Control Fertility

The guideline reported test validity criterion for fertility is ≥95% for eggs produced in the 

control(s). As depicted in Figure 14, in 17 of the studies, the overall mean fertility was ≥95% in 

the controls. In the other four studies, the overall mean fertility ranged from 90-94%. 
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Variation in Measured Test Concentrations

The validity criterion for the CV in a measured test concentration over the duration of the 

exposure period is reported as 20% in the guideline where the reported CVs are based on the 21-

day treatment mean or time-weighted mean test concentration and its associated standard 

deviation. There was a wide range of test chemical solubility and/or stability across the test 

chemicals evaluated. In some cases, a chemical’s low solubility and/or instability influenced its 

overall ability to be maintained at a consistent concentration in the water column. As depicted in 

Figure 14, 12 of the 21 studies maintained CV’s within 20% during the exposure period. In the 

other nine studies, CVs ranged from slightly greater than 20% to up to 80% (Figure 16). A 

solvent carrier was used in eight of the 21 studies, whereas a saturator column was used to 

generate test solution in three other studies. CV values higher than 20% were generally 

attributed to poor water solubility or the adsorptive properties of the test chemical (e.g. sorbed to 

aquaria glass). Generally, laboratories took steps to reduce the variation in the overall measured 

concentrations. These steps included increasing the delivery flow rate of the test solutions and 

cleaning the tanks more frequently. Some laboratories included additional analytical 

measurements to better characterize the actual test concentrations over time. However, CV 

values that exceed the stated performance criterion will likely continue to be associated with 

chemicals with limited water solubility, adsorption and/ or degradation properties. CV values 

greater than 20% may be considered acceptable on a case-by-case basis if the time-weighted 

average can be estimated and demonstrated not to overlap between test levels.

Figure 16. Summary of the Range of Reported Coefficient of Variation (CV) for the Studies

 

0 

2 

4 

6 

8 

10

12

14

≤ 20% up to 30% up to 40% up to 50% up to 80%

# of Studies

Range of Reported CVs
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(iv) Adequate Dosing and Overt Toxicity 

The guideline states that the highest concentration tested should be one that does not 

result in increased mortality or signs of morbidity compared to the control. The use of 1⁄3 of a 

derived 96-hr LC50, using fish of similar age and size as those used in the FSTRA, is suggested as 

an option for establishing the high test concentration, with a 10-fold decrease between 

subsequent test concentrations. It is important to demonstrate though that the study was 

conducted at a high enough concentration to allow for the possible observation of effects. The 

guideline alternatively suggests that, in the absence of observed toxicity, the highest 

concentration may be set at the solubility limit of the chemical in water or at the historical limit 

test concentration of 100 mg/L (part per million; ppm). Twelve of the 21 evaluated studies used, 

at least in part, 1⁄3 of an LC50 value when setting the highest test concentration (Table 28). Other 

studies used a variation of a reported maximum tolerated concentration, solubility limit or the 

maximum test concentration of 100 mg/L to set the high concentration. When evaluating the 

results, the use of a 1⁄3 LC50 value sometimes resulted in substantial mortality as well as effects 

on physical appearance, behavior and/or changes in body weight. The LC50 value used in dose 

selection was sometimes generated using fish of similar age and size, but other times was from 

studies with juvenile fish and in some cases even a different species. In 17 of the 21 studies, 

effects on survival, decreased body weight and/or observation of clinical toxicity were reported 

for at least one test concentration (Table 28). For some sublethal effects (e.g., body weight) it is 

not always clear if the observed effect is due to overt toxicity, but was included in the table to 

illustrate the types of lethal and sublethal effects observed. 
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Table 28. Summary of the Types of Overt Toxicity Observed in the 20 Assays 

Chemical

Code

Type of Effect Observed (test levels affected1) Method Used to Set High 

Survival Test Concentration 2 Sublethal clinical signs of toxicity3 Body 

Weight2,4 

O − − − Max. necessary conc. (100 

mg/L)

E High − − 1⁄3 96h LC50

I − High (discoloration, loss of 

equilibrium) High 14-d MTC 

C − − High Solubility

A − High (abnormal swimming) Mid, High Not reported

H − High (discoloration) − Not reported

Q −5 − − 1⁄3 96h NOAEC

J − − High 1⁄3 96h LC50

K High6 Mid, High (loss of equilibrium, 

lethargy , deformed) − 1⁄3 96h LC50

G − − Low, 

High8 1⁄3 LC50 (unknown duration)

L − − − ELS

T − − Low, Mid, 

High 1⁄3 96h LC50

M High − − 1⁄3 96h LC50

U − − Low, Mid, 

High Max. necessary conc.

S High Not well reported − 1⁄3 LC50 (duration unknown)

N − High (decreased feeding, emaciated) High 1⁄3 96h LC50 & ELS

P High High (decreased feeding, abnormal 

swimming) − 1⁄3 96h LC50 

D −7 − − 1⁄3 96h LC50 & NOAEC

B − High (decreased feeding) Mid, High Solubility

F − High (loss of equilibrium, 

discolored, paralysis) High8 1⁄3 96h LC50

T − Not well reported − Not well reported

Total # of 

Studies 

7 8 10 --

13 (reported effects on survival or clinical signs of 

toxicity)

-- --

17 (reported effects on survival, clinical signs of toxicity, or 

decreased body weight)

--
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‘—‘ = no effects observed; MTC = maximum tolerated concentration; ELS = fish early life-stage test 1 The three test concentrations in the assays referred to as low, mid, and high

2 Indicates a significant (p≤0.05) overall effect at the listed test concentration

3 Clinical signs of toxicity (other than survival and growth) were visually evaluated.

4 Changes in body wt. may or may not be due to overt toxicity. Other lines of evidence from the study should be used to 

distinguish between overt toxicity and endocrine mediated changes

5 While overall survival was not significantly (p>0.05) decreased, one replicate at high concentration had >2 mortalities

6 Additionally, one replicate at low concentration had >2 mortalities 7 While overall survival was not significantly (p>0.05) decreased, where was a significant decrease in female survival at the high 

concentration

8 Significant increase in body weight compared to control (p≤0.05), so likely not indicative of overt toxicity.

Overt toxicity is described in the guideline (pg 18) as “>2 mortalities in any replicate that 

can only be explained by toxicity rather than technical error” (pg 18). Other signs of overt 

toxicity identified in the guideline include “hemorrhage, abnormal behaviors, abnormal 

swimming patterns, anorexia, and any other clinical signs of disease”. According to the 

guideline, if such signs of overt toxicity are observed then the replicate or possibly the entire test 

concentration should be removed from statistical analysis. This definition of ‘overt toxicity’ is 

broad and may be interpreted differently among laboratory investigators or reviewers. 

Additionally, if overt toxicity is observed, and a 10-fold decrease in test concentration is used, 

the large differences between concentrations may limit the ability to observe potential chemical 

interactions with the endocrine system as the next lower concentration may result in responses 

that are statistically indistinguishable from control(s). It is recognized that changes in some 

endpoints (e.g., fecundity) in this assay may be due to non-endocrine mediated pathways, and the 

observance of clinical signs of toxicity should be used to help distinguish those pathways. 

However, for some sublethal endpoints (e.g., changes in body weight), it is not definitively 

known if the observed effect was due to a non-endocrine mode of action or possibly the result of 

a downstream effect from a modification to the endocrine system. 

Survival 

In 21 of the evaluated studies, there were reported effects on survival in seven studies. 

Five studies (29%) reported statistically significant effects (p≤0.05) on overall survival (males 

and females combined) at the highest concentration tested. In four of the five studies, the percent 

mortality in the highest treatment group at test termination ranged from 29 to 92%, and in the 

fifth study, the highest treatment group was terminated during the study due to >30% of the fish 

showing signs of overt toxicity (i.e., mortality, loss of equilibrium). In the sixth study, there was 

a significant decrease in female mortality at the high concentration compared to the control. 

Lastly, in the seventh study, at the highest concentration tested, there was one replicate with 

more than 2 mortalities and per the guideline, this replicate may be considered for exclusion in 

the data analysis; it is expected that inclusion or exclusion of replicates/treatments will be analyst 

dependent. In addition to the seven studies with effects on survival at the highest concentration 

tested, six other studies reported sublethal clinical signs of toxicity at the highest concentration 
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tested. The reported sublethal effects ranged in severity and included decreased feeding and 

lethargy, abnormal swimming and loss of equilibrium to complete paralysis. 

Body Weight 

Body wet weight and length are measured in the surviving fish at test termination. In 

only one study were length and weight significantly decreased (p≤0.05) at the highest 

concentration compared to the control. In 5 of the 21 studies, significant (p≤0.05) changes in 

male or female body weight were observed at concentrations where other sublethal and/or lethal 

effects were also reported. However, in four other studies, significant (p≤0.05) decreases on 

body weight were reported at concentrations where there were no other sublethal or lethal effects 

reported. Another study reported a significant increase in body weight compared to the control 

with no reported overt toxicity; whereas there was one study that reported a significant increase 

in weight in the presence of observed overt toxicity. The magnitude difference in body weight in 

the studies that reported a significant difference relative to controls varied but was generally 

between 10 and 30%. 

For females, the timing of the last spawning event relative to test termination may 

influence overall body weight for an individual. Evaluation of body weight was not based on 

changes in body weight over the course of the study but rather was based on the absolute body 

weights at test termination. The test is not designed for evaluating changes of an individual fish’s 

body weight throughout the exposure period; although, tracking body weight by gender in a 

replicate is possible, it would require more effort. This could be accomplished, for example, by 

recording not only the female body weight but also which replicate tank it was placed into during 

the pre-exposure phase and then using the test termination body weights to calculate a change in 

body weight for females for that replicate. However, this value would represent both the preexposure as well as the treatment group exposure which could confound the interpretation. Prior 

to distribution to the pre-exposure tanks, each fish is weighed and the goal is to keep the wet 

weight with ±20% of the arithmetic mean wet weight for the same gender. This mean wet 

weight is determined from a sub-set of the population of fish to be used in the study. While most 

of the studies reported that the initial wet weight was within 20%, there were four studies that 

reported a range of approximately 30%. As there is allowed variability in the initial wet weight 

of the fish and weights of individual fish are not tracked throughout the exposure, anticipating 

whether a given magnitude of change will result in a statistically significant difference may not 

be feasible. In terms of whether a change in body weight, compared to the control, is the result 

of generalized overt toxicity or an alteration in an endocrine mediated pathway is not always 

clear. The collective evaluation of the other measured endpoints, especially the endocrine 

specific endpoints, as well as reported feeding behavior, if available, is key toward determining 

the potential cause for the change in body weight. 
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As lethal and/or sublethal effects (not including changes in body weight) were reported in 

at least the highest test concentration in 13 of the 21 evaluated studies, this could lead to the 

exclusion of at least one treatment concentration from the analysis. Furthermore, in an additional 

four studies, fish exhibited decreases in body weight which may or may not be due to endocrine 

interactions. Given that there were generally three test concentrations, the interpretation of 

potential endocrine interactions is then limited to two concentrations which are generally 10 and 

100 times lower than where the lethal and/or sublethal effects were observed. Additionally, 

given that there would only be two test concentrations for data analysis, the ability to observe 

potential increasing or decreasing trends compared to the control for an endpoint is more limited.

The Use of Solvent Carriers in the FSTRA

The use of a solvent carrier (co-solvent) to facilitate dosing is discouraged in the FSTRA 

test guideline. Co-solvents are discouraged as they have the potential to interfere with the 

interpretation of the results by potentially modifying the fish’s response to a chemical or 

increasing the microbial community in a tank which may influence water quality, food 

availability, and/or potential degradation of the test chemical. Co-solvents were used in seven of 

the 21 evaluated studies. Acetone and dimethyl-formamide (DMF) were the reported solvents, 

with all solvent concentrations at or below the maximum recommended concentration of 0.1 

mL/L in regulatory fish chronic toxicity guidelines (OCSPP 850.140018; OECD 21019). In five 

of the seven studies where co-solvent was used, the solvent concentration was consistent (i.e., at 

the same concentration) between the solvent control and all the treatment groups. The other two 

studies utilized proportional diluters and only the highest test chemical concentration had the 

same solvent concentration as the solvent control. 

If a co-solvent was used, an evaluation of potential solvent interferences is conducted by 

considering results from a concurrent solvent control group. If there are statistically significant 

differences detected between the negative (dilution water only) control and the solvent control 

for a given endpoint(s), then best professional judgment may be used by Agency reviewers to 

evaluate if the study is valid. In one of the seven studies which utilized a co-solvent, there was a 

significant (p≤0.05) reduction of 34% in fecundity between the solvent (DMF 0.04mL/L) and 

negative control; female body weight was also significantly (p≤0.05) increased by 11% relative 

to the negative control. Reduced dissolved oxygen (<60% saturation) was reported in four of the 

seven studies which utilized solvents (3 used DMF and one acetone), an observation perhaps 

reflective of enhanced microbial growth. However, the decreases in DO appeared to be relatively 

short in duration and the laboratories did take measures to rectify the situation. Overall, it 

 

18 USEPA. 1996. Ecological Effects Test Guidelines OPPTS 850.1400 Fish Early-Life Stage Toxicity Test. Office of Chemical 

Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 7120C096-113. 

April 1996. 

http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850-1400.pdf 19 OECD 1992. Test No. 210: Fish, Early-life stage toxicity test. Organisation of Economic Co-operation and Development. 

http://www.oecd-ilibrary.org/docserver/download/9721001e.pdf?expires=1361831113&id=id&accname=guest&checksum

=8F099EABD91311E2B51F27DA93614DE4
USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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appears that in one of these studies that utilized a co-solvent, that there was a potential solvent 

interference. 

Incorrectly Sexed Fish in the FSTRA

In this test, fish are distributed into the test chambers in a pre-determined sex ratio of two 

males and four females per replicate. The guideline recognizes the potential for incorrectly 

identifying the sex of a fish (referred to as a mis-sexed fish) and recommends that additional 

tanks be established in the pre-exposure phase to ensure adequate sample size at test initiation. 

Upon evaluation of the 21 FSTRAs, six of the 21 studies had at least one male fish mistaken for 

a female but the error was not caught until test termination. Three of these studies contained one 

misidentified fish, with the other three studies resulting in 2, 3, and 5 replicates with a 

misidentified fish. In all of these studies, calculation of fecundity in these replicates with the 

mis-sexed fish was adjusted to account for the additional male. However, for the other genderspecific endpoints, some laboratories included measurements from the mis-sexed fish in the 

analyses while other labs excluded the fish. As these fish were most likely not expressing male 

characteristics (i.e., may have been immature males) prior to test chemical exposure, the Agency 

considered it appropriate to exclude them from data analyses. 

Reasons for mis-sexed fish vary but may include technician inexperience and/or the use 

of insufficiently mature fish. The guideline states that the fish should be within the range of 4.5 

to 6 months old and reproductively mature; this assumes the fish were cultured at 25±2°C with 

adequate feed. The OECD 229 FSTRA also provides guidance on fish selection, including 

recommended mean wet weights20. Given that there may be slight differences in time to 

maturation between batches of fish in a laboratory and among laboratories, fish between 4.5 to 6 

months old for a given cohort may not always be in appropriate reproductive status necessary for 

this assay. However, as there are well-defined culturing protocols for fathead minnows (ASTM 

200521, Benoit 198222, Brauhn, and Schoettger 197523, and USEPA 198724), the likelihood of 

this occurring is expected to be low. In 18 of the 21 evaluated assays, the reported age of the fish 

at the beginning of pre-exposure was within the 4.5 to 6 months age range, with all but one 

ranging from 5 to 6 months. The age for two other studies was between 6 – 7 months, and the 

final study indicated that the fish were at least 120 days old. For the assays with the mis-sexed

fish, the reported age ranged from 4.5 to 7 months with no discernible pattern. To increase the 

 

20 OECD. 2009. Fish Short Term Reproduction Assay. Test No. 229. Organisation of Economic Co-operation and Development . 

http://miranda.sourceoecd.org/vl=68881146/cl=11/nw=1/rpsv/cgi-bin/fulltextew.pl?prpsv=/ij/oecdjournals/1607310x/v1n2/s30/p1.idx

(accessed February 6, 2013). 

21 American Society for Testing and Materials. ASTM E 1241-05, Standard Guide for Conducting Early Life-Stage Toxicity 

Tests with Fishes. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved 

2005.

22 Benoit, D.A., 1982. User’s guide for conducting life-cycle chronic toxicity tests with fathead minnows (Pimephales promelas). 

Environ. Res. Lab - Duluth, Duluth, MN. EPA 600/8-81-011. 23 Brauhn, J.L. and Schoettger, R.A., 1975. Acquisition and Culture of Research Fish: Rainbow trout, Fathead minnows, Channel 

catfish and Bluegills. p. 54, Ecological Research Series, EPA-660/3-75-011, Duluth, MN 24 Denny, J.S..1988 Guidelines for the culture of fathead minnows, Pimephales promelas for use in toxicity tests U.S 

Environmental Protection Agency, Environmental Research Laboratory. Duluth, MN. EPA/ 600/ S 3-87/001
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likelihood of success, it is considered important to have a well-established healthy culture of 

fathead minnows (i.e., proper density, adequate feed and environmental conditions) from which 

to draw upon for each test. Having a culture that is sufficient in numbers as well as old enough 

to establish fully mature females (i.e., the presence of a fully-developed ovipositor) should help 

reduce the incidence of mis-sexed fish. As investigators gain experience with this assay, the 

incidence of mis-sexing fish may diminish; however, it still may occur from time to time. While 

there is potential for incorrect sexing, the presumption was that either the mis-sexed fish would 

be identified during the pre-exposure period and excluded from further chemical exposure or, if 

they were not identified in the pre-exposure period, that the occurrence would be minimal and 

not have a substantive impact on the study conclusions. However, if the presence of mis-sexed 

fish occurs, it will influence the analysis of the data for endpoints such as fecundity and gender -

specific endpoints (i.e. VTG and GSI) as well as potentially increase the variability for a 

measured endpoint. 

(v) Measured Endpoints in the FSTRA

The FSTRA is intended to provide insight on whether a chemical may interact with the 

HPG axis. The FSTRA has been validated using chemicals with known modes of action (MOA, 

for discussion see Ankley et al. 2010) and is expected to provide information on a chemical’s 

potential to disrupt any of several endocrine pathways (e.g., estrogen, androgen, steriodogenesis) 

related to the HPG axis (USEPA 2007a). Reviewing the results from a chemical with a known 

mode of action may be useful when evaluating the results a chemical with an ‘unknown’ 

potential for endocrine interaction. This review included chemicals with previously unknown 

potential for endocrine interaction. This section will discuss each of the endpoints in the assay 

and the preliminary results of these endpoints for each of the 21 available assays. In some cases, 

significant differences (p≤0.05) were reported for endpoints at concentrations where effects on 

survival or other clinical signs of toxicity were reported. Additionally, effects on endpoints were 

reported at concentrations that also reported effects on body weight. 

Fecundity and Fertility: As pointed out previously, the 2008 SAP noted that the fecundity 

observations were “an essential part of apical analysis of HPG activity” (USEPA 2008, pg 13). 

Significant (p≤0.05) reductions in fecundity were reported in 16 of the 21 assays in at least one 

test concentration. In eight of the studies, there were statistically significant (p ≤ 0.05) effects on 

fecundity only at the same concentration as reported effects on survival, female body weight or 

measures of clinical toxicity. However, in four of the 16 studies, there was a significant decrease 

in fecundity without a reported lethal or sublethal effect in the study. Lastly, in the four other 

studies, overt toxicity was observed in some, but not all, of the concentrations that reported a 

significant decrease in fecundity. 
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Fertility was reported to be significantly decreased in 9 of the 21 assays for at least one 

test concentration. In seven of these studies, effects on fertility were only reported at 

concentrations that had effects on survival, male body weight or other clinical signs of toxicity. 

Secondary Sex Characteristics: Evaluation of secondary sex characteristics in the FSTRA are 

evaluated both qualitatively (e.g., presence of vertical banding, fat pad (dorsal nape pad), 

ovipositor, body coloration) and semi-quantitatively (i.e., nuptial tubercles and sometimes fat 

pad) in males and females. Nuptial tubercles are located on the head (dorsal pad) of 

reproductively active male fathead minnows, and are not exhibited by control females and 

juvenile males and females (Jensen et al. 2001). However, exposure to certain types of endocrine 

disrupting chemicals may cause the appearance of nuptial tubercles in females (androgen) or 

diminish them in males (estrogens). In this assay, if present on a fish, the nuptial tubercles are 

enumerated and graded based on prominence, and the overall scores between the control and 

treatment groups are compared. In the 21 assays evaluated, no nuptial tubercles were observed 

in either control females or in females exposed to the test chemicals. Control male fish did 

exhibit nuptial tubercles, expect for the four fish that were mistakenly identified as females (missexed) during the distribution of the fish to the pre-exposure tanks. Eight of the 21 assays 

reported a statistically significant decrease (p≤0.05) in male nuptial tubercle scores compared to 

the control, and these effects were only reported at test concentrations with overt toxicity for six 

of these studies. In an additional study, a decrease in tubercles was reported at test 

concentrations both with and without reported overt toxicity. In the final study, all reported 

effects were in the absence of overt toxicity.

Gonado-somatic Index (GSI): The gonado-somatic index (GSI) is calculated for each fish at test 

termination. As with the other endpoints identified as core endpoints in the test guideline, GSI is 

evaluated in concert with other endpoints to determine potential chemical-induced effects on the 

reproductive system of a fish. The guideline states that typical GSI values for reproductively 

active fathead minnows range from 8 to 13% for females and from 1 to 2% for males. For the 21 

assays, the range of male GSI in the controls was 0.98 – 1.75% with a median value of 1.3% and 

was 11.1 – 16.4% with a median value of 13% for control females. Therefore, the reported GSIs 

appear to be generally within typical values. For males, there were three studies out of the 21 

which reported a statistically significant (p<0.05) decrease in GSI for at least one test 

concentration compared to the control with another four studies reporting a statistically 

significant (p<0.05) increase in GSI. In three of these seven studies, the change was reported 

only at concentrations where there was an effect on survival, male body weight or other clinical 

signs of toxicity were reported; however, this was not the case for other concentrations in four of 

the studies. For females, a statistically significant (p≤0.05) decrease in GSI was reported for 

three studies as well as a significant increase in three other studies. In two of these studies, the 

effect was not dose-dependent, and signs of potential overt toxicity were reported in 

corresponding concentrations in three of the studies. 
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Vitellogenin (VTG): Vitellogenin, an egg yolk precursor glycolipoprotein, is an important 

endpoint in the FSTRA for evaluating if a chemical is potentially interacting with the endocrine 

system and has been characterized as the most valuable biochemical endpoint in this assay 

(USEPA 2007a). VTG levels are normally very low in healthy male fathead minnows, but VTG 

production can be induced in both male and female fathead minnows when exposed to estrogenic 

chemicals, whereas other types of endocrine disrupting chemicals (e.g., androgens) can cause a 

decrease in female VTG (USEPA 2007a). Upon review of the 21 assays, a significant (p≤0.05) 

increase in VTG in a treatment group compared to the control was observed in males for two 

studies whereas a significant decrease (p≤0.05) in VTG was reported in another study. In two of 

these three studies, effects on survival or other clinical signs of toxicity were reported at the 

same concentration as the significant change in VTG. For females, a significant decrease

(p≤0.05) in VTG was reported in four of the 21 studies, and a significant increase (p≤0.05) was 

reported in another study. In three of these studies, effects on female VTG were only reported in 

the same concentrations that had survival effects or other clinical signs of toxicity. 

When reviewing the VTG results, occasionally the mean VTG value for a test 

concentration visually appeared to be substantially greater or lower than the control mean VTG 

(treatment mean based on the average of the four replicate means). This was observed in nine of 

the studies for male VTG and in two of the studies for female VTG. However, none of these 

cases was not statistically significantly different, and may not have been dose-responsive. As 

male VTG is normally very low and the measured concentration can be near the limit of 

detection in an assay, considerable variation in plasma VTG can occur. Examination of the CVs 

for these treatments also provided insight on the variability within a treatment group. 

Additionally, plotting the data is an effective means of determining whether an observed increase 

or decrease in mean VTG in a treatment group was due to a marked change in a limited number 

of fish or if it represented an overall change in response in the exposed fish. Data from one of 

the 21 studies demonstrating where male VTG was statistically significant in a treatment group 

compared to the control as well for another study in which it was not significant is shown in 

Figure 17. As depicted in the Figure 17B for Chemical A, all of the male VTG values are similar 

except for two values in the mid concentration (L2), illustrating the magnitude of variability 

sometimes observed in the 21 evaluated assays. For Chemical L (depicted in Figure 17A), the 

highest treatment group was significantly increased (p≤0.05) compared to the control, likely due 

to two outliers, with greater variability observed in this treatment compared to the control and 

lower treatment groups. Generating a simple plot of the data and looking for general trends is 

not limited to VTG and may be beneficial for other endpoints as well. 
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Figure 17. Male VTG data. 

Figure 17.A demonstrates male VTG data (ng/mL) from Chemical L where the high 

treatment group was statistically different (p≤0.05) than the control. 

A (Chemical L).
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Figure 17.B is presenting data from Chemical A where there were no significant effects 

(p>0.05). The median of the individual data values is represented by the solid line, with the 

box representing the 25th and 75th percentile and the whiskers are the data range, with 

suspected outliers denoted by a small open circle next to the data point.

B (Chemical A)

Sex Steroids 

Currently, analysis of plasma sex steroids (17β-estradiol and testosterone) is an optional 

measurement endpoint in the test guideline. Due to the small blood volumes available from 

fathead minnow, an individual sample of adequate volume is not always available after removing 

a sample for VTG analysis. While routine in many mammalian toxicology studies, sex steroid 

measurements have not historically been required in most regulatory ecotoxicity studies and labs 

performing the FSTRA may have limited experience with performing the immunoassays and 

reporting the relevant data. In the 21 studies evaluated, only three analyzed sex steroids with 

effects reported for females in one study and for males in another study. In these cases, 

availability of the sex steroid data improved the Agency’s ability to interpret any effects on the 

other endpoints. 

11,700
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Gonadal Histopathology 

Histopathology is another important endpoint in the FSTRA because it provides a direct 

evaluation of the gonads, and changes observed can encompass the integration of several 

molecular, cellular and physiologic processes. In doing so, histopathology can provide insight 

on the potential reproductive impacts of chemical disruption, and it is useful to confirm changes 

in other assay endpoints (USEPA 2007). Histopathological evaluation of the gonads of surviving 

male and female fish is conducted on fixed specimens collected at test termination. 

Histopathology guidelines for the fathead minnow (Pimephales promelas) are provided in 

Appendix E of the guideline. The guideline is intended to provide general direction on 

evaluating gonadal tissue sections, promote awareness of potential pathological findings, and to 

foster consistency in diagnostic terminology. 

Gonadal staging as well as individual pathological findings were reported in the studies. 

Increase in oocyte atresia and changes in sperm maturation were the two main histopathological 

observations reported in the evaluated data set. Ten studies reported an increase in oocyte atresia 

and 8 studies reported changes in sperm maturation. In eight of these studies, effects on survival 

and/or other clinical signs of toxicity were recorded in the same treatments as the reported 

histopathological observations. In a few other studies, the observed findings did not appear to be 

treatment-related. 

(vi) Statistical Approach for the FSTRA

Statistical analysis of the data for 20 assays generally followed the statistical path 

outlined in Figure 1 in the guideline and its supporting text (USEPA 2009, pg 17); one other 

assay used slightly different statistical tests than outlined in the guideline. All statistical tests 

were performed at the 0.05 significance level (α = 0.05). The guideline states that “for all 

continuous quantitative endpoints (i.e., fecundity, VTG, GSI) consistent with a monotonic doseresponse, the Jonckheere-Terpstra test should be applied in step-down manner to establish a 

significant treatment effect” (USEPA 2009, pg 15). For the endpoints in the evaluated assays, 

monotonicity was visually assessed using professional judgment by examining the treatment 

and/or replicate means or medians. Generally, a trend test was used by reviewers when the mean 

or median values, as applicable, for each test concentration followed a clear increasing or 

decreasing pattern. Per the guideline, the non-parametric Jonckheere-Terpstra test was used for 

monotonic data sets regardless of whether the data met the assumptions for normality and 

variance homogeneity. The test was run under a one-sided hypothesis, with the direction based 

on whether the data was monotonically increasing or decreasing.

For all endpoints, if the data were not judged to be consistent with a monotonic doseresponse, the data were assessed for normality and variance homogeneity. If the data met these 

assumptions for parametric data, Dunnett’s test was used to analyze the data; if not, then the nonparametric Mann-Whitney Wilcoxon U test was used. Both tests were run under a two-sided 
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hypothesis in order to detect both increases and decreases in the data. Per current Agency 

practice, when data do not meet normality or homogeneity assumptions for parametric tests, 

transformations are not applied and analysis proceeds directly to non-parametric methods. The 

statistical approach for the FSTRA and the potential impact of alternative statistical methods are 

discussed in more detail in Appendix 1.

(vii) Overall Conclusions for the FSTRA

In terms of the test validity criteria depicted in Figure 14, the FSTRA is generally being 

performed successfully. Four of the 21 reviewed assays met all the validity criteria, whereas 

another 11 assays deviated from only one of the six identified criteria. In the cases of DO, if 

decreases were observed, the laboratories appeared to be responsive to rectifying the situation. 

This was also the case for measured concentrations of some chemicals which are considered 

more difficult to test due to their physio-chemical properties, as investigators generally took 

measures to try to regulate exposure. Maintaining consistent test concentrations is expected to 

be challenging for difficult to test substances and as laboratories gain experience with nonsolvent delivery methods, this observed variability should decrease, but is not expected to be 

eliminated. It is re-iterated, that a deviation from a defined test validity criterion does not 

necessarily indicate that the study cannot provide useful information, but rather, the conduct and 

results of each study are evaluated as a whole to determine the degree of confidence in the ability 

to discern the potential for endocrine interactions.

Statistically significant (p≤0.05) effects on survival and/or the observation of overt 

toxicity were observed in the highest concentration tested in 13 of the submitted studies with 

another four studies reporting a decrease in body weight. Evaluation of potential endocrine 

interaction needs to be considered in the context of whether overt toxicity was present in the 

study and the degree to which is was observed.

Thirty percent of the studies contained at least one replicate with incorrectly sexed fish. 

If a misidentified fish is observed, while adjustments were made to endpoints such as fecundity, 

it may also be appropriate to exclude the misidentified fish from the data analysis for the gender 

specific endpoints. If there are numerous misidentified fish in a study, the potential implications 

in the interpretation of the results should be addressed. Ultimately, as laboratories gain 

experience with this assay, the occurrence of mis-sexed fish is expected to decrease.

Although the measurement of plasma sex steroids is an optional endpoint, the endpoints 

are endocrine specific and are anticipated to provide a very useful line of evidence on whether a 

chemical is potentially impacting the HPG axis. Therefore, the Agency emphasizes the 

importance of measuring sex steroids when the blood sample volume is sufficient.
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f. Amphibian Metamorphosis Assay 

(i) Background 

The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to 

empirically identify substances which may interfere with the normal function of the 

hypothalamic-pituitary-thyroid (HPT) axis (Fort et al. 2007). The AMA represents a generalized 

vertebrate model to the extent that it is based on the conserved structures and functions of the 

HPT axis. It is an important assay because amphibian metamorphosis provides a well-studied, 

thyroid-dependent process which responds to substances known to be active along the HPT axis, 

and it is the only assay in the EDSP Tier 1 battery that detects thyroid activity in an animal 

undergoing metamorphosis. As described in the test guideline (OCSPP Guideline 890.1100) and 

the Standard Evaluation Procedure (SEP) for the AMA (USEPA 2011a), each of the Tier 1 

EDSP assays, including the AMA, is intended to be interpreted within a battery of in vitro and in 

vivo tests to identify substances with potential to interact with the endocrine system.

The general experimental design entails exposing Nieuwkoop-Faber (NF; Nieuwkoop 

and Faber 195625) stage 51 African clawed frog (Xenopus laevis) tadpoles, in separate treatment 

groups, to a minimum of three concentrations of a test chemical or a negative (clean water) 

control for 21 days. There are four replicates 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 are hind limb length (HLL), snout-to-vent length 

(SVL), developmental stage, body weight, thyroid histopathology, and daily observations of 

mortality and clinical signs. (Fort et al. 2007, OCSPP Guideline 890.1100).

This chapter uses data submitted in response to Tier 1 EDSP test orders to explore 

whether Test Order Recipients and their contracted laboratories are able to successfully execute 

the AMA. Evaluation criteria presented in the test guideline are perhaps the most 

straightforward benchmarks for comparison. Previously acknowledged limitations and 

uncertainties of the assay are summarized. These uncertainties are not considered “fatal” flaws 

of the AMA; rather, they underscore the utility of the holistic, battery-type approach that has 

been implemented in Tier 1 for evaluating the potential for endocrine interaction. Consideration 

is given to observations and recommendations from the previous peer reviews of the AMA, 

which culminated in the 2008 SAP review of the EDSP Tier 1 battery. Finally, issues identified 

during the preliminary review of the submitted studies are discussed. Again, these issues do not 

suggest critical flaws of the AMA experimental design, but they may indicate that additional, 

targeted guidance could improve study conduct and interpretation.

 

25 Nieuwkoop, P.D., and J. Faber. 1956. Normal table of Xenopus laevis. Amsterdam: North Holland Publishing.
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(ii) Strengths and Limitations 

The AMA provides direct evidence of the potential for effects of chemical exposure on 

aquatic-phase amphibians. More specifically to the purposes of Tier 1 EDSP, it allows 

inferences to be drawn about potential chemical-induced endocrine interactions which may have 

consequences for development and general fitness in vertebrate taxa. The developmental and 

histological endpoints in the AMA may reveal effects which are reflective of perturbations in 

physiological pathways which are known to be conserved across amphibians and mammals: the 

best understood of these is likely the negative feedback loop through which thyroid hormone 

(TH) levels are regulated via the controlled release of TSH (see Brown and Cai 2007).

Since the assay is conducted on intact larvae, it includes toxicokinetics and 

toxicodynamics that may impact the potential exposure of a compound in terms of absorption, 

distribution, metabolism and excretion of the test chemical and the effects of the chemical 

throughout various levels of biological organization, i.e., biochemical (thyroxine; T4), tissue 

(thyroid histology), and the whole animal (phenotype, survival). The study is conducted under 

flow-through conditions and measured concentrations are reported to document and ensure 

exposure over the 21-day test period. Exposure concentrations typically span a broad range with 

a factor of 3-to-10 between test concentrations. Study conditions are optimized to ensure that 

environmental conditions promote development and do not obscure potential effects from the test 

chemical.

The degree of homology among the amphibian and mammalian HPT axes is somewhat 

uncertain. Many aspects, including specific enzymes, synthetic pathways, regulatory feedback 

mechanisms, cell types, and transporter proteins are known to be conserved (Fort et al. 2007, Shi 

2000), but some degree of divergence is expected in downstream phenotypic responses to HPT 

perturbations. For example, goitrogens (iodine uptake inhibitors) are known to be active in both 

mammals (Brucker-Davis 1998, Green 1996) and amphibians (Kaltenback 1996); whereas in 

mammals, the primary biomarkers of goitrogenic effects are circulating thyroid hormone levels 

and neurological endpoints, the AMA is potentially more diagnostic of this mechanism in that 

exposed tadpoles exhibit inhibited development (OECD No. 77, 2007). Such differences in 

potential HPT-related responses between amphibians and mammals are not determined but rather 

underscore the value of the AMA as a unique screening tool. 

The AMA is expected to be sensitive to HPT perturbations which may not be detected in 

the mammalian assays with pubertal rats or non-EDSP guideline studies with maternal exposure 

to the test substance. For example, a recent study by Tietge et al. (2013) showed that 2-

mercaptobenzothiazole (MBT) was a potent inhibitor of thyroid peroxidase (TPO) activity in the 

AMA, whereas MBT had not been identified as a thyroid toxicant in mammals in studies 

conducted by the National Toxicology Program. The reasons for such potential differences in 
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sensitivity across taxa are varied but may include differences in route of exposure and 

metabolism which result in different doses in a given tissue. Additionally, the peripheral tissue 

changes associated with metamorphosis provide a suite of bioindicators in the AMA that allow 

for observation at multiple scales of biological organization, from organismal to histological, and 

the test substance exposure in the AMA occurs independent of any maternal signal. Therefore, 

the AMA is a useful counterpart to the thyroid endpoints in the Tier 1 assays with male and 

female pubertal rats. 

Based on validation studies, the AMA has demonstrated sensitivity to various MOAs. 

Reference chemicals have included T4 (thyroid agonism; OECD No. 76 and No. 77, 2007), 

sodium perchlorate (inhibition of the sodium-iodide symporter (NIS); OECD No. 77, 2007), 

iopanoic acid (Type I, II, and III deiodinase inhibition; OECD No. 77, 2007), 2-

mercaptobenzothiazole (thyroid peroxidase inhibition; Tietge et al. 2013), propylthiouracil 

(inhibition of iodine organification; OECD No. 76, 2007), and to the putatively weakly antithyroid substance benzophenone-2 (OECD No. 91, 2008). The native estrogen 17β-estradiol 

(E2) has also been tested in the AMA and was determined not to be a thyroid disruptor; a single 

exposure at high concentrations relative to background levels resulted in slight developmental 

delay, based on NF stage, with only a slight reduction in thyroid follicular cell height (OECD 

No. 91., 2008). Results from these studies are also published in the traditionally peer-reviewed 

literature (Degitz et al. 2005, Tietge et al. 2005). 

As with any assay, limitations of the AMA were identified through the research and 

development and the validation of the test design (OECD, 2004; 2007a). These limitations were 

discussed by the 2008 FIFRA SAP, by prior Federal Advisory Committee Act (FACA) 

Committees, and in supporting documents from Agency and independent peer reviews of the 

AMA. The conclusions regarding these potential limitations are reiterated in the test guideline 

and in various other documented in publicly available records. They are highlighted here for 

transparency and because they are relevant when considering the performance and interpretation 

of the AMA. Generally, the findings include technical performance issues and uncertainties in 

interpretation due to an incomplete understanding of amphibian physiology and endocrinology; 

broadly speaking, similar types of caveats are common to most ecotoxicology assays. 

Importantly, the acknowledged limitations did not detract from the overall SAP conclusion that 

the AMA was a critical component of the proposed Tier 1 screening battery (USEPA 2008). 

Relevance to Other Taxa

Amphibian metamorphosis, as a life stage, has been compared to the postembryonic (preand postnatal) stage in mammals, when thyroid hormone levels first become elevated and the 

organs are rapidly developing (Shi 2009, Tata 1993, Hetzel 1989). Nonetheless, our 

understanding of the relevance of the amphibian metamorphosis model for understanding 

mechanisms of HPT disruption in other taxa, including fish and mammals, continues to evolve. 

Genomic and transcriptomic approaches are revealing multiple functions of the thyroid hormone 
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receptor (TR) in amphibians: Shi (2009) and others have proposed that unliganded and liganded 

TH, respectively, impact the timing of metamorphosis in X. laevis by recruiting protein 

complexes which act either as co-repressors or co-activators of TH-dependent genes. It is 

unknown whether mammalian TR function similarly in post-embryonic development. There is 

an even greater paucity of information with respect to how peripheral [i.e., other than thyroid 

hormone receptor (TR)-mediated)] mechanisms of effect may compare across taxa. Examples of 

such effects include tissue-level TH regulation, TH transport in the circulatory system, and 

hepatic metabolism. Certain differences have been identified: with respect to binding of thyroid 

hormones to transport proteins, T3 has a higher binding affinity for the transthyretin (TTR) in 

amphibians, whereas T4 has the higher TTR affinity in mammals. Thus, test compounds which 

affect TTR binding may elicit a different response in amphibians than in mammals. 

Differences in Sensitivities Based on Life-Phase and Developmental Stage 

In amphibians and other tetrapods, the thyroid axis of a developing animal is regulated 

and responds differently than in the adult-life phase. The regulatory relationships between and 

amongst the different juvenile organs and hormone action on tissues vary with the stage of the 

life cycle. Since the morphological and molecular signatures of thyroid axis perturbation can 

differ markedly from larval phase to adult, extrapolating effects of a substance on larval phase 

amphibians to effects of exposure on juvenile and adult life phases is difficult. This is 

particularly true since for many amphibians, the juvenile and adult phases are adapted to the 

terrestrial environment as opposed to the aquatic environment. Given the purely aquatic nature 

of X. laevis, concerns have been expressed regarding its relevancy to amphibian species that have 

a distinct terrestrial phase (USEPA 200726). The amphibian life stage recommended for testing 

in the AMA was selected to maximize sensitivity to potential HPT interaction and not 

necessarily to predict adverse consequences of exposure in adult organisms.

Even within the larval phase, the dynamic regulatory environment of tissue formation, 

growth, and remodeling (morphogenesis), accompanied by programmed cell death (apoptosis), is 

a moving target for endocrine stressors. The potential consequences of disruption along this 

spectrum of development range from subtle to profound and from reversible to fatal. The 

developmental trajectory of the recommended test species, X. laevis, is extremely wellcharacterized; its use in the AMA allows investigators to test specific hypotheses about whether 

the test substance may behave agonistically or antagonistically vis-a-vis particular HPTdependent tissues or processes at a specific developmental stage. While a response at one time 

point or in one tissue may be suggestive or even diagnostic of an endocrine interaction, it is not 

necessarily informative of whether exposure at a different developmental or life stage would 

result in the same type of interaction (e.g., apparent TH agonism) or a similar functional response 

(e.g., advanced development).

 

26 USEPA. 2007, Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held October 9 – 11, 2007 

on the Potential for Atrazine to Affect Amphibian Gonadal Development. 

http://www.epa.gov/scipoly/sap/meetings/2007/october/finalminutes.pdf 
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Logistical Considerations for Aquatic Exposure Methods 

The suitability of the aqueous exposure method recommended in the AMA, as with other 

aquatic toxicity test methods, may be limited by the physico-chemical characteristics of the test 

substance. Aquatic testing of substances with low solubility, high octanol:water partition 

coefficients (Kow), high volatility, rapid hydrolysis, and photolysis under ambient laboratory 

conditions is challenging and sometimes uninformative. Methods for delivering such materials 

have evolved and are discussed in the OECD Guidance Document on Aquatic Toxicity Testing 

of Difficult Substances and Mixtures (OECD 2000) and OCSPP Guideline 850.1000 (USEPA 

199627). The AMA test guideline directs the investigator to this OECD (2000) guidance 

document, but the concepts therein were not directly incorporated into validation work with the 

AMA. Delivery of test substances via food for difficult test materials has been proposed by 

OECD, and may be considered an appropriate alternative approach in the future if adequately 

demonstrated. 

Non-Thyroidal Toxicity 

Non-thyroidal toxicity of test materials has the potential to affect some of the endpoints 

included in the AMA. For example, changes in body weight and organism length can be altered 

by processes independent of the thyroid axis. The use of multiple endpoints is intended to 

provide a WoE to identify cases where HPT-mediated effects may be occurring while accounting 

for other forms of toxicity (e.g., mortality, malformation, other clinical signs).

(iii) Observations from Initial Screening of Case Study Chemicals

Results from 18 industry-submitted studies with chemicals previously untested using the 

AMA method and with unknown potential for endocrine interaction are reviewed and discussed 

within the context of experimental performance, consistency of approach, and interpretation of 

results. For the purposes of this review, the chemicals were selected to be illustrative, but not 

necessarily all-encompassing, of a range of pesticide product types, physico-chemical 

characteristics, technical performance issues, and study outcomes. Given the preliminary nature 

of these reviews, actual chemical names are omitted.

Quality Control 

The AMA test guideline presents a series of validity elements (Table 29) based upon the 

recommended experimental design. These validity elements provide an indication of the general 

performance of the AMA under test conditions, e.g., control mortality that exceeds the reference 

value may be an indication of underlying problems associated with husbandry, environmental 

conditions, or the particular strain of test organisms used in the study. Alternatively, high 

 

27 USEPA. 1996. Ecological Effects Test Guidelines OPPTS 850.1000 Special Considerations for Conducting Aquatic 

Laboratory Studies. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic 

Substances (7101). EPA 7120C096-113. April 1996.

http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850-1000.pdf
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treatment mortality may limit or preclude the ability to detect potential endocrine-mediated 

effects in the remaining dataset. When met, the validity elements are used to support the 

scientific soundness of a given study and the degree of confidence in its results. 

Similarly, performance criteria (Tables 30 and 31) are identified in the AMA test 

guideline to promote consistent and biologically appropriate environmental conditions and to 

support the interpretation of results. Performance criteria are considered when evaluating each 

study but may be especially useful when deviations from the guideline are reported, as satisfied 

criteria may demonstrate that the deviations had a minor impact on study outcome. That said, a 

study that did not meet one or more performance criteria may still provide useful information 

and is not necessarily rejected. In some cases, performance criteria overlap with validity 

elements; for example, a threshold for control mortality is presented in both categories in the 

AMA test guideline. 

Table 29: Validity Elements for the Amphibian Metamorphosis Assay.

Element Reference Value(s)

Mortality (each treatment, including controls) < 10%

Mortality (each replicate)

 If positive for thyroid interaction

 If negative for thyroid interaction

< 2 tadpoles/replicate (<10%)

< 3 tadpoles/replicate (<15%)

Uncompromised treatment levels (all 4 replicates available 

for analysis)

> 2, in addition to controls

Treatment levels without overt toxicity (mortality) > 2, in addition to controls

Table 30: General Performance Criteria for the Amphibian Metamorphosis Assay,

when Conducted Under Flow-Through or Static-Renewal Conditions

Criteria Reference Value(s)

Measured test concentrations CV < 20% over 21 days

Control mortality < 2 tadpoles/each replicate (<10%)

Median NF stage in controls at test termination > 57 

Difference between 10th and 90th percentile of 

NF stage distribution in controls at test 

termination

< 4 stages

Dissolved oxygen > 40% air saturation

pH 6.5-8.5 (< 0.5 difference between replicates, 

treatments)

Water temperature 22±1°C (<0.5°C difference between replicates, 

treatments)
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Table 31: Additional Performance Criteria for the Amphibian Metamorphosis Assay,

when Conducted Under Static-Renewal Conditions

Criteria Guideline Recommendation(s)

Measured test concentrations Analysis immediately prior to and immediately 

following renewal 

General water quality parameters, including 

ammonia Analysis immediately prior to renewal

Renewal period < 72 hours (< 24 hours preferred)

Feeding schedule Adjusted as necessary (e.g., 50%) based on 

renewal scheme

When a study is reviewed by the Agency, the Data Evaluation Record (DER) notes 

whether each validity element and performance criterion was met. The DER includes a 

discussion of any rationale provided in the study report for any deviations or deficiencies. This 

includes identification of whether, to what extent, and in what way an inability to meet the 

criteria has an impact on the quality or utility of the study. Figure 18 illustrates the proportion of 

preliminarily reviewed AMA studies which met each of the major validity elements and 

performance criteria outlined in the test guideline. No studies using a static-renewal exposure 

method were submitted; therefore, those criteria are not evaluated.

Figure 18. The majority of AMA study submissions reviewed to date have met the validity 

elements and performance criteria outlined in the test guideline.

0 3 6 9 12 15 18

Control and treatment mortality ≤ 10%

Replicate mortality ≤ 10%

Replicate mortality ≤ 15%

Uncompromised treatments ≥ 2

Treatments without overt toxicity ≥ 2

Test concentration CV ≤ 20% over 21 days

Median Day 21 NF stage in controls ≥ 57

Control Day 21 NF stage (10th - 90th ...

Dissolved oxygen ≥ 40%

pH 6.5-8.5 (≤ 0.5 difference)

Water temperature 22 1 C (≤0.5 C ...

Met

Not Met

Not reported

# Studies
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Consistency in exposure levels, demonstrated by a test concentration CV < 20%, has 

been the most difficult performance criteria for laboratories to meet; only 11 of 18 (61%) 

submissions satisfied this criterion across the 21-day exposure period. It is uncertain to what 

extent the sample of existing study submissions is biased toward higher performing studies since 

time extensions have been granted in some cases due to technical challenges. “Not reported” 

indicates that the study report did not directly address the validity element or performance 

criterion or did not provide enough information (e.g., individual data points) for the Agency 

reviewer to determine if it was met. 

Uncertain Association between Alternative Feeding Approaches, Developmental 

Progression, and Spinal Curvature in X. laevis

The AMA test guideline recommends the use of Sera Micron feed for tadpoles during 

the in-life portion of the test. A specific feeding regime is recommended, with the quantity of 

feed increasing as tadpoles develop. The guideline recommendations to use Sera Micron and at 

these particular rates are based upon experience gained during the development and validation of 

the AMA, and because the iodide content in this product has been documented. Other diets and 

feeding regimes may be equally or more appropriate for X. laevis husbandry within the context 

of the AMA, but publicly available documentation of the performance of such alternatives is 

minimal. Therefore, the Agency recommends that test submitters provide information 

supporting the performance of the feeding regime, if the feed or amount and frequency of 

feeding differ from the guideline recommendations. In addition to general nutritional 

information, analyses of iodide content and for potential contaminants are particularly important.

In the initial data submissions, some labs have reported challenges regarding tadpole 

development in controls that advances beyond the recommended stage at test termination by Day 

21 in the AMA when using the guideline recommended feeding regime. For example, the 

guideline recommends that the minimum median stage in controls at test termination be NF 

Stage 57, when X. laevis tadpoles are undergoing toe differentiation and hind limb growth during 

prometamorphosis, and endogenous TH levels are rising (Shi 2000, Nieuwkoop and Faber 1956). 

Tadpoles which have reached NF Stage 61 or beyond are routinely excluded from analyses other 

than the analysis of NF stage; these tadpoles have entered metamorphic climax and TH levels 

have peaked and drastic tissue remodeling is occurring (Shi 2000, Nieuwkoop and Faber 1956). 

Although a precise threshold has not been calculated, the test guideline concludes that the 

development of more than 20% of control tadpoles beyond this stage may compromise the ability 

to detect treatment-related effects in the study.

The cause of the more rapid control development observed in some AMA study 

submissions, as compared to the validation studies, is not well understood. Similarly, there 

appears to be high variance in the incidence of spinal curvature observed in X. laevis tadpoles 

across studies and laboratories (Table 32). It is uncertain whether these phenomena are 

independent of one another or if, and to what extent, they are both dependent upon the same 
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causal factors. Such factors may include differences in the strain or even individual parentage of 

test specimens, environmental conditions such as temperature and photoperiod, iodide 

availability in the water and feed, nutritional variables other than iodide (Crawshaw 2003, 

Whitaker 2001), or a combination of these elements. Experimental work by the USEPA Office 

of Research and Development demonstrated that excessive feed can be a particularly strong 

factor in both outcomes (Olmstead et al., unpublished manuscript). Generally, the Agency 

recommends examining whether any patterns of spinal curvature are consistent between 

treatments and controls or whether there is evidence of a potential treatment-related induction of 

spinal curvature (USEPA 2011b). 

As shown in Table 32, the initial data submissions reflect that some labs are exploring the 

use of different feeding regimes in an attempt to synchronize the developmental stage of control 

tadpoles with that which is expected for the 21-day timeframe of the guideline AMA. 

Specifically, certain studies are being conducted with feeding rates that are approximately 50% 

of the rates recommended in the test guideline. While this appears to slow the developmental 

rate of control tadpoles in these studies so that guideline performance criteria for developmental 

stage are met, without elevating mortality, it is unclear whether such a drastic reduction may 

have other adverse effects on the health or endocrine status of the test specimens which may 

limit the sensitivity of the AMA.

Based on the initial data submissions, musculo-skeletal deformities (e.g., spinal curvature 

or “bent tail,” as it is typically confined to the tail region) have been reported in fifteen of the 

eighteen studies reviewed by the Agency, but the incidence varies widely across studies. The 

remaining three studies did not indicate whether spinal curvature was observed. The deformities 

have been reported both in studies which observe the guideline-recommended feeding regime 

and in studies which deviate from the guideline by reducing the amount of feed by 

approximately one-half. Therefore, a hypothesis of excessive feed as the primary factor in the 

development of spinal curvature does not appear to be supported in these cases. However, these 

observations are anecdotal; the Agency is unaware of any intra-lab comparisons from private 

laboratory facilities regarding feeding rates and musculo-skeletal deformities within a single 

population of X. laevis. There is no clear pattern in the metadata for the initial data submissions

that reveals a single, predictive factor for the prevalence of spinal curvature (Table 32). 

The submitted studies vary with respect to the amount of detail provided regarding 

observations of spinal curvature. In ten cases, observations were reported as the number of 

surviving tadpoles with spinal curvature at test termination. Three studies reported the number 

observed and the first onset of noticeable spinal curvature, e.g., beginning day 8. Three studies 

reported only an approximation (“a few”) or a range (“one-to-five per replicate”). In one study 

(Chemical U) that reported the number of observations at test termination, investigators assigned 

a ranked severity score (0, 1, 2, or 3) for spinal curvature to each test specimen at test 

termination and provided these individual-level data as an appendix to the study report. Absence 

of spinal curvature was graded as 0, and 3 represented the most severe form of spinal curvature 
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observed. The criteria used to assign the numerical grades were not further described. The 

investigators then reported incidence of spinal curvature of severity grade 2 and 3, together, on a 

percentage basis per treatment level. Using this approach, the incidence of spinal curvature 

(falling within either grade 2 or 3) was 40%, 27%, 21%, and 7% in the negative control and low, 

medium, and high treatment levels, respectively. When grade 1 observations are included with 

grades 2 and 3, the incidence rises to 74%, 52%, 59%, and 10% in the negative control and low, 

medium, and high treatment levels, respectively.

In this example, Sera Micron feed was supplied during the exposure period at rates 

approximately half of the guideline recommended rate; no additional iodide supplementation was 

provided. The report contained insufficient information to determine whether test specimens 

were selected from the “best single spawn.” There was no correlation between NF stage at test 

termination and incidence of spinal curvature. With the exception of tadpole mortality in one 

replicate, all validity elements and performance criteria were met. Similar and apparently 

inverse concentration-related trends in the occurrence of spinal curvature were observed in some 

other studies; however, the significance and reproducibility of these findings is unclear.
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Table 32: Prevalence of Spinal Curvature in the Amphibian Metamorphosis Assay.

Lab Test Chemical Animals Feeding Add’l Sources

of Iodide Prevalence of Spinal Curvature (%)

ID ID Product 

Type

Suppplierh

Location Product Reduced Iodide (ug/g)

Wateri

(ug/L)

Supplem

. 

Negative 

Control

Solvent 

Control

Trtmt. 

1 

Trtmt. 

2 

Trtmt. 

3 

10 A Insecticide Massachusetts Xenopus 

Express® No .

a

7 No 12 NA 17 29 12

Q Fungicide Massachusetts Xenopus 

Express® No .

a

7.4 No 41 NA 30 37 20

K Insecticide Wisconsin Xenopus 

Express® Yes .

a

7.8 No 41 NA 49 46 30

Gb Acaricide Wisconsin Xenopus 

Express® No .

a

7 No . NA . . . 

9 Vc Insecticide . Sera Micron® No . 13 No . NA . . .

Sd Acaricide . Sera Micron® No . 13 No . . . . .

3 M Insecticide Wisconsin Nasco Frog 

Brittle® Yes 48e

1.2 No 29 29 21 16 26

6 C Herbicide . Sera Micron® Yes . 4.5 No 43 53 39 40 36

U Insecticide . Sera Micron® Yes . 4.5 No 74 NA 52 59 10

P Herbicide Michigan Sera Micron® No . 4.5 No 66 NA 85 98 48

B Herbicide Michigan Sera Micron® Yes . 4.5 No 87 74 92 87 60

5 H Herbicide .

f Sera Micron® No 40 3.6 Yes 22 28 23 23 13

2 Od Herbicide Florida Sera Micron® No 54 <50 No . NA . . .

Ed Herbicide Florida Sera Micron® No 40 <10 No . . . . .

I

g Insecticide Florida Sera Micron® No 40 <10 No . . . . .

 

Color Key: Prevalence 0 to 25% 26 to 50% 50 to 100%

a Iodide content was not addressed in the study report. The Agency has requested information from the manufacturer of Xenopus Express® Tadpole Diet and is awaiting a 

response.

b The study report described “several” tadpoles exhibiting spinal curvature beginning on Day 10 in controls and the lowest treatment, Day 15 in the intermediate treatment, and 

Day 19 in the highest treatment level.

c The incidence of spinal curvature was reported as one to five tadpoles per replicate between Day 8 and test termination.

d The study report did not provide information on observations of spinal curvature.

e Reported on manufacturer’s website as 48 mg iodine/kg. http://www.enasco.com/page/xen_brittle f X. laevis strain: Daudin. Supplier not reported. No in-house parental stock; embryos were purchased for the selection of test specimens.
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Lab Test Chemical Animals Feeding Add’l Sources

of Iodide Prevalence of Spinal Curvature (%)

ID ID Product 

Type

Suppplierh

Location Product Reduced Iodide (ug/g)

Wateri

(ug/L)

Supplem

. 

Negative 

Control

Solvent 

Control

Trtmt. 

1 

Trtmt. 

2 

Trtmt. 

3 

N Inert . Sera Micron® Yes 54 <50 . 17 NA 20 29 17

J Funcigide Michigan

Florida Sera Micron® No 40 7 No 27 NA 8 11 19

F Herbicide Florida Sera Micron® No 50 <50 No 2 5 2 13 2

 g Spinal curvature was observed in a “few” tadpoles but incidence values were not reported.

h Original source of parental stock.

i

Background iodide in laboratory water.
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Interpreting Growth Effects within an Endocrine Context

Morphogenesis (development) and somatic growth in amphibians are mediated through only 

partially overlapping hormonal pathways (Denver 1997); although both events are directed through 

the hypothalamic-pituitary axis, the cascades of chemical messengers, their proximate ligands, and 

the phenotypic responses can differ markedly. Moreover, there is some indication that 

morphogenesis, and specifically amphibian metamorphosis, may be altered (either promoted or 

inhibited) based on body condition, effects on growth at critical phases of development, and 

ecological factors such as intraspecific competition and hydroperiod (e.g., Wissinger et al. 2010, 

Downie et al. 2004). Various explanations for these sometimes contradictory phenomena have been 

postulated and tested across scales of biological organization, but our understanding of the interplay 

between the molecular and, specifically, the hormonal pathways of growth and development remain 

incomplete.

Based on preliminary analysis, 12 of the 18 studies demonstrated some effect on growth 

endpoints, such as body weight or SVL, in at least one treatment level. In 10 of these studies, the 

effects on growth accompanied effects on at least one developmental endpoint (NF stage, normalized 

HLL) or thyroid (histo)pathology, although these effects were not always observed at the same 

treatment level, and the direction of the effect (i.e., increasing or decreasing) was occasionally 

different. In two cases, a promotion of SVL was observed at one treatment level, but an inhibition of 

SVL was observed at another treatment level. Both of these studies also showed evidence of a 

solvent effect based on comparison (Student’s t-test) of the solvent control to the negative control. 

As an example, results from one of these studies, Chemical F, are shown in Tables 33 and 34 below 

(solvent: DMF, at a concentration of up to 0.1 mL/L). The study report did not state whether 

proportional or serial diluters were used; therefore, it is unknown whether all treatments used equal 

concentrations of solvent. 
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Table 33: Developmental and Thyroid Gross Pathology/Histopathology Endpoints35,36 in the 

AMA with Chemical F. 

Table 34: Growth Endpointsa,b in the AMA with Chemical F. 

Color Key Promotion Inhibition

 

Abbreviations: Diff. Difference. NA Not applicable.

35 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 36 Unless otherwise specified, effects are considered statistically significant at p < 0.05. 37 Hind-limb length is normalized to snout-vent length (SVL). 38 p values for solvent control represent the p value from a two-sided t-test assuming unequal variance.

Treatment

(mg a.i./L)

[TWAmeasured]

NF Developmental Stage Hind Limb Length37

Thyroid Gross 

and

Histopathology

Day 7 Day 21 Day 7 Day 21 Day 21

Median p Median p 

% 

Diff. p38 % Diff. pd

TreatmentRelated Effects? 

(Yes/No)

Negative 

Control

54 -- 58 -- N/A -- N/A -- --

Solvent Control 54 -- 59 -- 0.80 0.84 16.1 0.05 No

0.003 54 >0.99 59 0.10 3.05 0.77 25.4 0.01 No

0.03 54 >0.99 59 0.10 -0.76 0.99 17.9 0.04 No

0.23 54 0.48 57 0.10 -1.53 0.96 -19.6 0.02 No

Statistical Test Mann-Whitney Mann-Whitney Dunnett’s Dunnett’s N/A

Treatment

(mg a.i../L)

[TWA-measured]

Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. pd % Diff. pd % Diff. pd % 

Diff. pd

Negative Control 0 N/A 0 N/A N/A -- N/A --

Solvent Control 6.90 0.04 1.00 0.35 18.6 0.08 10.0 0.05

0.003 5.63 0.22 1.80 0.24 14.8 0.31 12.6 0.18

0.03 8.79 0.04 -0.17 >0.99 23.3 0.07 -5.23 0.77

0.23 -16.9 <0.01 -5.76 0.08 -38.5 <0.01 14.4 0.11

Statistical method Dunnett’s Mann-Whitney Dunnett’s Dunnett’s
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In the case of Chemical F, the presence of a solvent effect similar in direction and magnitude 

to effects in the lower treatment levels, without any corresponding effect on thyroid 

(histo)pathology, suggests that the effects observed are driven by the solvent and not the test 

substance. Conversely, the statistically significant reductions in Day 21 normalized HLL, Day 7 

SVL, and Day 7 body weight at the highest treatment level (p<0.05), without any corresponding 

effects on the thyroid gland, are consistent with generalized toxicity of the test substance. Overall, 

the growth effects observed in this study suggest that the range of concentrations tested was 

adequate to challenge the organism, but the presence of solvent effects makes it difficult to interpret 

the findings. 

Apparent Absence of Effect with Difficult-to-Test Substances

Of the 18 studies in this preliminary analysis, seven did not meet the performance criterion 

for maintaining consistent measured test concentrations over the entire exposure period, as indicated 

by the percent coefficient of variability (%CV) (i.e., the %CV was > 20%). Across studies, the CVs 

tend to vary either directly or inversely with increasing test concentration. For example, in cases 

where the variability is driven by difficulties testing or quantifying very low concentrations of a test 

substance, the CVs are greater at lower treatment levels. Conversely, where the variability is driven 

more by difficulties testing near the limit of solubility of the test substance, the CVs tend to increase 

with increasing test concentration. Measured test concentrations are used to place the biological 

results of the study in context of aquatic exposure to the test substance. When recoveries are 

variable but treatment-related effects are observed within a given study, there may still be enough 

information within that study to determine whether exposure has the potential to elicit an effect. 

However, when test substance recoveries are poor and no effects are observed within the study, it 

may be questionable whether the assay was successfully performed. A different exposure method 

may be better suited for highly unstable compounds. Two examples follow from the initial 

screening of pesticides with the AMA.

One study (Chemical M) reported an error in stock solution preparation that increased the 

variability in test substance recovery: the initial stock solution concentration was inadvertently 

doubled prior to study initiation. As a result, percent recovery in individual samples at Day 0 ranged 

from 79% of the intended nominal concentration in a low treatment replicate to 161% in an 

intermediate treatment replicate. By Day 7, recovery ranged from 29% in a high treatment replicate 

to 44% in a low treatment replicate. Recovery at Day 14 was similar, ranging from 31% in a high 

treatment replicate to 57% in a low treatment replicate. At test termination on Day 21, recovery was 

only 20% in the high treatment and up to 37% in the low treatment. (Recovery percentages in this 

example are all based on comparison to the intended nominal concentrations.) Given the low and 

declining recoveries throughout the exposure period, it is unlikely that the study would have met the 

performance criterion for %CV even if the stock solution had been correctly prepared at test 

initiation. The biological results of this study included the advancement of median NF stage by one 

stage in the intermediate and high treatment levels; an initial increase and subsequent decrease of 

SVL at the intermediate and high treatment levels, respectively; and the increased body weight in 
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both the intermediate and high treatment levels, all based on comparison to the negative control. 

The biological results are difficult to interpret within the context of actual exposure to the test 

substance, which is known to be a low solubility, highly adsorptive compound. The use of a solvent 

(DMF, up to 0.1 mL/L) and the presence of effects on NF stage and growth parameters in solvent 

controls further complicates interpretation.

In the study with Chemical S, mean %CVs for test substance concentrations were 69% in the 

lowest treatment level, 33% in the intermediate treatment level, and 49% in the high treatment level. 

Test substance recovery was only 9% of nominal in the high treatment level at test termination. 

Other than %CV, all validity elements and performance criteria were met. No biological effects 

were observed. The treatment concentrations were based on three 24-to-96 hour range-finding 

studies with X. laevis tadpoles (NF stage not reported). However, given the high %CVs and poor 

recovery in the definitive test, it is questionable whether the investigators achieved and maintained a 

high enough exposure level to elicit a potential response in the 21-day AMA. A dietary exposure 

approach, which is mentioned in the test guideline as a potential alternative test method, may have 

been more useful in this case. 

Interpreting Amphibian Thyroid Histopathology Diagnoses 

Thyroid gross pathology and histopathology observations are critical components of the 

AMA, particularly in cases when other biological effects are absent or difficult to interpret. To assist 

investigators in consistently preparing, analyzing, and reporting an assessment of thyroid 

histopathology diagnoses, the Agency served as a lead in the preparation of a Guidance Document 

on Thyroid Histopathology (OECD No. 82, 2007), which was informed by international expertise in 

pathology and experience gained during the validation of the AMA (Grim et al. 2009). The guidance 

document includes micrographs of “normal” X. laevis thyroid glands and recommends a four-point 

(0, 1, 2, or 3) severity grading scale for assessment, with 0 representing findings that are “not 

remarkable,” and 3 generally representing findings that are “severe.”

With the exception of gross pathology findings (thyroid gland atrophy and hypertrophy), 

severity grades are generally assigned based on comparison to “normal” thyroid findings depicted in 

the guidance or based on the professional opinion of the pathologist conducting the assessment, and 

not in comparison to control findings from a given study. The severity grades are to be assigned 

based on a reading of at least two-to-three slides per specimen. Pathologists are expected to have 

experience or have received training in amphibian and specifically X. laevis thyroid histopathology; 

otherwise, the guidance document suggests additional oversight or peer review of the findings. The 

study reports reviewed thus far by the Agency indicate that histopathology analyses were performed 

by a certified histopathologist, but the level of individual experience with amphibian histopathology 

is generally not described. Finally, consistent with practice in the discipline, the guidance document 

recommends that samples be evaluated initially in a non-blinded manner, with possible confirmation 

of results by a subsequent, blinded reading (Crissman 2004). The degree of compliance with this 
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recommendation varies based on the AMA histopathology reports submitted to date; when a nonblinded reading has been used, a subsequent blinded reading is rarely reported. 

The AMA test guideline recommends that five of the fifteen tadpoles remaining per replicate 

(assuming no mortality) at test termination (Day 21) be selected for thyroid histopathology. To the 

extent possible, the guideline states that these specimens should be stage-matched to the median NF 

stage in controls. If precise stage-matching is not possible because of an apparent treatment-related 

effect on NF stage development, the investigator is encouraged select specimens as closely matched 

as possible to the median NF stage in controls, but consistent with the directionality of any apparent 

treatment-related effect. For example, if there are less than fives tadpoles in a treatment replicate 

which are matched to the median NF stage in controls, and the data suggest a possible treatmentrelated developmental delay, then the investigators would select tadpoles of the next lower NF stage 

when compared to controls. The Agency considers stage-matching of histopathology specimens to

be important to the interpretation of histopathology findings because tadpoles undergoing 

metamorphosis (i.e., at different NF stages) are expected to have different histological presentations 

(Grim et al. 2009). In the absence of significant effects on developmental biomarkers (NF stage or 

normalized HLL), the inability to stage-match thyroid specimens may reduce the likelihood of 

detecting a treatment-related lesion caused by a less potent thyroid inhibitor. 

Thyroid histopathology was performed in all eighteen of the studies reviewed here. Seven of 

these eighteen studies reported that tadpoles selected for histopathology were stage-matched to 

controls. Two studies reported that specimens were not stage-matched but instead were impartially 

selected. The remaining nine study reports did not provide specific information on specimen 

selection, although some inferences may be made by comparing information in the pathology report 

to the raw data from the study, when available.

Other Guideline Recommendations

Use of the Best Single Spawn 

With respect to embryo selection for the AMA, the test guideline recommends that two-tothree of the best individual spawns from in-house adults be retained to evaluate the quality of the 

spawns. From these two-to-three spawns, it is recommended that the test organisms originate from 

the best single spawn, based upon embryo viability/appearance and the presence of an adequate 

number (>1,500) of embryos. The guideline recommendation to concurrently evaluate multiple 

spawns was intended to increase the likelihood of identifying a single spawn with an adequate 

number of viable embryos in a time-efficient manner (i.e., instead of evaluating spawns serially) 

(pers. comm., J. Tietge, March 2013). The guideline recommends against co-mixing spawns 

because this has been demonstrated to increase variability and decrease the statistical power of the 

test (OECD 2007). 
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In its review of the data, the Agency considers and documents in the DER how many spawns 

were evaluated. The DER also includes details of the evaluation method (e.g., timing of evaluation 

and number of eggs evaluated per sample) and whether the organisms selected for the definitive test 

originated from the best single spawn, when this information is provided in the study report. Of the 

18 studies in the initial analysis, only seven reported using the best single spawn consistent with test 

guideline recommendations. Seven studies reported using another method, either co-mixing 

embryos from different spawns based on NF stage or using embryos from a single (but not 

necessarily the best of two-to-three) spawn. Four study reports did not contain enough information 

to determine the method of embryo selection. 

Optional Size Selection Method 

The exposure is initiated when a sufficient number of tadpoles in the pre-exposure stock 

population, which are less than or equal to 17 days of age post-fertilization, have reached NF stage 

51. The test guideline includes additional guidance on optional size selection, based on total length 

(not snout-vent length), that complements the stage selection. The optional addition of size selection 

further reduces variability by ensuring that the NF stage 51 tadpoles fall within a specified size 

range. None of the 18 studies reviewed thus far have implemented the optional size selection 

method. 

Iodide Measurement and Supplementation 

Sufficient iodine intake, generally in the form of iodide, is necessary for normal functioning 

of the HPT axis because iodine serves as a substrate for the synthesis of thyroid hormone (Shi 2000). 

Iodination of thyroglobulin produces T4, and partial deiodination of T4 produces the more 

biologically active T3 (Dodd and Dodd 1976). The AMA was successfully performed in validation 

studies when test water iodide (I-) concentrations ranged between 0.5 and 10μg/L. This is in 

addition to dietary iodide exposure at levels present in the guideline recommended feeding regime 

using Sera Micron. Therefore, the test guideline recommends that the iodide concentration in test 

water be determined. Iodide supplementation is recommended if the concentration is naturally 

below 0.5 μg/L in the test water or if test water is reconstituted from deionized water. It is 

recommended that iodide supplementation not exceed 2 μg/L. The Agency considers both dietary 

and test water sources of iodide that are reported for a given study when reviewing the data. For the 

eighteen studies reviewed thus far, these values are summarized previously in Table 32. Iodide 

supplementation was reported in only one study (Chemical H).

(iv) Statistical Approach and Considerations

The Agency routinely verifies the statistical conclusions of industry data submissions in 

response to AMA test orders. Agency reviewers typically re-analyze individual-level data using 

statistical methods recommended in the AMA test guideline, as appropriate. Some differences 

between the AMA test guideline recommendations for statistical analyses and current Agency 

practice have emerged. Numerous reasons exist for these deviations, such as differences in 
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analytical software capability, characteristics of a given dataset, historical practice, uncertainty 

regarding the ability of a particular statistical test to detect an effect given the guideline 

recommended-experimental design, and occasionally perceived ambiguity in the guideline 

recommendations. 

Exclusion of Late Stage (>NF Stage 60) Tadpoles 

Sixteen of the eighteen AMA studies which have undergone preliminary review reported that 

some proportion of tadpoles had moved beyond NF stage 60 by test termination (Day 21). At this 

stage of development, X. laevis tadpoles are shifting from prometamorphosis to metamorphic 

climax; the regression of tissues (e.g., tail resorption) and overall changes in size and shape during 

metamorphic climax are reflected in changing morphometric ratios. For this reason, the test 

guideline recommends that measurements from individual tadpoles which have advanced beyond NF 

stage 60 (termed “late stage”) be censored from the analysis of continuous endpoints (i.e., SVL, 

HLL, body weight). When more than 20% of tadpoles are classified as “late stage” at test 

termination, the Agency currently recommends conducting two analyses for each continuous 

endpoint: one on the “non-late stage” tadpoles and a second analysis on the “late stage” tadpoles. 

Note that in this case the guideline recommends a two-factor analysis of variance (ANOVA) with a 

nested variance structure be undertaken on all tadpoles to assess growth effects due to chemical 

treatments while taking into account the effect of late stage development on growth. However, the 

Agency’s attempt at this approach was unsuccessful due to the greater variance between individuals 

than between replicates. Four of the eighteen studies reviewed reported “late stage” in more than 

20% of tadpoles in at least one treatment group. All data points are considered together when 

analyzing non-continuous endpoints (i.e., NF stage).

Normalization of Morphometric Measurements

The test guideline recommends that each individual hind limb length (HLL) measurement be 

normalized to the snout-vent length (SVL) of the same individual (i.e., HLL:SVL) prior to analysis 

of the dataset for an HPT-mediated effect. The resulting, calculated value is referred to simply as 

“normalized hind limb length.” The normalization is intended to control for effects on growth which 

are analyzed separately and may be due to non-specific toxicity. Before evaluating the statistical 

conclusions of a given AMA study submission, the Agency confirms whether the HLL data are 

normalized consistent with guideline recommendations. 

In some cases, the investigators are applying a different transformation to HLL data. Rather 

than reporting and analyzing normalized HLL as the ratio of HLL-to-SVL, some investigators are 

calculating the ratios and then multiplying the original HLL values by these ratios prior to statistical 

analysis (i.e., (HLL/SVL)*HLL). While there is nothing inherently flawed in the alternate approach, 

for consistency, the Agency currently abides by the test guideline recommendations and uses only 

the HLL:SVL ratios when analyzing normalized HLL. 
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(viii) Overall Conclusions for the AMA

The preliminary analysis of 18 AMA studies submitted by industry test order recipients 

suggests that laboratory performance of the AMA generally meets expectations. Previous peer 

review conclusions during the development and validation of the AMA test design are reinforced by 

the initial data submissions. Briefly, these conclusions were that (1) the AMA provides useful, 

complementary, and sometimes unique information about potential perturbations of the HPT axis in 

amphibians, (2) conclusions regarding the potential for a test substance to interact with the HPT axis 

in amphibians and, more broadly, in vertebrate taxa need to be based on a holistic examination of all 

endpoints in the AMA and within the context of other available data (i.e., the Tier 1 EDSP battery), 

(3) in some cases, it may be difficult to discern HPT-specific effects in the AMA from other types of 

toxicity, e.g., when growth is inhibited or with only weakly active compounds, and (4) certain test 

substances may not be amenable to the 21-day aquatic exposure design of the AMA. The previously 

acknowledged limitations and uncertainties do not suggest critical flaws of the AMA experimental 

design as presented in the test guideline, but they may indicate that additional, targeted guidance 

could improve study conduct and interpretation. 
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IV. Performance of Battery 

In 1998, when developing the Tier 1 battery, the EDSTAC considered screening endpoints 

for their utility in screening chemicals substances or mixtures for their potential to interact with the 

endocrine system. The goal of Tier 1 assays is to detect chemical substances or mixtures capable of 

have the “potential” of interacting with estrogen, androgen, or thyroid hormone systems. Assessing 

these activities is important as changes may adversely affect the development, reproductive function, 

or chronic health status of humans or wildlife. The objective of the Tier 1 assays is not to determine 

dose-response relationships, confirm the mechanisms of action, or determine the adverse outcome(s). 

The screening assays, however, must be sensitive enough to detect all known xenobiotics that act via 

the mechanism of action each assay is designed to detect. The Tier 1 screening battery as a whole

should maximize sensitivity to minimize false negatives, while recognizing some acceptable level of 

false positives. Based on EDSTAC conclusions in 1998, when the Tier 1 screening battery is 

validated, it should include a combination of in vitro and in vivo studies that are not weighted 

equally, but when combined together, should detect all known modes of action for the endocrine 

endpoints of concern, include sufficient range of taxonomic groups among the test organisms, 

incorporate sufficient diversity among endpoints and assays and overall, it should minimize missing 

potential endocrine active chemicals. (EDSTAC, 1998)

A decade after the publication of the EDSTAC report, the SAP in 2008 reviewed the Tier 1 

proposed battery of 11 assays and concluded that “the proposed set of Tier 1 assays are appropriate 

to begin screening for disruptors of the EAT axes.” The 2008 SAP panel also concluded that “[t]he 

ability to identify endocrine active substances is enhanced in the Tier 1 battery because the tests 

provide adequate replication and redundancy.” and should “provide safeguards against false 

positive or false negatives.” The 2008 SAP did recommend that the agency “will continue to 

develop, refine, and review the battery.” 

Introduction

This chapter describes the performance of the Tier 1 Battery using data from a subset of 

chemicals issued Test Orders (List 1) beginning in October 2009. This analysis used the 2008 

FIFRA SAP report39 as a guide, borrowing the concepts of “complementarity” and “redundancy” as 

defined by the SAP in 2008; terms such as “complementarity” describes the concordance of 

endpoints within an assay and “redundancy” describes the concordance of endpoints across the 

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

Complementarity and redundancy will be evaluated in the context of different “modalities” that span 

multiple levels of biological organization, including molecular interactions, and cellular, organ and 

 

39 March 25-28, 2008 Session of the FIFRA Scientific Advisory Panel entitled “Review of the Endocrine Disruptor Screening 

Program (EDSP) Proposed Tier 1 Screening Battery”. 
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organism responses for each hormonal class (i.e., E, A, and T hormonal systems) that the battery 

was expected to assess. The 2008 SAP defined these different biological levels of organization as 

consisting of “receptor/target binding, cell responses, enzyme activity, organ response, males and 

female system integrity, development (thyroid) and comparative system integrity”. Thus, the 

purpose of this preliminary battery review is to: 1) evaluate the complementary relationships among 

responses within the assays in the Tier 1 battery, and 2) evaluate the redundancy of responses across 

the Tier 1 battery. Subsequently, this review will also consider whether a different combination of 

assays, reduced in number, can adequately ensure the identification of potential interactions with the 

E, A, or T hormonal pathways.

For this systematic preliminary assessment, a subset of List 1 chemicals with submitted Tier 

1 data were selected as a representative group of chemicals to provide a broad range of responses 

across 11 Tier 1 assays. This subset of Tier 1 battery results allowed the agency to assess the 

performance of the battery in terms of the complementary relationships within an assay (namely the 

multi-parameter in vivo assays) and redundancies across assays. The battery performance analysis is 

also separated into the three hormone classes (i.e., E, A, or T) to evaluate the extent of 

complementary relationships within assays and redundancy across assays for each of the hormonal 

pathways using the subset of List 1 chemicals whose identity has been coded.

Individual Tier 1 Assay Assumption: Observations of Overt Toxicity 

The review of the battery performance is based on the assumption that individual assays are 

performing adequately. As noted in Section III, overt toxicity (e.g., mortality, reduction in body 

weight, behavior) was reported in some assays. Effects on endocrine-related endpoints observed in 

the Hershberger, male and female pubertal rat assays, and FSTRA were in some cases coincident 

with signs of overt systemic toxicity at some doses; however, at this time such effects were not 

excluded from the battery performance analysis. When positive Tier 1 findings are observed in the 

presence of overt toxicity, the agency is aware that there is uncertainty as to whether the responses 

are mediated through an endocrine pathway. The agency, however, will evaluate the degree of 

confidence for endocrine responses that co-occur with systemic toxicity in subsequent weight of 

evidence determinations, on a case by case basis. To ensure full transparency, cases where overt 

toxicity was observed are clearly identified and are noted in all tables included in the battery review 

section. 

1. Interaction with the HPT Axis

Three assays in the Tier 1 battery are intended to detect potential interaction with the HPT 

axis or its downstream thyroid hormonal pathways. These assays include the Female and Male 
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Pubertal rat assays, and the AMA. Table 35 lists the endpoints from these guideline assays which 

the 2008 FIFRA SAP40 identified as biomarkers of potential thyroid interaction.

Table 35. Endpoints for Detecting Potential Effects on the Thyroid Hormonal Pathway 

According to the 2008 FIFRA SAP. 

Assay Endpoint(s)

Male Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T4) 

• Thyroid gland histology 

Female Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T4) 

• Thyroid gland histology

AMA • Amphibian (tadpole) development

• Thyroid gland histology

In its preliminary analysis of Tier 1 battery performance, the Agency considers a treatmentrelated effect on thyroid stimulating hormone (TSH), thyroxine (T4), or thyroid weight or histology 

in the male or female pubertal rat to indicate a potential interaction with the thyroid hormonal 

pathway; however, any observations of overt toxicity are noted and will be considered in the future 

WoE determinations. The 2008 FIFRA SAP recommended, “[a] significant effect of test substance 

on serum T4 must be considered a positive finding, whether or not serum TSH is affected, and 

whether or not the AMA is positive” (USEPA 2008a, p. 31). For the AMA, the Agency is following 

the decision logic outlined in the AMA test guideline (OCSPP 890.1100) and Standard Evaluation 

Procedure (SEP), which was informed by the results of earlier validation studies using chemicals 

with known thyroidal MOAs (USEPA 2008b). This decision logic is illustrated in Figure 19. Using 

this approach, the direction of effect is considered when evaluating whether treatment-related effects 

on amphibian development are indicative of thyroid activity: Developmental delay is considered a 

thyroid finding only if corroborated by treatment-related effects on thyroid gland histology, whereas 

advanced development, asynchronous development, or effects on thyroid gland histology may each

be considered to be diagnostic findings independently. 

 

40 USEPA. 2008a. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25-26, 2008, to 

review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008-

03. 11 June 2008. Washington, DC.
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Figure 19. Suggested Decision Logic for the Conduct and Interpretation of the AMA (OCSPP 

890.1100, OECD 2009). 

Sixteen case study chemicals were used in the preliminary analysis of performance of the 

battery for HPT/thyroid hormonal pathway screening. In 13 of these cases, Tier 1 guideline data 

showed indications of potential thyroid activity in one or a combination of assays (Table 36). 

Responses in HPT-relevant endpoints were identified in seven of 16 guideline Male Pubertal studies, 

in 9 of 16 guideline Female Pubertal studies, and in seven of 16 submitted AMA studies, and in 4 

instances where all 3 assays had effects noted in the studies that have been reviewed. Of these, one 

Male Pubertal study and three Female Pubertal studies only showed effects at the same dose level 

where overt toxicity was recorded. Potential solvent interference was identified in two of the three 

AMA studies for chemicals which only showed responses in this particular assay. Overall, all 3 

assays for a given chemical reported effects in three cases. Submissions of Other Scientifically 

Relevant Information (OSRI) which were used to potentially satisfy guideline requirements are 

noted in the table. 
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Table 36. Preliminary Results of Tier 1 Assays Relevant to the HPT Axis or Thyroid 

Hormonal Pathway, by Case Study Chemical.

Chemical Code Male Pubertal Female Pubertal AMA

A x

B x x 

C x x x

E x x

F x x x

G x x

a x 

H x

b

I

J x

K x

a xa

M x

b

N

P x x 

S x

a c

Td

V x

x: =Effects were seen on thyroid-related endpoint(s); 

Blank cell = no thyroid-relevant effects observed a

= Effects on thyroid-related endpoints only observed at doses where overt toxicity was observed

b

= Potential solvent interference made results difficult to interpret c = Did not achieve adequate exposures

d = Pubertal rat assay Test Orders were satisfied by OSRI; effect only on T3 (endpoint not included in the guideline) in 

the male rat. 

(i) Complementarity of Results Within Each Tier 1 Assay 

When considering the possible complementarity of responses among endpoints within an 

assay, the following general patterns were noted in the initial data submissions (Table 36). A more 

detailed examination of the direction and magnitude of each effect will be performed in the future 

when evaluating complementarity within the chemical-specific WoE analyses.

Male Pubertal Rat 

• A response in T4 was accompanied by a response in TSH in only one case (Chemical E). An 

effect on thyroid weight, but not histology, was also observed in this case.

• A response in T4 occurred without a response in TSH in three cases (Chemicals C, F, and G).

• A response in TSH occurred without a response in T4 in two cases (Chemicals J and P).
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• The response in TSH was accompanied by a difference in thyroid weight in only one case 

(P).

• The response in TSH was accompanied by an effect on thyroid histology in only one case 

(Chemical J).

• In one case, an effect on thyroid histology occurred without responses in any other diagnostic 

endpoints (Chemical K). However, overt toxicity was observed at the same dose levels as 

histological effects.

• Effects on thyroid histology were not observed in association with effects on T4 in the male 

rat.

Female Pubertal Rat 

• A response in T4 was accompanied by a response in TSH in two cases (Chemicals E, and F).

• In six cases, T4 was affected without a concomitant response in TSH (Chemicals A, B, C, G, 

K, P).

• In four of the cases where an effect on T4 was observed, an effect on thyroid histology was 

observed (Chemicals B, G, K, and P). Effects on thyroid histology only occurred 

concomitant with alterations in T4 in the female pubertal rat.

• In two of these cases, the effects on T4 and thyroid histology occurred only in the presence of 

overt toxicity (Chemicals G and K).

• In one case, TSH was altered in the absence of any other effects (Chemical Q).

• In one case, thyroid gland weight was affected in the presence of overt toxicity (Chemical S). 

No other effects were observed.

Amphibian Metamorphosis 

• Observations of advanced development (based on developmental stage and/or normalized 

hind limb length) were complemented by thyroid histopathology diagnoses in three cases 

(Chemicals C, H, and V). In one of these cases, potential solvent interference was identified 

(Chemical H).

• Advanced development was observed in the absence of effects on thyroid histopathology in 

two cases (Chemicals F and M). In one of these cases, potential solvent interference was 

identified (Chemical M).

• Observations of developmental delay (based on developmental stage and/or normalized hind 

limb length) were complemented by effects on thyroid histopathology in only one case 

(Chemical G).

• Developmental delay occurred in the absence of effects on thyroid histopathology in five 

cases (Chemicals I, J, K, P and T). According to the AMA test guideline decision logic 

outlined in Figure 19, these cases are considered “likely thyroid inactive.”
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• Notably, one of these putatively inactive cases (Chemical T) represents a special data 

submission based on examination of preserved specimens at a later developmental stage (NF 

stage 66). This special submission lacks observations at the interim time point (Day 7 of the 

AMA) which are expected to be more sensitive to potential thyroid agonism (e.g., advanced 

development). 

The variety of responses observed among the HPT-relevant assays illustrates two main 

points: 1) Less complementarity is built into the individual Tier 1 assays for detecting potential 

thyroid effects, as compared to the estrogen and androgen hormonal pathways. Therefore, all three 

thyroid-relevant assays and each of their endpoints need to be considered when screening for 

potential interaction. 2) The AMA decision logic (Figure 19) is a simple but useful and important 

tool for interpreting the results of this assay. Although developmental delay was observed in six 

cases (Chemicals G, I, J, K, P, and T), only one of these cases (Chemical G) is characterized as 

potentially thyroid-active in the AMA when the decision logic is implemented. 

Table 37 summarizes the preliminary diagnostic endpoint responses in each assay. Effects 

determined to be indicative of potential thyroid interaction are shown with an “x”. Effects on 

amphibian development are identified consistent with the AMA decision logic (i.e., they are not 

marked in the table if the effect was a delay and there were no corroborating histological effects). 

The responses will be considered using a WoE approach to determine potential for interaction along 

the thyroid pathway; therefore, the binary responses shown in this table (effect: “x”, no effect: blank 

cell) are only preliminary indicators and do not reflect any overall chemical conclusions.

Table 37. Associations between Endpoint Responses in Tier 1 Assays Relevant to the HPT and 

Thyroid Hormonal Pathway, by Case Study Chemical (Preliminary). 

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4

Amphibian 

Development

A 

Male Pubertal

Female Pubertal x 

AMA

B 

Male Pubertal

Female Pubertal x x 

AMA x

C 

Male Pubertal x 

Female Pubertal x 

AMA x x

E 

Male Pubertal x x x 

Female Pubertal x x 

AMA

F 

Male Pubertal x 

Female Pubertal x x 

AMA x
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Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4

Amphibian 

Development

G 

Male Pubertal x 

Female Pubertal x

a xa

AMA x x

H 

Male Pubertal

Female Pubertal

AMA x x

b

I 

Male Pubertal

Female Pubertal

AMA

J 

Male Pubertal x x 

Female Pubertal

AMA

K 

Male Pubertal x

a

Female Pubertal x

a xa

AMA

M 

Male Pubertal

Female Pubertal

AMA x

b

N 

Male Pubertal

Female Pubertal

AMA

P 

Male Pubertal x x 

Female Pubertal x x

AMA

S 

Male Pubertal

Female Pubertal x

a

AMA c

T 

Male Pubertald

Female Pubertale

AMAf

V 

Male Pubertal

Female Pubertal

AMA x x

x = treatment-related effect; Blank cell=no thyroid-relevant effect; shaded cell: not measured in the assay a

= Effects were observed only at dose levels with overt toxicity

b = Potential solvent interference

c = Did not achieve adequate exposure

d = Test order requirement satisfied by OSRI (Stoker et al. 2000); dose-dependent effects were observed on T3 but no effects were 

observed on T4, TSH, or thyroid histopathology e = Test order requirement satisfied by OSRI (Laws et al. 2000); no effects on thyroid hormones or thyroid gland morphology

f

= Special data submission: observations were based on preserved NF stage 66 specimens
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(ii) Redundancy Across Tier 1 Assays

As shown in Table 36 and Table 37, there was general consistency in the overall responses 

between the Male and Female Pubertal assays, but not necessarily complete agreement between the 

individual endpoints that were affected. The AMA was sometimes responsive when the pubertal 

assays were not, and vice versa. 

Comparisons of Male and Female Pubertal Rat

• Both the male and female pubertal rat assays were responsive in six cases (Chemicals C, E, 

F, G, K, and P). However, Chemical K caused overt toxicity in both the male and female rat.

• Six chemicals showed no response on guideline endpoints in either the male or female 

pubertal rat (Chemicals H, I, M, N, T, and V). However, an OSRI submission showed that 

Chemical V caused an effect on T3 in the male pubertal rat. T3 is not typically included as an 

endpoint in the guideline studies.

• Chemical J caused an effect in the male rat but not the female rat. 

• Three chemicals caused an effect in the female rat but not the male rat (Chemicals A, B, and 

S). Chemical S was considered overtly toxic in the female rat. 

Comparisons of Pubertal Rats and Amphibian Metamorphosis 

• For chemicals having all three guideline studies available, a relevant effect in the AMA was 

accompanied by a relevant effect in at least one pubertal rat study in four of seven cases 

(Chemicals B, C, F, and G).

• In three of these cases, HPT-relevant responses were observed in each of the male pubertal, 

Female Pubertal, and Amphibian Metamorphosis Assays (Chemicals C, F, and G). 

• Chemical S, which was overtly toxic in the female rat and caused no effect in the male rat, 

was not successfully tested in the AMA. 

• Chemical B, which caused an effect in the female rat, also affected thyroid histopathology in 

the AMA.

• In two cases, a test chemical did not cause a significant effect on any diagnostic endpoint in 

any of the three guideline assays (Chemicals I and N). 

• The special data submission for Chemical T showed no significant effects in the frog at NF 

stage 66. The OSRI for this chemical showed an effect on a non-guideline endpoint (T3) in 

the male rat only.
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(iii) Discussion

Visually, the summary results illustrated in Table 36 show a degree of consistency between 

the overall results in the Male and Female Pubertal rat studies. At a high level, the assays both 

showed either some effect or no effect on guideline endpoints in 12 of 16 cases where both Male and 

Female Pubertal rat studies were available for review. Similarly, the specific endpoint responses in 

male and female rat mirror one another in many, but not all, cases (Table 37). This observation 

underscores the intended redundancy of these assays with respect to mechanisms and downstream 

effects which may be conserved across sexes. However, sex-specific differences in the preliminary 

overall results for guideline endpoints were observed in the remaining four of 16 cases. In its review 

of the proposed Tier 1 battery, the 2008 SAP advised, “Replacement of the pubertal male or female 

assays with an alternative assay is not considered a reasonable alternative because it is possible to 

miss gender specific effects from certain chemicals” (USEPA 2008a, p. 23). The preliminary results 

from Tier 1 studies provide support for the assertion that the Male and Female Pubertal rat assays are 

both useful: in some cases, they show redundancy in responses which increases confidence in the 

overall findings, and in other cases, they provide information on possible sex-specific effects which 

may be missed if one of the assays were excluded.

This preliminary review shows that thyroid-relevant results in the rat are not necessarily 

predictive of results in the amphibian, and vice versa. The 2008 SAP observed that the AMA was 

the only assay in the Tier 1 battery that provided an evaluation of an early developmental timeframe. 

The AMA may provide information on the downstream effects of potential thyroid pathway 

interaction, as captured by peripheral tissue endpoints associated with amphibian metamorphosis, 

whereas the Male and Female Pubertal rat assays provide information on circulating TSH and T4

levels. The three assays all include examinations of thyroid gland histology; however, different 

effects across assays may be observed due to differences in exposure route, metabolism, life stage, 

and taxonomic differences in downstream responses to certain types of perturbations. Such 

differences will need to be considered when interpreting the nature, magnitude, and relevance of 

various endpoint responses when the chemical-specific WoE analyses are conducted in the future. 

At a high level, the preliminary results reinforce the importance of including both taxa in the 

screening battery to reduce the potential for false negatives. 

• In one case (Chemical A), an effect on T4 was observed in the female rat, but no effects were 

observed in the male rat, and no effects were observed in the amphibian. In the absence of 

other information, the screen would have missed a potential interaction if the Female 

Pubertal assay were dropped from the battery. 

• In the second case (Chemical B), effects were observed on T4 in the female rat and on thyroid 

gland histology in both the female rat and the amphibian, but no effects were observed in the 

male rat.
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• In the third case (Chemical S), thyroid gland weight was affected in the female rat, but only 

at overtly toxic doses. No effects were seen in the male rat, and the chemical was not 

amenable to aquatic exposure testing in the AMA. The latter case illustrates the importance 

of considering overt toxicity and general experimental performance when interpreting results, 

especially when results across assays do not appear to be concordant.

• The male rat showed greater sensitivity in only one case (Chemical J) wherein both thyroid 

gland histology and TSH were affected; no significant diagnostic effects of this chemical 

were observed in the female rat or in the amphibian.

• Using the test guideline decision logic depicted in Figure 19, the AMA showed meaningful 

responses to three chemicals which caused no relevant effects in either male or Female 

Pubertal rat assays (Chemicals H, M, and V). However, potential co-solvent interference 

was identified in the AMA in two of these three cases (Chemicals H and M). 

• In the third case (Chemical V), effects on amphibian thyroid histology and developmental 

biomarkers were observed in the absence of any potential confounding effects, while no 

effects were seen in the rat. Such results may support the 2008 SAP prediction that “... 

weakly acting chemicals that affect thyroid hormone receptor or deiodinase activity in 

peripheral tissues would score positive only in the AMA” (USEPA 2008a, p. 23).

Therefore, in the evaluation of battery performance, it is useful to consider the inherent 

differences between each assay, its biological domain of applicability, and the physiology and life 

stage of its test species, in addition to any confounding factors in individual studies. A thorough 

understanding of these issues will be helpful in preparing for the future WoE determinations for 

individual chemicals subjected to Tier 1 EDSP screening.

Across the domain of chemicals evaluated, less complementarity and redundancy in results 

was observed for endpoints within and across Tier 1 assays with respect to the HPT/thyroid 

hormonal pathway than was observed with the estrogen (E) and androgen (A) hormonal pathways 

(Sections b and c below). This observation is likely reflective of the design of the battery; as 

compared to the E and A pathways, fewer assays and biomarkers are available in the battery for the 

detection of thyroidal MOAs. Accordingly, this preliminary review agrees with the 

recommendations of the 2008 SAP that all three thyroid-relevant assays, and each of their diagnostic 

endpoints, need to be considered when screening for potential thyroid activity.
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2. Interaction with the Androgen Pathway

Six assays in the Tier 1 battery are designed to, either specifically or at least in part, screen 

for potential androgen interaction. These assays include three in vitro assays: the androgen receptor 

binding cytosol assay; the aromatase assay; and, the steroidogenesis assay. Additionally, three in 

vivo assays are included in evaluating interaction with the androgen pathway: the Hershberger assay; 

the male pubertal assay; and the fish short-term reproduction assay (FSTRA). The 2008 FIFRA 

SAP41 did not specifically identify the aromatase and steroidogenesis assays when discussing the 

androgen pathway; however, they noted that “[a]ndrogens could also influence enzyme activity in 

the aromatase and H295R cell line [steroidogenesis] assays”. Therefore, all six assays were 

considered when evaluating the complementary and redundant nature of the assays relative to 

potential androgen interactions. Table 38 lists the endpoints from these guideline assays which the 

2008 FIFRA SAP42 identified as indicators of potential androgen interaction. In some cases, the 

endpoints are indicative of a potential androgen agonist (e.g., development of male secondary sex 

characteristics, such as tubercles, in female fish). However, for other endpoints, the observed effect 

may be due to a potential androgen antagonism or an estrogen agonist (e.g., attenuated tubercle 

expression in male fish). This is consistent with the 2008 SAP which stated, “The comparative fish 

short-term reproduction assay would also be sensitive to the effects of an androgen. In this assay, 

androgen would affect organ responses, male and female system integrity with the apical endpoints 

being manifest on development and comparative reproductive system integrity.” The panel also 

stated that “[a]ndrogens will affect organ responses and male system integrity in the male pubertal 

assay,” and that the effects are mediated through sub-organ activities. The Panel also pointed out 

that apical responses such as those in the FSTRA are critical components to the battery’s consistency 

and redundancy, i.e., “the role of fecundity . . . is paramount for evaluations of the HPG axis” 

recognizing that “EPA should be alert to possible non-endocrine mechanisms” (pg. 27). 

 

41 USEPA. 2008. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25-26, 2008, to 

review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008-

03. 11 June 2008. Washington, DC.

42 USEPA. 2008. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25-26, 2008, to 

review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008-

03. 11 June 2008. Washington, DC.
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Table 38. Endpoints for Detecting Potential Effects on the Androgen Hormonal Pathway. 

Assay Endpoint(s)

AR Binding • Receptor binding

Aromatase • Enzyme activity (steroidogenesis)

Steroidogenesis • Enzyme activity (steroidogenesis)

Hershberger • Changes (increase or decrease) in weight of androgen-dependent tissues

Male Pubertal

HPG

• Sexual developmental characteristic (age at preputial separation (PPS))

• Weight and/or histology of androgen-dependent reproductive organs or tissues

• Total serum concentrations of testosterone

Fish Reproduction

HPG

• Fecundity 

• Fertilization success

• Secondary sex characteristics

• Gonadal size (gonado-somatic index [GSI]) and histopathology

• Plasma concentrations of vitellogenin [VTG] in females and males

• Plasma concentrations of testosterone in males (optional)

In the current analysis of Tier 1 battery performance, for the androgen receptor binding and 

aromatase in vitro assays, the Agency considers the extent to which a chemical elicits a response, 

based on the chemical’s percent activity under assay conditions surpassing the threshold established 

in the guideline. A statistically significant (p≤0.05) difference in a chemical’s response compared to 

the control was used in the steroidogenesis assay to evaluate responsiveness. Similarly for the 

Hershberger assay, response was defined when any two of the five measured androgen-dependent 

tissue weights in the treated animals were statistically different (p≤0.05) from the control animals. 

In the male pubertal assays, a change in any of the three endpoint categories listed in Table 38 could 

be considered as indicative of a response with the androgen pathway. Finally, for the FSTRA assay, 

a statistically significant differences (p≤0.05) from the control was found for on fecundity, 

fertilization success, tubercles, GSI, VTG or sex steroids and thus could be considered indicative of 

a response in androgen dependent tissues/processes. 

(i) Complementarity of Results Within Each Tier 1 Assay

Results for each of the six identified assays are presented in Table 39. It is noted that for the 

in vivo studies summarized in Table 39, the determination of a treatment-related effect reflects a 

range of responses over what may be multiple endpoints for a chemical. There are chemicals that 

exhibited multiple treatment-related effects across several different modalities as described by the 

2008 SAP within and across assays. While for other chemicals, a single significant effect was 

observed within a multiple endpoint assay. Additionally, effects may have been detected in the 

presence of overt toxicity; however, at this time such effects were not excluded from the analysis. To 

help in evaluating if the Tier 1 screening assays provide redundancy and complementarily for 

androgenic responses, examples of the types and number of responses observed are discussed below. 
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Male Pubertal Assay: 

• Six chemicals (E, F, J, N, G, and S) resulted in alterations in two or more androgenresponsive endpoints (e.g., testosterone and multiple organ weights); whereas, 

• Three other chemicals (B, C, and P) only reported a single type of androgen-responsive effect 

(e.g., multiple organ weights or testosterone); 

• Chemicals G and S, an alteration on PPS as well as effects in testosterone and androgendependent organ weights was observed but in the presence of overt toxicity (salivation and/or 

decreased body weight). 

Hershberger Assay 

• Three chemicals (Chemical A, G, and H ) exhibited significant alterations in two or more of 

the measured androgen responsive tissues within the assay; whereas,

• Chemicals E, K and S elicited a response by alterations in androgen responsive tissues, but in 

the presence of overt toxicity. 

• Chemical B also reported effects on both androgen-dependent tissues and on testosterone, but 

in the presence of overt toxicity. 

FSTRA

• With the FSTRA, for Chemical J, there were complementary responses in the endpoints 

which included alterations in fecundity, female GSI, female VTG and E2, and female 

testosterone with alterations in female gonads; additionally there was a change in male GSI 

and alterations in male gonads. 

• Chemical V reported an effect on tubercles in males along with alterations in fertility. 

Effects on fecundity were also observed, however, an effect on female body weight (i.e., 

overt toxicity) was reported at the same test concentrations. 

• For Chemical A, while there were responses in two or more endpoints which include 

fecundity, fertilization success, male tubercles, male and female GSI, female and male 

gonadal histopathology, and female VTG, overt toxicity was observed at some, but not all of 

the concentrations where the responses were observed. 

• Additionally, Chemicals B and Q also reported alterations in fecundity, gonadal 

histopathology, and/or fertility and male tubercles, but also at test concentrations both with 

and without overt toxicity. 

• For Chemical H, a reported effect in male tubercles, as well as on fecundity and female 

histopathology, was reported at test concentrations both with and without overt toxicity. 

• While the responses were of a complementary nature (fertility, male tubercles and male 

gonadal histopathology or male GSI and male gonadal histopathology) among endpoints for 

Chemicals K and M, these responses were observed in the presence of overt toxicity. 

• Additionally, effects on fertility or alterations in male gonadal histopathology were also 

observed in the presence of overt toxicity for Chemicals C, D, N, and P. 
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(ii) Redundancy Across Tier 1 Assays 

An examination among the three in vivo assays and three in vitro assays was conducted to 

look for potential redundant responses across the nine different modalities for the assays, with some 

examples presented in Table 39 and discussed below. These examples though should not be 

construed as an interpretation of whether the androgen pathway has been perturbed since such a 

determination would require a WoE analysis. 

Based on the evaluation of redundancy in identifying potential interactions across the assays 

using the available dataset and consistent with input from the SAP it appears that the in vitro assays 

do not consistently exhibit significant effects when effects were observed in the in vivo assays. For 

the in vitro assays, given the specificity and different mechanisms of action represented in the three 

in vitro assays, the mode of action by which the interaction may be occurring may not be consistent 

or necessarily redundant across the in vitro assays. The AR binding assay evaluates binding 

potential whereas the aromatase and steriodogenesis assays examine enzymatic activity. This is 

consistent with the Panel comments that the in vitro assays represent “specificity and 

sensitivity...and do not provide complementary or redundant assessments of androgenic activities”.

• Chemical J reported responses in sex steroids and organ responses (androgen-dependent 

tissue weight and female VTG levels) in both the male pubertal and fish assays as well as 

responses in the in vitro aromatase and steroidogenesis assays and an equivocal response in 

the androgen binding assay. 

• For Chemical A, organ responses were observed in both the Hershberger and FSTRA; 

however, the organ response in the FSTRA (effects on female VTG) was observed in the 

presence of overt toxicity. Additionally, effects on cell response, male and female systems

and fertility were also observed in the fish assay, but again some of those responses were 

observed in the presence of overt toxicity. For Chemical A, there were also responses in the 

androgen binding and steroidogenesis assays and an equivocal response in the aromatase 

assay. 

• For Chemical E, effects on androgen-responsive organ weights were observed in the 

Hershberger and male pubertal assays with effects on testosterone observed in the pubertal 

assay; overt toxicity was observed in the Hershberger. 

• In Chemical B, effects on androgen-dependent organs weights were observed in the 

Hershberger and male pubertal, but overt toxicity was observed in the Hershberger. 

Additional effects including male tubercles and alterations in male gonads were affected in

the FSTRA assay for Chemical B, but there may be potential solvent interferences with this 

assay.
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Table 39. Summary of Results in Tier 1 Assays Relevant to the Androgen Hormonal Pathway 

by Chemical. 

Chemical 

Code ARB Aromatase Steroidogenesis Hershberger Male Pubertal FSTRA

A x equivocal x x x 

B xa x xc

C U x x xb

D equivocal xa

E xa x 

F NA x x 

G x xa xd

H x equivocal x x 

I equivocal x xa

J equivocal x x x x 

K x x xa xa

M equivocal equivocal x 

N x x 

P equivocal equivocal x xa

S equivocal xa xa xa

T NA NA NA x I 

V x 

x = Treatment-related effect(s) on measured endpoint(s) indicative of potential androgen interaction reported

Blank cells represent the absence of a statistically significant effect).

U = Chemical could not be tested under assay conditions 

I = Inconclusive

NA = Data submission not yet reviewed (OSRI) a 

= Effects only observed at test levels with overt toxicity (e.g., substantial decrease in body weight in rats; for fish - 

reduced survival, abnormal swimming, reduced feeding)

b = Changes in fish body weight reported at concentrations were an effect was also reported c = Potential solvent interferences

d = Study conduct questionable, results possibly not reliable

(iii) Discussion

The six assays discussed are intended to provide insight on a chemical’s potential to interact 

with the androgen pathway as measured through different levels of biological organization. The 

identified assays are intended to provide complementary and/or redundant assessments of 

androgenic activity. This is reflected in the 2008 SAP comment that “the in vivo assays included in 

the Tier I battery provide for both redundancy and complementarily for androgenic responses”, and 

that the aromatase and steroidogenesis assay “complete the complement of in vitro assays in the Tier 

I battery”. However, the in vitro assays may not provide the same level of redundancy, nor are they 

expected to provide the same level of data compared to the in vivo assays. 
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As discussed above and as summarized in Table 39, some concordance was observed across 

the in vitro and in vivo assays with the chemicals evaluated in support of this white paper. 

In terms of responses across the in vitro assays, there were three (Chemicals A, J, and K) out 

of the 17 cases that had effects in two in vitro assays which were considered to have potential 

activity where two of those cases (Chemicals A and K) included the AR binding assay (Table 39). 

For Chemicals M and P, there were two of the three in vitro assays that had equivocal results. For 

Chemicals A, H, I, and J there an equivocal result for one in vitro assay along with an effect in at 

least one other in vitro assay. 

Based on the evaluation of responses within the more complex assays (i.e., FSTRA and the 

male pubertal), two or more endpoints were altered indicating the in vivo assays provide 

concordance with which to evaluate androgenic responses.

For the three in vivo assays (Hershberger, Male Pubertal, and FSTRA), there were alterations 

in an endpoint (s) among them providing sufficient information to evaluate potential androgenic 

responses. However, evaluation of responses among the three in vivo assays is complicated by the 

observation of overt toxicity in some of the assays. Overt toxicity was observed for at least one 

study in each of the three in vivo assays for chemicals where effects were reported, with six of the 

FSTRAs reporting significant effects only where overt toxicity was also observed. Additionally,

another FSTRA study had potential co-solvent interferences and in another, the results of the study 

were technically deficient and could not be readily evaluated. A discussion on the observation of 

overt toxicity in the FSTRA is presented in a previous section (Section 2e). 

Although the in vitro assays did not exhibit effects as frequently as the in vivo, there are a 

number of plausible biological reasons why this may occur and does not necessarily refute the 

intended redundancy of the assays within the battery. The importance of the intentional redundancy 

is illustrated where of seven chemicals (Chemicals A, B, E, G, H, K, and S) that elicited significant 

results in the Hershberger assay, four (B, E, G, and S) also produced a significant response in the 

male pubertal assay; for Chemicals A, J and K effects were observed in the in vitro assays as well. 

For the three chemicals that did elicit a response in the Hershberger but not the male pubertal assay

(Chemicals A, H, and K), a response was observed in the FSTRA. However, overt toxicity was 

observed in some of the in vivo assays. Therefore, the range of endpoints evaluated in the Tier 1 

battery provides complementary response within the in vivo assays and redundancies across the 

relevant in vitro and in vivo assays to evaluate androgenic responses. 

To what extent is this level of redundancy among the in vivo studies necessary for the Tier 1 

assay is a question posed to the SAP panel. The agency will seek guidance from the 2013 SAP on 

whether the Hershberger assay is a necessary redundancy in the current battery given that the 

Hershberger typically showed responses in androgen sensitive tissues when the male rat pubertal 

and/or FSTRA also exhibited effects (Table 39). 
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3. Interaction with the Estrogen Hormonal Pathway 

The EDSP Tier 1 battery covers the Estrogen hormonal pathway more extensively (i.e., has 

more responsive endpoints) than the Androgen or Thyroid pathways. However, battery performance 

for the estrogen pathway is difficult to fully assess because of the apparent absence of case studies 

with unequivocal estrogen receptor agonist activity (as indicated by positive ER binding and ERTA, 

and/or Uterotrophic assay results) in this initial review of 18 chemicals. 

Seven assays in the Tier 1 battery are designed to identify potential interactions on the 

estrogen hormonal system and with different sexes and taxa. These include four in-vitro assays: the 

ER binding assay; ERα transcriptional activation (ERTA) assay; the aromatase assay; and the 

steroidogenesis assay. Additionally, three in vivo assays are useful in evaluating interaction with the 

estrogen pathway: the uterotrophic assay; the female rat pubertal assay; and the FSTRA. With

regard to the female pubertal assay, the 2008 FIFRA SAP identified vaginal opening and onset of 

first estrus as suitable endpoints for estrogen-dependent responses, but the panel noted that ovarian 

cyclicity may not be a useful endpoint because of high variability caused by other factors. The 2008 

FIFRA SAP considered these seven assays as a means of evaluating the complementary and 

redundant nature of the assays relative to potential estrogen interactions. Endpoints from these 

guideline assays which the 2008 FIFRA SAP43 identified as biological indcators of potential 

estrogen interaction are presented in Table 40. 

In this preliminary analysis of Tier 1 battery performance, for the estrogen receptor binding 

and aromatase in-vitro assays, the Agency considers a chemical to have the potential to interact with 

the estrogen pathway based on the chemical’s percent activity under assay conditions surpassing the 

threshold established in the guideline. A statistically significant (p≤0.05) difference in a chemical’s 

response (alteration in estradiol levels) compared to the control was used to determine a potential 

estrogen interaction in the steroidogenesis assay. In the female pubertal assay, a change in any 

endpoint listed in Table 40 could be considered and indication of a response in estrogen dependent 

tissues/ processes. Finally, for the FSTRA assay, a statistically significant differences (p≤0.05) from 

the control was found for fecundity, fertilization success, tubercles, GSI, VTG or sex steroids and 

thus could be considered indicative of a response in estrogen dependent tissues/processes. 

 

43 USEPA. 2008. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25-26, 2008, to 

review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008-

03. 11 June 2008. Washington, DC.
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The inclusion of the FSTRA assay was reported by the 2008 SAP Panel as “an important 

component of the estrogenicity screen”, with a specific mention of the measurement of plasma VTG. 

Ultimately, the degree to which the responses across a pathway are complementary and redundant 

are considered in the context of any observations of overt toxicity and procedural adequacy to 

support the WoE determination for each chemical, which will be the subject of a future SAP. 

Table 40. Endpoints for Detecting Potential Effects on the Estrogen Hormonal System.

Assay Endpoint(s)

ER Binding • Receptor binding

ERTA • Cell Signalling

Aromatase • Enzyme activity (steroidogenesis)

Steroidogenesis • Enzyme activity (steroidogenesis)

Uterotrophic • Increased uterine weight

Female Pubertal

HPG

• Sexual developmental characteristics (vaginal opening and onset of 

first estrus) 

• Weight and/or histology of estrogen-dependent reproductive organs 

or tissues 

Fish 

Reproduction

HPG

• Fecundity 

• Fertilization success

• Secondary sex characteristics

• Gonadal size (gonado-somatic index) and histopathology

• Plasma concentrations of vitellogenin in females and males

• Plasma concentrations of sex steroids (optional)

(i) Complementarity of Results Within Each Tier 1 Assay 

Results for each of the seven identified assays are presented in Table 41. It is noted that for 

the in vivo studies summarized in Table 41, the determination of a treatment-related effect reflects a 

range of responses over what may be multiple endpoints for a chemical. There are chemicals that 

exhibited multiple treatment-related effects across several different modalities as described by the 

2008 SAP within and across assays. For other chemicals, a single significant effect was observed 

within a multiple endpoint assay. Additionally, effects may have been detected in the presence of 

overt toxicity; however, at this time such effects were not excluded from the analysis. To help in 

evaluating if the Tier 1 screening assays provide redundancy and complementarity for estrogenic 

responses, examples of the types and number of responses observed are discussed below.
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Female Pubertal Assay:

• Chemical E resulted in changes in VO and first estrus as well as effects on estrogen 

dependent tissues;

• Chemical Q had effects on VO and first estrus; 

• Chemical G, effects were reported in the presence of overt toxicity (mortality, decreased 

body weight and clinical signs) and included alterations in VO and effects on ovarian and 

uterus (wet, and blotted ) weights; 

• For Chemical K, effects observed in the presence of overt toxicity (tremors) were ovarian 

follicle changes; 

• Chemical M, effects reported in the presence of overt toxicity (salivation) were alterations in 

VO and first estrus; and 

• For Chemical S, effects reported in the presence of overt toxicity (decreases in body weight 

and food consumption) included changes in VO and in ovarian and uterus (wet and blotted) 

weights. 

FSTRA Assay:

• Chemical V reported effects on fecundity, fertility and male tubercles; however, there was an 

effect on female body weight reported. 

• Chemical H had alterations in fecundity, male tubercles and female gonadal histopathology, 

although overt toxicity was observed at some, but not all, concentrations with the reported 

effects. 

• Chemical M reported alterations in fecundity, male and female GSI and gonads; however, the 

histopathology and male GSI effects corresponded with observed overt toxicity along with 

some of the effect concentrations for fecundity. 

• Chemical F reported effects on fecundity, fertility, male tubercles, GSI, estradiol levels and 

male and female gonads; however, except for fertility and male GSI, all the remaining effects 

were only observed in the presence of overt toxicity.

• Similarly, for Chemical S, alterations in fertility, male tubercles, male GSI, and male VTG 

were observed only at levels with observed overt toxicity. 

• In Chemical N, alterations in fecundity, fertility, female GSI and gonadal histopathology 

were only observed in the presence of overt toxicity, except for female VTG. 

• Chemicals C, D, I, K, and P also reported alterations in fecundity and/or gonadal 

histopathology, fertility, male tubercles, GSI or VTG only in the presence of overt toxicity. 

Uterotrophic Assay: 

• None of the chemicals were active in the in vivo uterotrophic assay. However, the results in 

the ER binding and the uterotrophic assay demonstrated concordance in that there was a lack 

of response in both assays which suggests a lack of estrogenicity among the case studies. 
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(ii) Redundancy Across Tier 1 Assay 

An examination among the four in vivo assays and three in vitro assays was conducted to 

determine potential redundant responses across the nine different modalities for the assays, with 

some examples presented in Table 41 and discussed below. These examples should not be construed 

as an interpretation of whether the estrogen pathway has been perturbed since such a determination 

would require a WoE analysis. 

In general, concordance of receptor-mediated in vitro responses (ER binding and ERTA) and 

in vivo assays including HPG effects (female pubertal rat and FSTRA) results was limited, 

suggesting that estrogen pathway effects observed in the evaluation of battery performance are likely 

mediated through mechanisms other than receptor binding. Potential effects of enzymatic 

conversion of androgens to estrogens are evaluated directly by the aromatase assay (inhibition only) 

and indirectly by the steroidogenesis assay (induction or inhibition relative to controls of 

testosterone and estradiol from cholesterol). Results of these two assays were biologically consistent 

for the chemicals included in this evaluation; Chemical J significantly inhibited aromatase activity 

and estradiol production in the steroidogenesis assay and all other chemicals with statistically 

significant (p ≤ 0.05) steroidogenesis assay results (Chemicals B, C, D, F, I, O, P, and V) appeared 

to induce estradiol production and had no significant effect on aromatase activity. 

• Chemical Q reported responses in female estrogen-dependent systems (i.e., alterations in 

vaginal opening/estrus or fecundity) in the female pubertal and FSTRA; however, an effect 

on fecundity was only reported in the presence of overt toxicity.

• Chemical M reported responses in female estrogen-dependent systems (i.e., alterations in 

vaginal opening/estrus or fecundity) in the female pubertal and FSTRA; however for the 

pubertal assay, these effects were only observed in the presence of overt toxicity. Equivocal 

results were reported for the aromatase and steroidogenesis assays.

• In Chemical S, organ responses were observed in both the pubertal and FSTRA. But only in 

the presence of toxicity. An equivocal result was reported for the aromatase assay.

• Similarly, for Chemical K, alterations in cell response (gonadal histopathology) and female 

estrogen-dependent systems (vaginal opening and fecundity) were observed in the female 

pubertal and FSTRA in the presence of overt toxicity. An effect in the aromatase assay was 

reported as well. 

• In Chemical T, there were effects reported on female estrogen-dependent systems in the 

pubertal but were equivocal in the FSTRA. 

It may be worth noting that if studies with overt toxicity are excluded from consideration, 

statistically significant (p ≤ 0.05) estrogen responses observed in the female pubertal rat (Chemicals 
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E, T, and Q) and FSTRA (Chemicals A, F, H, J, N, Q, and V) possibly represent two distinct 

chemical sets and may indicate different sensitivities of these two test species or different chemical 

actions due to oral (rat) versus aquatic (fish) exposure routes. Though significant (p ≤ 0.05) estrogen 

pathway effects were observed in in vivo assays without accompanying in vitro effects, for no 

chemical were significant in vitro effects observed without accompanying in vivo effects.

Table 41. Summary of Results in Tier 1 Assays Relevant to the Estrogen Hormonal Pathway, 

by Chemical.

Chemical 

Code ERB ERTA Aromatase Steroidogenesis Uterotrophic

Female 

Pubertal FSTRA

A equivocal equivocal x 

B x X xc

C equivocal X x

b

D equivocal X xa

E x x 

F x X x

G xa x

d

H equivocal x

I x X xa

J x X x

K x xa xa

M equivocal equivocal xa x

N x 

P equivocal X xa

Q I x x 

S equivocal xa xa

T NA NA x I 

V X x

x = Treatment-related effect(s) on measured endpoint(s) indicative of potential estrogen interaction reported

I= Inconclusive

NA = Data submission not yet reviewed (OSRI) a

= Effects observed only in the presence of overt toxicity (e.g., substantial decrease in body weight in rats; for fish - reduced survival, 

abnormal swimming, reduced feeding)

b = Changes in fish body weight reported at concentrations were an effect was also reported c = Potential co-solvent interferences

d = Study conduct questionable, results possibly not reliable
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(iii) Discussion

The seven Tier 1 screening assays discussed above are meant to provide insight on a 

chemical’s potential to interact with the estrogen hormonal system as measured through different 

levels of biological organization. The identified assays are intended to provide complementary or 

redundant assessments of estrogenic activity. The 2008 SAP stated that the “in vivo assays are well 

represented with a breadth of complementary endpoints. In vitro assays on the other hand have few 

or no complements such as indicated for receptor binding or transactivation assay.” However, the 

Panel did comment that the estrogenic pathway for each modality did appear to retain at least some 

redundancy within the battery. 

In conclusion, the range of endpoints evaluated in the Tier I battery is consider to provide the 

necessary redundancies to evaluate estrogenic responses. The different mechanisms of action and 

exposure routes among assays relevant to the estrogen hormonal pathway were designed to minimize 

false negatives and detect effects of chemicals at multiple levels of biological organization. It 

should be noted that the set of chemicals on which this initial evaluation of battery performance does

not include estrogen receptor agonists, thus making it difficult to assess the performance of the 

battery for this mode of action. Nonetheless, in 2008, the battery of assays was validated for 

estrogen agonism using known reference chemicals. 

Based on the results of this subset of chemicals as reported in Table 41, the uterotrophic 

assay did not appear responsive for this subset of chemicals. While acknowledging that there does 

not appear to be estrogen agonists in this subset of chemicals, it is important to note that there were 

no cases where the uterotrophic assay was positive and pubertal and/or FSTRA was negative. To 

what extent is this level of redundancy across the in vivo assays necessary for the Tier 1 screen will 

be posed to the SAP panel. The agency seeks advice on whether the uterotrophic assay provides a 

necessary redundancy in the battery for the estrogen signaling pathway given the mechanistic 

specificity of the in vitro assays and the multi-parameter female pubertal assay and FSTRA. 

V. Overall Summary and Conclusions 

In 1999, following the EDSTAC recommendations to develop a two-tiered screening and testing 

program, a joint subcommittee of the Agency’s Science Advisory Board and FIFRA Scientific 

Advisory Panel recommended to the Agency, after review of the initial set of Tier 1 data, to subject 

that data to external scientific peer review for consideration to further optimize the Tier 1 screening 

battery. The scientific analyses included in this white paper are intended to evaluate the performance 

of the individual Tier 1 assays and the overall battery. 

Individual Assay Performance Conclusions: 

EDSP Tier 1 assay data were reviewed on a subset (up to 21 test chemicals) of the List 1 

chemicals (consisting of 50 pesticides and 2 inerts). The subset represents a broad range of physical-
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chemical properties (e.g., water solubility, octanol-water partition coefficients, volatility), as well as 

a range of chemical classes and pesticidal biological activities. The purpose of the analysis was to 

evaluate: 

• How well each Tier 1 assay test guideline can be consistently executed based on defined 

performance criteria;

• Issues associated with interpretation of responses within each assay;

• Complementary relationships within the multi-parameter in vivo assays in the Tier 1 battery 

which may be indicative of potential interactions with the estrogen, androgen or thyroid 

hormonal pathways; and, 

• Redundancy across the in vitro and in vivo assays that comprise the Tier 1 battery.

Additionally, this review considered whether a modified battery of assays, could reduce 

animal usage and lower costs, while adequately ensuring the identification of potential interactions 

with the E, A or T hormonal pathways. 

Based on the case studies evaluated, the Tier 1 assays provide useful information to evaluate 

potential interactions with the E, A, and T signaling pathways. There were no major problems 

identified with the Tier 1 assays; in general, contributing laboratories were able to execute each 

assay protocol in accordance with the respective test guidelines and achieve the specified 

performance criteria. Although there were some minor deviations from the performance criteria, 

these differences were not substantial so as to render the study unreliable in the vast majority of 

cases. Although recommended CV values for test concentrations were not always achieved with the 

in vivo assays using aquatic organisms, it is not unexpected for those test substances that are poorly 

soluble or prone to absorb to surface matrices within these experimental test systems.

Battery Performance Conclusions:

In evaluating the battery performance with the current subset of test order chemicals, there 

appears to be reasonable representation of endpoint alterations intended to evaluate potential 

interactions on the androgen hormonal pathways, the HPG axis and HPT axis. However, based on 

this initial evaluation, the subset of List 1 chemicals may be limited with respect to estrogenic 

modes of action (particularly, agonism). It should be noted, that the Tier 1 assays were validated 

against compounds that act directly on the estrogen receptor. The discussion below will present the 

analysis for the battery performance by each hormonal pathway.

Thyroid Pathway: 

There are three Tier 1 assays (i.e., the male and female pubertal assays, and AMA) relevant 

to evaluating the HPT pathway. There is less complementarity built into the design for the T 

pathway compared to the E- and A-pathways which is reflected in the results of the current analysis. 

In the rat pubertal assays, thyroid hormone changes were the most common observation which was 
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accompanied by thyroid histology only in a few cases. In the AMA, histology was generally the 

most sensitive endpoint. There is also less redundancy built into the battery for the thyroid pathway. 

Thyroid-related results in the AMA were not necessarily predictive of the rat findings and vice versa. 

This initial analysis supports the view of the 2008 SAP that the AMA and the rat pubertal studies can 

provide different types of information and it reinforces the importance of including both taxa in the 

screening battery to reduce the potential for false negatives. 

Androgen Pathway: 

There are six Tier 1 assays relevant to evaluating the potential of a chemical to interact with 

the A pathway (i.e., the androgen receptor binding cytosol assay, aromatase assay, steroidogenesis 

assay, Hershberger assay, male pubertal assay, and the fish short-term reproduction assay. The 

degree of complementary findings relevant to the androgen hormonal pathway was evaluated for the 

more complex multi-parameter in vivo assays. In the male pubertal assay, responses were generally 

observed for two or more different types of endpoints or for two or more of the androgen-dependant 

organs. With the FSTRA, effects were observed for all the chemicals and complementarity was 

generally found with two or more endpoints affected. It should be noted that about a third of the 

time the findings in the FSTRA were at exposure concentrations that also elicited signs of overt 

toxicity, and thus, distinguishing effects due to generalized toxicity versus potential interaction with 

the androgen (or estrogen) pathway for each chemical will be an important consideration in future 

weight of evidence determinations. The male pubertal also showed a number of responses but overt 

toxicity did not appear to be a major issue. 

Consistent with the 2008 SAP, the in vitro assays do not consistently exhibit significant 

effects when effects are observed in the in vivo assays. But this analysis did find redundancy across 

the in vivo assays (Hershberger, male pubertal, and FSTRA) with this subset of chemicals, albeit 

some in vivo responses were complicated by the observation of overt toxicity. A number of the 

chemicals that elicited responses in the Hershberger assay also produced a response in the male 

pubertal assay. For the chemicals that elicited a response in the Hershberger but not in the male 

pubertal assay, a response was observed in the FSTRA indicating that the in vivo redundancy for the 

androgen signaling pathway enables the battery to serve as an effective means of minimizing false 

negatives. The agency will seek guidance from the 2013 SAP on whether the Hershberger assay is 

a necessary redundancy in the current battery given that the Hershberger typically showed responses 

in androgen sensitive tissues when the male rat pubertal and/or FSTRA also exhibited effects.

Estrogen Pathway: 

There are seven assays in the Tier 1 battery designed to screen for potential interaction with 

the E pathway (i.e., the estrogen receptor (ER) binding assay, ERa transcriptional activation (ERTA) 

assay, aromatase assay, steriodogenesis assay, uterotrophic assay, the female rat pubertal assay, and 

the FSTRA). As indicated earlier, the current case studies may be limited in potential modes of 

action relevant to the E pathway. Again, the FSTRA showed a number of responses complicated by 
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overt toxicity about one third of the time. Effects found in the female rat pubertal assay were 

generally associated with responses in the fish, but not always. Based on this data set, the female rat 

pubertal and fish assays are important in evaluating the E pathway across taxa, and therefore are 

important components to the battery. The uterotrophic assay was consistently negative for all the 

chemicals examined, an effect which would be viewed as consistent with the lack of responses found 

for the in vitro estrogen receptor binding assay. The lack of response in the uterotrophic assay, ER 

binding assay and ERTA, and the discordant results with ER binding and ERTA, suggests that there 

are no ER agonists in this subset of chemicals. There are also some examples of responses in the in 

vitro steroidogenesis assay that were associated with the female rat pubertal and/or fish assays. The 

agency seeks advice on whether the uterotrophic assay represents an unnecessary redundancy in the 

battery for estrogen signaling pathway given the mechanistic specificity of in vitro assays and the 

multi-parameter femal pubertal assay and FSTRA, noting that the results of the uterotrophic assay 

were consistently negative and that there were no cases when the uterotrophic was positive but not in 

the pubertal or FSTRA. 
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