# Section 2.2.3 : 

# Comparison of ToxCast data and Tier 1 ER assays for

List 1 chemicals 

# Section 2.2.4 : Demonstration of a CompTox Approach to Evaluating ER 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 17

# Presentation Roadmap

Estrogen Receptor Bioactivity Introduction Androgen Receptor

Bioactivity Exposure



## Toxicokinetics/Dosimetry

Integrated Bioactivity Exposure Ranking

Future Directions FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 2 of 17

# Major Points of Section  ER computational model is most mature and used as an example • EDSP originally established to evaluate environmental 

# chemicals with estrogen agonist activities • Consistent with this, Tier 1 battery has more extensive coverage of estrogen agonist than other bioactivities 

# or endocrine pathways • ToxCast assays are similarly weighted towards ER activity 

#  EPA evaluated each proposed application of computational tools in a fit-for- purpose manner
FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 3 of 17

# Introduction
 Follow- up of the January 2013 SAP meeting “Prioritizing the 

# Universe of Endocrine Disruptor Screening Program (EDSP) Chemicals Using Computational Toxicology Tools” SAP report commented: • The approach presented is appropriate for prioritizing the EDSP 

# chemical inventory to identify chemicals that may initiate ERmediated events. • Screening techniques can be conducted relatively quickly and efficiently and reduce unnecessary animal testing 

#  Further refinement to the approach based on SAP recommendations • 18 (vs 8) assays integrated into a network model 

# • More extensive set of reference chemicals

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 4 of 17

# Objectives: ER Pathway
 Before using the model of ER bioactivity in the 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 17

# Proposed Methodological Approach  Use computational tools to model ER bioactivity – focus on agonism • 16 ToxCast assays relevant to agonist 

# pathway • Model AUC for R1 (agonist) and R2 (antagonist) activities 

#  Prioritize chemicals in EDSP universe for ER agonist bioactivity

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 6 of 17

# Scientific Confidence Needed



# To Use [Any] New Assay  Are reference chemicals that include a range of structures and potencies accurately 

# detected? • in vitro reference chemicals 

# • In vivo reference chemicals 

#  How well do new methods compare with current methods? • ToxCast versus Tier 1 results

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 7 of 17

# Scientific Confidence: ER Reference Chemicals
 Excellent performance of ER AUC scores 

# against in vitro reference chemicals
 Excellent performance of ER AUC scores 

# against i n vivo reference chemicals Table 2.9 # True Pos 28 # True Neg 12 # False Pos 1 # False Neg 1 PPV 0.97 NPV 0.92 BA 0.95 Sensitivity 0.97 Specificity 0.92 # True Pos 28 # True Neg 12 # False Pos 0 # False Neg 4 PPV 1.0 NPV 0.75 BA 0.94 Sensitivity 0.88 Specificity 1.0

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 8 of 17

# Scientific Confidence: Current Methods  How do HTS results compare with ERmediated response in validated Tier 1 assays? Tier 1 Battery ER-specific endpoints Tier 1 Screening Battery Endocrine Pathway E + E - A + A - HPG Axis HPT

Axis in vitro ER Binding ■ ■ ERα Transcriptional Activation ■ In vivo Uterotrophic ■ Pubertal Female ■ ■ ■ ■ Fish Short-term Reproduction (male & female ) ■ ■ ■ ■ ■ 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. FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 9 of 17CODE ER AGONIST (R1) ER ANTAGONIST

(R2) T1 ER

BINDING T1 ERTA T1 UT 17 0.0438 0 Equiv Neg Neg 23 0.0413 0 Neg 19.10% Neg 28 0.0324 0 Neg 18.40% Neg 22 0.027 0 Neg 15.80% Neg 50 0.0221 0 Neg OSRI (P) Neg 8 0.0203 0 Equiv Neg Neg 9 0.0152 0 Equiv OSRI (P) Neg 40 0.00985 0 OSRI (N) Neg Neg 38 0.0095 0 Neg 24.70% OSRI (N) 11 0.00646 0 Neg Incl Neg 5 0.00357 0 OSRI (N) OSRI (N) OSRI (N) 51 0 0 OSRI (N) Neg Neg 12 0 0 Neg Incl Neg 20 0 0 Neg Neg Neg 10 0 0 OSRI (N) Neg Neg 13 0 0 OSRI (N) Incl OSRI (N) 29 0 0 Neg Neg Neg 26 0 0 OSRI (N) OSRI (N) OSRI (N) 31 0 0 Neg Neg Neg 32 0 0.000077 Neg Incl Neg 27 0 0.000133 Neg Neg Neg 33 0 0 Neg Incl Neg 34 0 0 Neg Neg Neg 35 0 0 Neg Incl Neg 36 0 0 Neg Neg Neg 16 0 0 Neg OSRI (N) Neg 37 0 0 Neg Neg Neg 39 0 0 Neg Neg Neg 41 0 0 OSRI (N) OSRI (P/N) OSRI (N) 42 0 0 Equiv Neg Neg 43 0 0 Neg OSRI (P/N) Neg 4 0 0 OSRI (N) Neg OSRI (N) 44 0 0 OSRI (N) Neg OSRI (N)



# Table 2.12 Slide 10 of 17

# Scientific Confidence: Current Methods  Excellent performance of ToxCast AUC scores 

# against draft results of Tier 1 ER binding, ERTA, and uterotrophic bioassay • 100% concordance

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 11 of 17

# HTP ER Agonist Bioactivity  Chemical Space:

~10,000 1444 98 

# 368 56 49 40 

# • EDSP Universe = ~10,000 chemicals



# - ~7000 discrete chemicals

- 52 List 1 chemicals

- 107 List 2 chemicals 

# • ToxCast Universe = ~1800 chemicals

- 1444 in EDSP Universe

- 49 List 1 chemicals

- 56 List 2 chemicals 

# • in vitro reference chemicals = 40

- 26 in EDSP universe 

# • Peer- reviewed “GL- like” uterotrophic studies = 144

- 98 in ToxCast Universe

- 6 List 1 Chemicals



# - 5 List 2 Chemicals FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 12 of 17

# ER Agonist Bioactivity

Figure 2.16 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 13 of 17

# ER Agonist Bioactivity

Figure 2.16 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 14 of 17

# Appendix 3: Supplementary Data

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 15 of 17

# Evaluating ER 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

– 18 assays detect multiple points in ER signaling pathway using multiple technologies 

# • ToxCast network model performance evaluated with a relatively large 

# set (45) of reference chemicals

– T1 ER binding: 23 (but 35% were not were not consistent with expected outcome)

– T1 ERTA: 12



# – T1 UT: 7 

# • Caveat of using uterotrophic results as “true”

– 26% of chemicals with >1 guideline- like study had contradictory uterotrophic results (RADAR plots) 

# • Caveat of using Tier 1 ER endpoints as “true”

– Tier 1 designed as a battery, and current analysis takes ER endpoints out of the larger Tier 1 context

– in vitro Tier 1 ER interactions determined from 1 ER binding and 1 ERTA FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 16 of 17

# Revisiting Objectives: ER Pathway
 Before using ER model 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 agonist, 0 antagonist; ~73% no bioactivity

– List 2: 0 agonist, 0 antagonist; ~86% no bioactivity

– Universe: 4.4% agonist, 0.6% antagonist; ~80% no 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

– Tier 1 ER binding

– Tier 1 ERTA

– Tier 1 Uterotrophic assay FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2-5, 2014 Slide 17 of 17