EDSP Tier 1 Battery 

Performance

Hypothalamic-Pituitary-Thyroid (HPT) Axis

Catherine B. Aubee, MPA

Environmental Fate and Effects Division

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
The Screening Battery

2

Screening Assays

Modes of Action

Receptor Binding Steroidogenesis Axis

E Anti-E A Anti-A E A HPG HPT

In vitro

ER Binding ■ ■2

ERα Transcriptional 

Activation ■

AR Binding ■ ■

Steroidogenesis H295R ■ ■

Aromatase 

Recombinant ■

In vivo

Uterotrophic ■

Hershberger ■ ■ ■

Pubertal Male ■ ■ ■ ■ ■

Pubertal Female ■ ■2 ■ ■ ■

Amphibian 

Metamorphosis ■

Fish Short-term 

Reproduction

(male & female)

■ ■2 ■ ■ ■ ■ ■

Abbreviations: E = estrogen, ER = estrogen receptor, A = androgen, AR = androgen receptor, T = thyroid,

 HPG = hypothalmic-pituitary-gonadal, HPT = hypothalmic-pituitary-thyroidal

1 Source: http://www.epa.gov/endo/pubs/assayvalidation/tier1battery.htm#guidelines

2 Assays are designed to detect anti-estrogens, but this test component has not been validated. 

Table. Complementary modes of action among screening assays

 in the EDSP Tier 1 battery 1
Assay Endpoint(s)

Male Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T4)

• Thyroid gland histology

• Thyroid weight

Female Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T4)

• Thyroid gland histology

• Thyroid weight

AMA • Amphibian (tadpole) development

 (NF stage and normalized hind limb length)

• Thyroid gland histology

Table. Tier 1 endpoints for detecting potential effects on the HPT axis

 or thyroid hormonal pathway, according to the 2008 FIFRA SAP.

Endpoints

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Interpreting the AMA

Figure. Suggested decision logic for the conduct and interpretation

 of the AMA (OCSPP 890.1100, OECD 231).

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Chemical Code Male Pubertal Female Pubertal AMA 

A x

B x x

C x x x

E x x

F x x x

G x xa x

H xb

I

J x

K x a x a

M xb

N

P x x

S x a Unstable

Tc

V x

Table. Preliminary results of Tier 1 assays relevant to the HPT axis

 or thyroid hormonal pathway, by case study chemical (16 cases).

a effect observed only at dose(s) with overt toxicity

b potential solvent interference c test orders satisfied by OSRI

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Nature of Responses Across Assays

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4 Amphibian Development

C

Male Pubertal x

Female Pubertal x

AMA x x

F

Male Pubertal x

Female Pubertal x x

AMA x

G

Male Pubertal x

Female Pubertal x a x a

AMA x x

Example 1: One or more responses in all three assays for a chemical (3 cases).

From Table 37 in the white paper.

a effect observed only at dose(s)with overt toxicity

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Nature of Responses Across Assays

Example 2: Responses in male and female pubertal rats but not in frog (3 cases).

From Table 37 in the white paper.

a effect observed only at dose(s)with overt toxicity

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4 Amphibian Development

E

Male Pubertal x x x

Female Pubertal x x

AMA

K

Male Pubertal xa

Female Pubertal x a x a

AMA

P

Male Pubertal x x

Female Pubertal x x

AMA

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Nature of Responses Across Assays

Example 3: Responses in frog but not in rat (3 cases).

From Table 37 in the white paper. b potential solvent interference with endpoint response

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4 Amphibian Development

H

Male Pubertal

Female Pubertal

AMA x xb

M

Male Pubertal

Female Pubertal

AMA xb

V

Male Pubertal

Female Pubertal

AMA x x

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Nature of Responses Across Assays

Example 4: Absence of response in guideline endpoints in all three assays (3 cases).

From Table 37 in the white paper.

c test order requirement satisfied by OSRI; dose-dependent effects

 were observed on T3 but no effects were observed on T4, TSH, or thyroid histopathology

d test order requirement satisfied by OSRI; no effects on thyroid hormones

 or thyroid gland morphology

e special data submission: observations were based on preserved NF stage 66 specimens

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4 Amphibian Development

I

Male Pubertal

Female Pubertal

AMA

N

Male Pubertal

Female Pubertal

AMA

T

Male Pubertalc

Female Pubertald

AMAe

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Nature of Responses Across Assays

Example 5: Other combinations (4 cases).

a effect observed only at dose(s)with overt toxicity From Table 37 in the white paper.

* unable to achieve adequate exposure in the AMA

Chemical 

Code Assay Thyroid Weight

Thyroid 

Histology TSH T4 Amphibian Development

A

Male Pubertal

Female Pubertal x

AMA

B

Male Pubertal

Female Pubertal x x

AMA x

J

Male Pubertal x x

Female Pubertal

AMA

S

Male Pubertal

Female Pubertal x a

AMA *

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Considerations for Interpretation

Inherent differences between assays

• Biological domain of applicability

• Physiology and life stage of test species

• Sex-specific differences

• Method of exposure

Study performance (case-by-case)

Any potential confounds

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Discussion

Generally less coverage in the existing battery 

for HPT/T pathway when compared to E and A 

pathways

Assays and endpoints are responsive

Some differences in response noted across 

sexes (rat) and taxa (rat and frog)

Must consider all three assays and their 

endpoints

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