EDSP Tier 1 Battery 

Performance

Evaluation of the Estrogen Pathway

Greg Akerman, Ph.D. 

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
Overview

Assays and Endpoints for Estrogen Pathway

Complementarity within an Assay

Redundancy across Assays

Observation of Results across Assays

Conclusions

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Assays that Inform the Estrogen Pathway

Assay Endpoint(s)

ER Binding 1. Receptor binding

ERTA 1. Cell signaling

Aromatase 1. Enzyme activity (steroidogenesis)

Steroidogenesis 1. Enzyme activity (steroidogenesis)

Uterotrophic 1. Increased uterine weight

Female Pubertal

HPG

1. Sexual developmental characteristic (vaginal opening (VO) and 

onset of first estrus)

2. Weight of estrogen-dependent reproductive organs

3. Histopathology of estrogen-dependent reproductive tissues

Fish Reproduction

HPG

1. Fecundity

2. Fertilization success

3. Secondary sex characteristics

4. Gonadal size (gonado-somatic index [GSI])

5. Gonadal Histopathology

6. Plasma concentrations of vitellogenin [VTG] in females and males

7. Plasma concentrations of sex steroids (optional)

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Overview of Results Across the Seven Assays

Chemical 

Code ERB ERTA Aromatase Steroidogenesis Uterotrophic Female Pubertal FSTRA

A Equivocal (E) E x

B x x xc

C E x xb

D E x xa

E x x

F x x x

G xa xd

H E x

I x x xa

J x x x

K x xa xa

M E E xa x

N x

P E x xa

Q Inconclusive x x

S E xa xa

T OSRI OSRI OSRI OSRI OSRI x Inconclusive

V x x

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From Table 41 in white paper a = change/alteration only observed at test levels with overt toxicity (e.g., substantial decrease

 in body weight in rats; for fish - reduced survival, abnormal swimming, reduced feeding) 

b = ↓in fish body weight reported at concentrations where a change/alteration was also reported 

c = Potential solvent interferences 

d = Study conduct questionable, results possibly not reliable 
Complementary Responses within an In Vivo Assay

Number of reported alterations out of the three identified 

estrogen-responsive endpoints for this assay

Total # of 

Chemicals

3 0

2 4

1 3

0 11

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Female Pubertal Assay

Endpoint Total # of Chemicals

Changes in uterine weight 0

Uterotrophic Assay

Example: Chemical E resulted in alterations in 2 complementary responses:

1. vaginal opening; and 

2. estrogen-dependent organ weights. 
Complementary Responses within an In Vivo Assay

Number of reported alterations out of the seven identified 

estrogen-responsive endpoints for this assay

Total # of 

Chemicals

7 0

6 2

5 3

4 3

3 7

2 3

1 1

0 1

Inconclusive 1

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FSTRA

Example: Chemical V resulted in alterations in 3 complementary responses:

1. fertility;

2. male tubercles; and

3. fecundity; however, there was a reported decrease in female body weight 
Redundancy in Modalities Across Assays

In vitro assays — limited redundancy and complementarity between in 

vitro assays due to the specificity and different mechanisms of action of each 

assay (ERB and ERTA different from aromatase and steroidogenesis)

In vivo and in vitro assay examples:

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Chemical 

Code Modality Female 

Pubertal

Uterotrophic FSTRA Aromatase

Steroidogenesis ERB ERTA

S Organ Response xa xa E

M Enzyme activity E E

Female System Integrity xa x

Q Enzyme activity I

Female System Integrity x x

K

Enzyme activity x

Cell Response xa xa

Male System Integrity xa xa

a = change/alteration only observed in presence of overt toxicity

I= inconclusive results 
Observation of Results Across the Seven Assays

Chemical 

Code ERB ERTA Aromatase Steroido. Uterotrophic Female Pubertal FSTRA

B x x xc

C E x xb

D E x xa

F x x x

I x x xa

J x x x

P E x xa

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Example 1: Responses in multiple in vitro assays (7 cases)

Example 2: Responses in multiple in vivo assays but NO response in in vitro (3 cases)

a = change/alteration only observed at test levels with overt toxicity (e.g., substantial decrease in 

body weight in rats; for fish - reduced survival, abnormal swimming, reduced feeding) 

b = ↓in fish body weight reported at concentrations where a change/alteration was also reported 

c = Potential solvent interferences 

d = Study conduct questionable, results possibly not reliable

Chemical 

Code ERB ERTA Aromatase Steroido. Uterotrophic Female Pubertal FSTRA

G xa xd

N x

Q Inconclusive x x
Observation of Results Across the Seven Assays

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Example 3: Responses in the in vitro and in vivo assays (10 cases)

Chemical 

Code ERB ERTA Aroma. Steroido. Utero. Female Pubertal FSTRA

B x x xc

C Equivocal x xb

D Equivocal x xa

E x x

F x x x

I x x xa

J x x x

K x xa xa

P Equivocal x xa

V x x

a = change/alteration only observed in presence of overt toxicity

b = ↓ in fish body weight reported at concentrations were a change/alteration was also reported

c = potential solvent interference
Conclusions

Complementary responses observed within an 

in vivo assay; and

Redundancy observed across Tier 1 assays

Observation of overt toxicity needs to be 

considered when evaluating for endocrine 

interactions

For all case chemicals, there was no response 

in the Uterotrophic and ERB assays

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