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 xa x xc

C Untestable x x xb

D E xa

E xa x

F NA x x

G x xa xd

H x E x x

I E x xa

J E x x x x

K x x xa xa

M E E x

N x x

P E E x xa

S E xa xa xa

T OSRI OSRI OSRI x Inconclusive

V x

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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 alterations out of the four 

identified androgen-responsive endpoints for this 

assay

Total # of 

Chemicals

4 0

3 2

2 4

1 4

0 7

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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 alterations 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:

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Chemical 

Code Modality Male 

Pubertal Hershberger FSTRA Aromatase Steroidogenesis ARB

E Organ Response x xa

G Organ Response xa x xd

J Enzyme activity x x x x

Organ Response x x

A Enzyme activity equivocal x

Organ Response x xa

S

Enzyme activity xa equivocal

Organ Response xa xa

Male System Integrity xa xa

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

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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 xa xa

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

Example 2: Responses in multiple in vivo assays but NO response in in vitro (4 cases)

Chemical 

Code ARB Aromatase Steroidogenesis Hershberger Male 

Pubertal FSTRA

B xa x xc

E xa x

G x xa xd

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 xa xa

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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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