# 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 Steroido

genesis Uterotrophic Female

Pubertal FSTRA

A Equivocal (E) E x

B x x x c

C E x x b

D E x x a

E x x

F x x x

G x a x d

H E x

I x x x a

J x x x

K x x a x a

M E E x a x

N x

P E x x a

Q Inconclusive x x

S E x a x a

T OSRI OSRI OSRI OSRI OSRI x Inconclusive

V x x

4 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 a lterations out of the three identified

estrogen

- responsive endpoints for this assay Total # of

Chemicals

3 0

2 4

1 3

0 11

5 

### 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 a lterations 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

6 

### 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:

7 Chemical

Code Modality Female

Pubertal Utero

trophic FSTRA Aroma

tase Steroido

genesis ERB ERTA

S Organ Response x a x a E

M Enzyme activity E E

Female System Integrity x a x

Q Enzyme activity I

Female System Integrity x x

K Enzyme activity x

Cell Response x a x a

Male System Integrity x a x a

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 x c

C E x x b

D E x x a

F x x x

I x x x a

J x x x

P E x x a

8 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 x a x d

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 x c

C Equivocal x x b

D Equivocal x x a

E x x

F x x x

I x x x a

J x x x

K x x a x a

P Equivocal x x a

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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