# 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

3

# Interpreting the AMA

Figure . Suggested decision logic for the conduct and interpretation of the AMA (OCSPP 890.1100, OECD 231).

4Chemical Code Male Pubertal Female Pubertal AMA

A x

B x x

C x x x

E x x

F x x x

G x x a x

H x b

I

J x

K x a x a

M x b

N

P x x

S x a Unstable

T

c

V x 

### T

able. 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 x a

Female Pubertal x a x a

AMA

P Male Pubertal x x

Female Pubertal x x

AMA

7

# 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 x b

M Male Pubertal

Female Pubertal

AMA x b

V Male Pubertal

Female Pubertal

AMA x x

8

# 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 Pubertal c

Female Pubertal d

AMA e

9

# Nature of Responses Across Assays

Example 5 : Other combinations (4 cases).

From Table 37 in the white paper. a 

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

* 

### 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 *

10

# 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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