Lessons Learned, Challenges, and Opportunities: The US EDSP | April 23 - 24, 2013| RTP, North Carolina Predictive Toxicology Summit | May 22 – 23 2012 | Boston MA FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

The Future of Endocrine Screening: 

An Animal Welfare Perspective

Catherine Willett, PhD

Director, Regulatory Testing

Risk Assessment and Alternatives

kwillett@humanesociety.org
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Outline

Animal use in the current EDSP design

Suggestion for an alternate approach:

– Begin with modified tiered system 

– + weight-of-evidence

– Move toward pathway-based approach
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Tier 1 Animal Use
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Putative Tier 2 tests

Assay # animals/test

Mammalian 2-Generation 1600 - 2400

Amphibian 2-Generation 400 P, plus progeny

Avian 2-Generation 1024

Fish Lifecycle ~1000*

Total > 4,000

*number varies by species
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Animals Used in Phase I

Data submitted and generated for 52 chemicals in Phase I: 

OSRI was submitted for all or some assays for 47 chemicals* 

* Bishop, Sullivan and Willett, submitted
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Need for a better approach

Current program design is resource, labor and animalintensive

Provides much data, only some of which may have have 

regulatory use

Provides insufficient opportunity for chemically-relevant 

tailoring 

Does not link mechanism to adverse outcome

Is not predictive 
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

First step: a more refined tiered system

Initial tier takes into account all existing information, 

including physicochemcial

Second tier assesses potential mechanisms of action

Following tiers address potential effects in more complex 

systems, on multiple modes of action

Highest tiers address adverse outcome and dose-response

WoE with clearly articulated criteria applied at each tier

• Defines positive and negative cut-offs at the outset for 

each test and each Tier or Level

• Results are used to design the strategy for further testing

Strategy is designed to address information needs
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

OECD Conceptual Framework

Level 1 Sorting and prioritization based on existing information 

•physical and chemical properties

•exposure

•available toxicological data

Level 2 In vitro assays providing mechanistic data 

•QSARs

•binding and transcription assays

•aromatase, steroidogenesis, thyroid function 

•high-throughput screens

Level 3 In vivo assays addressing single mechanisms and effects 

•uterotrophic

•Hershberger 

•amphibian metamorphosis

•fish VTG (e.g. TG 230)

Level 4 In vivo assays addressing multiple mechanisms and effects

•enhanced 407

•male and female pubertal assays

•adult intact male

•fish short-term reproductive assay (e.g. TG 299)

Level 5 In vivo assays providing data on effects from endocrine + other mechanisms 

•1 and 2 generation reproduction in rodents, birds, fish
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Level 3: Mechanistic in vivo screens/ 

metabolism considerations

• Negative

• Positive: MoA +

existing 

adverse effects 

data

• Positive and 

• More information required for HA /RA

Level 4: Targeted in vivo testing

to assess specific adverse 

effects 

Level 5: Targeted in vivo testing to 

address specific adverse effects 

more completely

More extensive 

characterization 

required for HA /RA

Clear positive 

or negative

Continue?

Yes

Level 1: Physical/chemical 

and known/estimated 

information

 

Level 2: Mechanistic in vitro

and in silico screens

Lower Priority for 

Further Testing

• Activity in more than one related assay

• Activity at low concentrations

Continue?

Yes

Continue?

Yes

 Higher Priority for 

Further Testing

• No activity or 

activity only at 

high concs.

• Activity in few, 

unrelated 

assays

Clear positive 

or negative

Refined tiered system with integrated assessment

Willett et al. 2011.

Toxicol. Sci.123(1):15–25.
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

The future of EDSP: a pathway-based approach

A conceptual construct that portrays existing knowledge of 

linkages between a direct molecular initiating event and an 

adverse outcome at a level of biological organization relevant 

to risk assessment

From: Ankley et al. Environ.Toxicol.Chem. 2010. 29 (3): 730–741.
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

ER-mediated reproductive impairment

Developed from:

• an understanding of the MIE plus

• a need for a strategy for measuring endocrine-mediated effects. 

• Includes use of biomarker (vitellogenin) that is (in the case of females) or is not (in the 

case of males) directly linked to the adverse outcome 
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

ER-mediated reproductive impairment 

integrated testing strategy

Adverse Outcome Pathway

Chemicals 

Receptor

Binding

ER Binding

Liver Cells

Altered

Protein 

Expression

Vitellogenin

Liver

Altered 

proteins

Gonad

Ova-testis;

Sex-reversed;

Fecundity

Sex 

reversal;

Altered 

behavior;

Repro.

In vivo

MOLECULAR

 Target

CELLULAR

Response TISSUE/ORGAN INDIVIDUAL

Skewed 

Sex Ratios;

Population

POPULATION

Toxicity Pathway

P. Schmieder, McKim conference 2008.

QSAR/

In vitro In vitro
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

ER and AR pathway efforts

US EPA:

• AOP development

• HTS and other in vitro assays

The Hamner Institutes: “Tier 1 and Done”

CAAT: Pathways of Toxicity 

OECD Validation Management Group – Non-animal

• Binding and TA assays

OECD Advisory Group on Molecular Screening and 

Toxicogenomics

• AOP development
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Thyroid pathway efforts

Development of AOPs and relevant assays

• OECD Thyroid Scoping Effort Expert Group

• OECD Validation Management Group – Nonanimal

• OECD Advisory Group on Molecular Screening 

and Toxicogenomics

• US EPA 
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

• Build Biological and adverse-outcome “pathways”

• OECD integration of AOPs into the Test Guidelines program

• Guidance

• Improve predictive tools

• NIH National Center for Advancing Translational Sciences 

• EPA’s Computational Toxicology Research

• OECD QSAR tool box

• Hamner Institute

• Develop assessment systems for complex endpoints 

• Reconstructed tissues and organ systems

• Human skin, eye, lung

• Liver-on-a-chip

• Stem-cell derived

• Integrate absorption, metabolism and distribution information

• QSAR QIVIVE -

• Liver cells, tissues, extracts, reconstructed tissues

• Integrated databases and “knowledge bases”

• ACToR and MetaPath: EPA – all available chemical toxicity data on over 500,000 

environmental chemicals searchable by chemical name and structure 

• Kegg pathway database: collection of manually drawn pathway maps representing current 

knowledge on the molecular interaction and reaction networks 

• AOP Wiki: EPA-European Commission joint project to house developing AOPs

Other necessary elements
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Conclusions

The current EDSP design could be improved in terms of 

efficiency and utility of data generated

Could begin with:

• a more refined organization of tiered tests

• combined with an iterative weight-of-evidence analysis

As AOPs and tests are developed, move toward pathwaybased approach
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Thank You

Catherine Willett, PhD

Director, Regulatory Testing

 Risk Assessment and Alternatives

Coordinator, Human Toxicology Project 

 Consortium

kwillett@humanesociety.org

www.humantoxicologyproject.org
FIFRA SAP to review EDSP Tier 1 Assays and Battery Performance| May 21 - 13, 2013| Arlington, VA

Main concerns from Animal Protection Perspective

High impact on animal use due to range finding

• Better prediction paradigms

> One assay to another

> From existing information (e.g. Part 158 data)

Need to move away from MTD

• Confounding of specific results

Use of human recombinant receptors in binding studies

Use of the BG1luc as alternate to STTA

• Both agonist and antagonist

• Concordance with uterotrophic

Elimination of the Hershberger and uterotrophic assays

• Not adding value

Need to tie results back to mechanistic interpretation 

• MoA or AOP

• To explain patterns of complementarity and redundancy

• As well as “non-concordance”