# EDSP Tier 1 Battery

Performance

Evaluation of the Androgen Pathway

Amy Blankinship, M.S.

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency

# Overview

 

# Assays and Endpoints for Androgen Pathway

 

# Complementarity within an Assay

 

# Redundancy across Assays

 

# Observation of Results across Assays

 

# Conclusions

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# Assays that Inform the Androgen Pathway

Assay Endpoint(s)

AR Binding 1. Receptor binding

Aromatase 1. Enzyme activity (steroidogenesis)

Steroidogenesis 1. Enzyme activity (steroidogenesis)

Hershberger 1. Changes (increase or decrease) in weight of androgen

- dependent

tissues

Male Pubertal

HPG 1. Sexual developmental characteristic (age at preputial separation

(PPS))

2. Weight of androgen

- dependant reproductive organs

3. Histopathology of androgen

- dependant reproductive tissues

4. Total serum concentrations of testosterone

Fish Reproduction

HPG 1. Fecundity

2. Fertilization success

3. Secondary sex characteristics

4. Gonadal size ( gonado

- somatic index [GSI])

5. Histopathology of androgen

- dependant reproductive tissues

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

7. Plasma concentrations of testosterone ( optional)

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# Overview of Results Across the Six Assays

Chemical

Code ARB Aromatase Steroidogenesis Hershberger Male

Pubertal FSTRA

A x Equivocal (E) x x x

B x a x x c

C Untestable x x x b

D E x a

E x a x

F NA x x

G x x a x d

H x E x x

I E x x a

J E x x x x

K x x x a x a

M E E x

N x x

P E E x x a

S E x a x a x a

T OSRI OSRI OSRI x Inconclusive

V x

4 From Table 39 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 four

identified androgen

- responsive endpoints for this

assay Total # of

Chemicals

4 0

3 2

2 4

1 4

0 7

5 

### Male Pubertal Assay

Endpoint Total # of

Chemicals

Changes in 2 or more androgen

- dependent tissues 7



### Hershberger Assay Example: Chemical E resulted in alterations in 2 complementary responses:

1. testosterone; and

2. androgen

- dependent organ wts

.

# Complementary Responses within an **In Vivo** Assay

Number of reported a lterations out of the seven

identified androgen

- responsive endpoints for this

assay Total # of

Chemicals

7 0

6 1

5 4

4 1

3 8

2 4

1 1

0 1

Inconclusive 1

6 

### FSTRA

Example: Chemical J resulted in alterations in 5 complementary responses:

1. fecundity;

2. female and male GSI;

3. female VTG;

4. female and male gonads; and

5. female E2 and testosterone

# Redundancy in Modalities Across Assays

 

# In vitro assays

— limited redundancy and complementarity between

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

assay (ARB different from aromatase and steroidogenesis)



##  

# In vivo and in vitro assay examples:

7 Chemical

Code Modality Male

Pubertal Hershberger FSTRA Aromatase Steroidogenesis ARB

E Organ Response x x a

G Organ Response x a x x d

J Enzyme activity x x x x

Organ Response x x

A Enzyme activity equivocal x

Organ Response x x a

S Enzyme activity x a equivocal

Organ Response x a x a

Male System Integrity x a x a

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

d = study conduct questionable, results possibly not reliable

# Observation of Results Across the Six Assays

8 Chemical

Code ARB Aromatase Steroidogenesis Hershberger Male

Pubertal FSTRA

A x Equivocal

(E) x x x

J E x x x x

K x x x a x a 

### Example 1: Responses in multiple **in vitro** assays (3 cases)

Example 2: R esponses in multiple **in vivo** assays but NO response in **in vitro** (4 cases)

Chemical

Code ARB Aromatase Steroidogenesis Hershberger Male

Pubertal FSTRA

B x a x x c

E x a x

G x x a x d

N x x

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

c = potential solvent interferences

d = study conduct questionable, results possibly not reliable

# Observation of Results Across the Six Assays

Chemical

Code ARB Aromatase Steroidogenesis Hershberger Male

Pubertal FSTRA

A x Equivocal

(E) x x x

H x E x x

J E x x x x

K x x x a x a

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### Example 3: Responses in multiple **in vitro** (including equivocal) and **in vivo**

assays (4 cases)

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

# 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

 

# Responses in Hershberger accompanied by a

response in the male pubertal and/or FSTRA

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