Endocrine Disruptor 

Screening Program:

Tier 1 Screening Battery

Background

Leslie Touart, Ph.D. 

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
Endocrine System

 Integrated

Homeostasis

Negative feedback

EAT subpart of broader endocrine axes

The endocrine system can be perturbed at multiple 

sites and by multiple mechanisms

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from Damstra et al. 2002
Screening Battery Considerations

Given the complex interactive nature of the 

endocrine system, it is clear that chemicals 

should be screened for their apical activity

(e.g., the ability to alter growth, development

or reproductive processes) rather than just for 

their sex steroid activity in in vitro assays if the 

objective is to effectively detect their potential

to disrupt endocrine regulated processes.

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EDSP Tier 1 Screening Battery

Designed to ensure detection of EAT hormonal 

systems interaction

Fulfills EDSTAC recommendation that a range of 

taxonomic groups be included 

Includes sufficient diversity of endpoints to 

maximize sensitivity and minimize false negatives

Emphasizes apical assays to provide more 

comprehensive assessment

Meets overall objective of detecting potential 

endocrine-mediated effects regardless of MOA

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Two-Tiered Approach to Screening

Tier 1

• Detects potential to interact with endocrine system (E, A, T)

• Includes both in vitro and in vivo assays 

• If positive, then Tier 2

Tier 2

• Corroborate interaction

• Characterizes adverse effects, if any 

• Provides dose-response data

• Consists of life-cycle studies

• Covers a broad range of taxa

(mammals, birds, frogs, fish, invertebrates)

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Tier 1 Assays

 In vitro assays

• Steroidogenesis

• Estrogen receptor binding

• Estrogen transcriptional activation

• Androgen receptor binding

• Aromatase

 HPG-interrupted assays

• Uterotrophic (female, no gonads)

• Hershberger (male, no gonads)

 Whole-animal assays

• Pubertal female

• Pubertal male

• Amphibian metamorphosis

• Fish short-term reproduction

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Tier 1 Battery of Assays

Designed to maximize sensitivity (minimize false 

negatives)

 In vitro assays provide sensitivity and mechanistic clues

 In vivo assays provide for integrative responses and 

metabolism and distribution considerations 

Relies on similarities of endocrine systems across 

taxa; includes “redundancies” across assays as well as 

assay-unique endpoints

 Interpreted as weight-of-evidence of the battery and 

relevant information, rather than sum of positive and 

negative assays

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Screens and EAT Systems Interaction

Steroid Synthesis

E E- A A- A E HPG HPT

In vitro

ER Binding X X

ER Transcriptional Activation X

AR Binding X X

Steroidogenesis (H295R) X X

Aromatase (Recombinant) X

In vivo

Uterotrophic X

Hershberger X X

Pubertal male X X X X X

Pubertal female X X X X X

Fish Reproductive Screen X X X X X X X

Amphibian Metamorphosis X
Estrogen Pathway

ER Binding In vitro; Detects chemicals that bind 

to ER

ER TA In vitro; Detects ER interaction and 

function, differentiates ER agonists

Uterotrophic (sc) In vivo; Detects chemicals that act 

through the ER; incorporates ADME.

Pubertal female In vivo; Detects chemicals that act on 

the mammalian estrogen system. 

Fish screen In vivo; Detects chemicals that act on 

the fish estrogen system

Assay Role
Androgen Pathway

AR Binding In vitro; Detects chemicals that bind to 

AR

Hershberger In vivo; Detects chemicals that act 

through the AR. Can distinguish 

agonists from antagonists. Incorporates 

ADME.

Pubertal male In vivo; Detects chemicals that act on 

the mammalian androgen system. 

Fish screen In vivo; Detects chemicals that act on 

the fish androgen system.

Assay Role
Thyroid Pathway

Pubertal female In vivo; Detects changes in hormones 

and/or thyroid histology. 

Pubertal male In vivo; Detects changes in hormones 

and/or thyroid histology. 

Amphibian metamorphosis In vivo; Detects changes in thyroid 

histology and in development.

Assay Role
Steroidogenesis

Recombinant Aromatase In vitro; Assay detects chemicals that 

inhibit aromatase

H295R Steroidogenesis In vitro; An adrenal carcinoma cell-based 

assay that detects chemicals that induce 

or inhibit enzymes in the steroid synthesis 

pathway 

Pubertal female In vivo; Detects chemicals that act on the 

mammalian estrogen system, adrenals.

Pubertal male In vivo; Detects chemicals that act on the 

mammalian androgen system, adrenals.

Fish screen In vivo; Detects chemicals affecting the 

estrogen or androgen systems in fish

Assay Role
EAT System Derivation of Detection Ability

Estrogenic Activity ER binding and ER TA

Uterotrophic

Female Pubertal

Fish

Anti-estrogenic Activity ER Binding

Female Pubertal

Fish

Androgenic Activity AR Binding

Hershberger

Male Pubertal

Fish

Anti-androgenic Activity AR Binding

Hershberger

Male Pubertal

Fish

Modulation of Steroidogenesis H295R and Aromatase

Male and Female Pubertals

Fish

Modulation of Aromatase H295R and Aromatase

Female Pubertals

Fish

Altered Hypothalamic-Pituitary 

Function

Male and Female Pubertals

Fish

Amphibian Metamorphosis

Anti-thyroid Activity Male and Female Pubertals

Amphibian Metamorphosis

Thyromimetic Activity Amphibian Metamorphosis

Strength of Detection Ability of Tier 1 Battery
Screening Battery Interpretation

Weight-of-evidence (WOE) review

• Uses professional judgment

• Some endpoints more diagnostic/specific than others

• Effects seen in multiple-endpoints and multipleassays carry most weight

Two possible interpretation outcomes:

1. The potential for EAT activity, which warrants 

analysis in Tier 2 testing 

2. Low or no potential for EAT activity

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FIFRA SAP

Review of the EDSP Tier 1 Screening Battery — 2008

 EPA Charge Question:

• Are there any unnecessary redundancies for MoAs across the battery?

 Panel Assessment:

• Considered each assay in reference to different modalities associated with 

the E, A, and T hormonal pathways

• Examined degree of “complementarity” or concordance of endpoints within 

an assay

• Examined degree of redundancy or concordance of endpoints across the 

assays

 Panel Conclusion:

• In vitro assays convey specificity and sensitivity but “provide no 

complementarity or redundancy within the battery”

• In vivo assays “provide both complementarity and redundancy within the 

battery”

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FIFRA SAP

Review of the EDSP Tier 1 Screening Battery — 2008

Panel Overall Recommendations:

• Based on the current state of the science, the 

proposed set of Tier 1 assays are an appropriate 

starting point to detect potential endocrine disruptors

• EPA should consider this set of assays to be a work 

in progress

• Panel expects EPA will continue to develop, refine, 

and review the battery

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Summary

Multiple assays required to comprehensively 

screen E, A, and T hormone systems

In vitro assays good for well understood 

mechanisms (e.g., receptor binding) 

In vivo assays with intact HPG/HPT axes good for 

efficiently screening complex processes

Multiple taxa and MOA endpoints provide range of 

metabolism and needed corroboration

Complete battery needed to support WOE finding 

of low or no potential for EAT activity

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