# Section 2.2.2: Comparison of ToxCast ER Agonist 

# AUC Data and Uterotrophic Studies

Warren Casey, PhD, DABT Director, NICEATM Division of the National Toxicology Program 

### National Institute of Environmental Health Sciences

Slide 1 of 22

#  The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods

 Provides support to federal agencies by facilitating the development, validation, and regulatory 

# acceptance of new test methods that reduce, refine, and replace the use of animals



# NICEATM

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 2 of 22

# EDSP Tier 1 Battery ER-Specific Endpoints

Tier 1 Screening Battery Endocrine Pathway E + E - A + A - HPG 1 Axis HPT 1

Axis In vitro ER Binding ■ ■ ERα Transactivation ■ In vivo Uterotrophic ■ Pubertal Female ■ ■ ■ ■ Fish Short -term Reproduction (male & female ) ■ ■ ■ ■ ■

Note: The 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 3 of 22

# Uterotrophic Bioassay Purpose  S hort term in vivo screen to evaluate the ability of a chemical to elicit a biological response similar to that of natural estrogens 

### Principle  U terus is under the control of estrogens to stimulate growth  P roduction of endogenous estrogens is prevented

- Ovariectomized (OVX)



### - Immature ( Imm )  Uterus becomes sensitive to external estrogenic substances

Billon -Galés A et al. PNAS 2011 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 4 of 22 

### Ctl E2

# Uterotrophic Bioassay Validation Organization of Economic Cooperation and Development (OECD) Guidelines OECD TG 440 / OCSPP 890.1600 

### Rodent models  OVX Rat  Imm Rat  OVX Mouse Dosing route  Oral gavage (Oral)  Subcutaneous injection ( Inj ) 

###  Intraperitoneal injection Duration of dosing  Minimum of 3 days FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 5 of 22

# Uterotrophic Bioassay

Adapted from Owens and Koëter , 2003 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 6 of 22 Relative increase in uterine weight



### Dose of ER agonist (mg/kg/day)

Bioactive Lowest Effect Level ( LEL) Inactive Max. Dose Tested ( MDT)

### OECD GD 34, Validation and I nternational Acceptance of New or 

### Updated T

est Methods Validation is a process by which the reliability and relevance of a test method are established for a specific purpose. Relevance and reliability should be characterized against data generated with a list of reference chemicals tested in the original method accepted by regulatory agencies (uterotrophic bioassay).



# Validation

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 7 of 22

## Uterotrophic Database 98 Chemicals tested in 442 uterotrophic bioassays considered to be “Guideline Like” (GL) 

# Developing a Database of Uterotrophic Studies Literature Searches 1500 Chemicals PubMatrix (keyword searches) FDA EDKB, other resources 

## Data Review 700 Papers 42 Descriptors Two reviewers Minimum Criteria High - Level Filter

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 8 of 22Chemical Name CASRN PMID Author Year Study Type Species Strain Target Route of Administration Age at 1st Dose Administration Age at OVX Elapsed time between OVX and RX Dosing Length # of doses per day # of animals in estrogen control group # of animals in RX group Reference Estrogen Vehicle/RX control? Diet Indicated that Diet is low- PE? necropsy time after last dose Additional Assay Info Source Name SID Chemical Tested Chemical Note Dose/Response (0 no, 1 yes) # of doses used Value Unit Response Value type LEL Max Conc Tested Descriptors captured for each

chemical - bioassay combination ~2400 entries 

# Developing a Database of Uterotrophic Studies

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 9 of 22

## Guideline- Like (GL) Study Criteria

1. Animal model 

### • Imm rats • OVX rats or mice • OVX performed between six and eight weeks of age 2. Group size 

### • Control group n >=3, test group n >=5 3. Route of administration • Oral gavage, subcutaneous or intraperitoneal injection 4. Number of dose groups 

### • Minimum of two dose levels plus positive control and vehicle control 5. Dose timing and duration • M inimum of three consecutive days • Immature rats: dosing should begin between post- natal day (PND) 18 and PND 21, and be completed by PND 25 6. Necropsy timing • Animal necropsy should be carried out 18 - 36 hours after the last dose Section 2.2.1, p. 42 

# Developing a Database of Uterotrophic Studies

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 10 of 22

# Uterotrophic Study Designs

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 11 of 22 Data from Table 2.6 

### # of Bioassays OVX_Mouse_Oral 

### OVX_Mouse_Inj OVX_Rat_Oral 

### OVX_Rat_Inj Imm_Rat_Inj Imm_Rat_Oral Uterotrophic

# Uterotrophic Reproducibility

Figure 2.9 (Table 2.7)



## Chemicals with >=2 GL Uterotrophic Bioassays

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 12 of 22 Rank Order ( Total # of Bioassays) Number of Bioassays Uterotrophic

# Uterotrophic Reproducibility

Figure 2.10 and Appendix 5



## Different Study Designs: Butylparaben

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 13 of 22 Active Inactive (LEL or MDT, mg/kg/day) 5 3 1

# Studies OVX_Mouse_Oral (1000)
OVX_Mouse_Inj (50) Imm_Rat_Inj (70) Imm_Rat_Oral (400)
OVX_Rat_Inj (800) 1
Uterotrophic

## Same Study Design ( Immature Rat): BPA

Data from Figure 2.11 and Appendix 5 

# Uterotrophic Reproducibility LEL or MDT (mg/kg/day)



### Injection Oral FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 14 of 22 Inactive Active Uterotrophic

### The relevance of a new test method should be characterized 

### against data generated with a list of reference chemicals in the original test method. Active – reported as active in two or more independent GL 

### bioassays, regardless of the number of inactive results Inactive – reported as inactive in two or more independent GL bioassays, with no GL studies reporting bioactivity 42 Reference Chemicals (29 Active, 13 Inactive) 

# Reference Chemicals

FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 15 of 22 Table 2.8FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 16 of 22 

# Reference Chemicals Average Uterotrophic Lowest Effect Level (29 Active)

Rank Order (Avg. LEL) Average LEL (mg/kg/day)

Most Potent Least Potent

# AUC Scales Monotonically With AC50 For A Pure ER Agonist Active In All Assays

AUC=0.1 Equivalent to AC50=100 μM

Section 2.1 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 17 of 22

Most Active

Least Active

1 0.1

0.001 0.01 AUC

# Reference Chemicals

See Figure 2.12 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 18 of 22 Active Inactive Uterotrophic



### Rank Order (ER Agonist AUC) ER Agonist AUC Most Active

Least Active

# Reference Chemicals

See Figure 2.12 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 19 of 22 Active Inactive Uterotrophic



### Rank Order (ER Agonist AUC) ER Agonist AUC Kaempferol

D4 Inactive

Active Uterotrophic Most Active

Least Active

# Performance of ER AUC Model

True Positive 28 True Negative 12 False Positive 1 

## False Negative 1 Accuracy 0.95 Sensitivity 0.97 

## Specificity 0.92

Table 2.9 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 20 of 22



### Uterotrophic reference chemicals (29 Active, 13 Inactive) ER AUC >= 0.1 as ActiveER Agonist AUC 

### Bioactive Reference chemicals 

# LEL and ER AUC

See Figure 2.13 FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 21 of 22 Average LEL (mg/kg/day)
Estrone Equilin 17 α- Estradiol Estriol Estradiol Ethinyl Estradiol Diethylstilbestrol Most Potent/Active Least Potent/Active

# Conclusions
 ER agonist (R1) AUC model, developed using HTS data, 

## demonstrated excellent performance against in vivo (uterotrophic) reference chemicals; balanced accuracy of 95%, positive predictive value of 97%, and negative predictive value of 92 %.
 There appears to be a functional relationship between in vitro AUC values and lowest doses causing in vivo 

## bioactivity, demonstrating both qualitative and quantitative utility for the ER (agonist) pathway model.


##  These results indicate that the ToxCast HTS data may provide a rapid and effective resource for identifying substances with potential hER bioactivity.
FIFRA SAP: Integrated Bioactivity Exposure Ranking Dec 2 -5, 2014 Slide 22 of 22