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 -

HPG1

Axis

HPT1

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

 Short term in vivo screen to evaluate the ability of a chemical to elicit a 

biological response similar to that of natural estrogens

Principle

 Uterus is under the control of estrogens to stimulate growth

 Production 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 International Acceptance of New or 

Updated Test 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 22
Chemical 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

• Minimum 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 2.8
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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 Active
ER 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

17α-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