Section 3.2.2: Curated Review of Hershberger 

Literature

Section 3.2.4: Demonstration of a CompTox Approach 

to Evaluating AR Agonist Bioactivity for EDSP Universe 

Chemicals

Patience Browne

Exposure Assessment Coordination and Policy Division

Office of Science Coordination and Policy

Office of Chemical Safety and Pollution Prevention

Slide 1 of 18
Presentation Roadmap

Androgen Receptor 

Bioactivity

Introduction

Estrogen Receptor 

Bioactivity

Exposure

Toxicokinetics/Dosimetry

Integrated Bioactivity 

Exposure Ranking

Future Directions

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Major Points of Section

 AR computational model is being developed

• Following a approach similar to ER model

• Expectation is that most androgen-active 

environmental chemicals will behave as antagonists

• Inherent difficulty discriminating antagonist 

response from cytotoxicity/overt toxicity

 EPA evaluated proposed application of 

computational tools in a fit-for-purpose 

manner

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Introduction

 Using the same template as ER using 

computational tools used to evaluate AR 

bioactivity

• 9 assays integrated into a network model

• 23 in vitro reference chemicals

 AR model will continue to develop as:

• In vitro reference chemical set is expanded

• In vitro reference chemical potency is included

• In vivo reference chemicals are defined

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Objectives: AR Pathway

 Before using the model of AR bioactivity in 

EDSP framework, EPA is reviewing the 

scientific strengths and limitation for each 

goal outlined in the white paper

1. Prioritize chemicals for further EDSP screening 

and testing based on estimated bioactivity,

2. Contribute to the weight of evidence evaluation 

of a chemical’s potential bioactivity,

3. Substitute for specific endpoints in the EDSP Tier 

1 battery

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 5 of 19
Proposed Methodological Approach 

 Use computational tools to model AR 

bioactivity – focus on antagonism

• 6 ToxCast assays relevant to antagonist 

pathway

• Model AUC for R1 (agonist) and R2 

(antagonist) activities

 Prioritize chemicals in EDSP universe for 

AR antagonist bioactivity

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Scientific Confidence Needed

To Use [Any] New Assay

 Are reference chemicals that include a range 

of structures and potency, accurately 

detected?

• In vitro reference chemicals

• In vivo reference chemicals 

 How well do new methods compare with 

current methods?

• ToxCast versus Tier 1 results

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Scientific Confidence: Reference Chemicals

 Excellent performance 

of ToxCast AR AUC 

scores against in vitro 

reference chemicals

 Performance of 

ToxCast AR AUC scores 

against in vivo 

reference chemicals -

TBD

# True Pos 14

# True Neg 8

# False Pos 0

# False Neg 1

PPV 1.0

NPV 0.89

BA 0.97

Sensitivity 0.93

Specificity 1.0

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Evaluation of Hershberger Assays

 Search published literature

 Compare studies with 7 minimum criteria to determine 

“guideline-like”

• Animal model: castrated male rats

• Sample size: >4 animals/group for positive results; >6 animals/group for negative

• Test chemical administration: oral gavage or subcutaneous injection.

• Dose levels: > 2 does of chemical for agonist portion, > 3 doses of test chemical 

for antagonist, and appropriate positive and controls for each activity. 

• Duration of exposure: begin PND 49 to PND 60 and extend for a minimum of 10 

days.

• Tissues examined: Absolute weights of 5 accessory sex tissue weights

 Agonist and antagonist portions of the assay evaluated 

independently

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Development of Hershberger Database 

 All identified chemical/manuscript pairs 

evaluated by 2 reviewers

• ~225 chemical/article combinations

 Currently EPA secondary review underway

• Hershberger bioassay relatively more complex 

than the uterotrophic bioassay

 Chemicals with >1 positive or negative 

“guideline-like” study result may be used to 

establish in vivo reference chemicals

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Scientific Confidence: Current Methods

 How do HTS results compare with ARmediated response in validated Tier 1 assays?

Tier 1 Battery AR-specific endpoints

NOTE: Full EDSP Tier 1 battery includes five in vitro and six in vivo assays that provide additional information on a chemical’s potential to interact 

with the androgen and thyroid, as well as alter steroidogenesis.

Tier 1 Screening 

Battery

Endocrine Pathway 

E + E - A + A -

HPG

Axis

HPT

Axis

In vitro

AR Binding ■ ■

In vivo

Hershberger ■ ■

Pubertal Male ■ ■ ■ ■

Fish Short-term 

Reproduction

(male & female)

■ ■ ■ ■ ■

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 11 of 19
Comparison of AR Model with Tier 1 

 Will be conducted when confidence in both in 

vitro and in vivo reference chemicals is well 

established

• AR antagonist response difficult to discriminate 

from non-specific toxicity

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 12 of 19
 Chemical Space:

23

410

1444

49

~10,000

56

AR Antagonist Bioactivity

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AR Antagonist Bioactivity
AR Antagonist Bioactivity

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Appendix 3: Supplementary Data

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Evaluating AR Model Performance

 New method must be fit for purpose and ideally, 

perform as well or better than existing method

• Possibility of greater sensitivity in HTS due to 

redundancy of assays

– 9 assays detect multiple points in AR signaling pathway using 

multiple technologies

• AR model performance evaluated with 23 reference 

chemicals

– T1 AR binding: 10

– T1 Hershberger: 13

• More reference chemicals will be include and potency 

will be defined

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 17 of 19
Evaluating AR Model Performance

 Current set of reference chemicals includes 

many pharmaceuticals designed to target AR

• 13 of 23 reference chemicals

• Expected to target the AR with high affinity

 AR model detects potent reference with a 

balanced accuracy of 97%

• Therefore, AR antagonist AUC scores in the range 

of reference chemicals very likely to be bioactive

• Strong confidence in using model for prioritization

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 18 of 19
Revisiting Objectives: AR Pathway

 Before using model of ER bioactivity in EDSP 

framework, EPA is reviewing the scientific 

strengths and limitation for each goal outlined in 

the white paper

1. Prioritize chemicals for further EDSP screening and 

testing based on estimated bioactivity,

– List 1: 0 agonists, 8 antagonists; ~82% no activity

– List 2: 0 agonists, 6 antagonists; ~91% no activity

– Universe: 0.1% agonists, 7.8% antagonists; ~88% no activity

2. Contribute to the weight of evidence evaluation of a 

chemical’s potential bioactivity,

3. Substitute for specific endpoints in the EDSP Tier 1 

battery

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 19 of 19