# 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

2 from Damstra et al. 2002

# Screening Battery Considerations

 

# G iven 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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# T wo - 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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# T ier 1 Assays

 In vitro assays

• S teroidogenesis

• E strogen receptor binding

• E strogen transcriptional activation

• A ndrogen receptor binding

• A romatase

 HPG

- interrupted assays

• U terotrophic (female, no gonads)

• H ershberger (male, no gonads)

 Whole

- animal assays

• P ubertal female

• P ubertal male

• A mphibian metamorphosis

• F ish short

- term reproduction

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# T ier 1 Battery of Assays

 

## D esigned to maximize sensitivity (minimize false negatives)

 

## I n vitro assays provide sensitivity and mechanistic clues

 

## I n 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

 

## I nterpreted 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 RoleEAT 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 multiple

assays 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:

• A re 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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