# EDSP

Weight - of - Evidence Process Case Study Chemicals: Chemical X

Patience Browne, Ph.D. Office of Science Coordination and Policy Office of Pesticide Programs Office of Chemical Safety and Pollution Prevention U.S. Environmental Protection Agency

# Case Study Chemicals

 

# Chemical A

 

# Chemical S

 

# Chemical J

 

# Chemical N

 

# Chemical X

2

# Chemical X

 

# Chemical Class: Widely used industrial alkylphenol 

# surfactant

 

# Multiple isomers with varying degrees of side chain branching

 

# Physical
- chemical Properties:

• Relatively low solubility in water

• Does not volatilize appreciably

• Resistant to abiotic and biotic degradation/metabolism

• Log K ow 

# 3.8 to 4.8

• Mildly bioaccumulative

3

# Chemical X

 

# Basis for selecting case study chemical

 T1 EDSP screening satisfied by OSRI

 Clearly interacts with estrogen pathway

 May interact with A and T pathway

 Many study methods were not identical to T1 assays

• 

## May not have been designed to detect endocrine interactions (especially for T)

• 

## Differences in samples sizes, dose selection methods, 

## number of doses tested, background/strains, endpoints measured, monitoring

• 

## No performance criteria

 Often dosing below levels inducing overt toxicity (not necessarily a problem)

4

# OSRI Considered

 

## ER Binding

• Rat uterine cytosol (Blair et al . 1999; Laws et al

. 2000)

• Rabbit uterine cytosol ( Danzo 1997)

• Human ER (Satoh et al . 2001)



###  

## ERTA

• HeLa stably transfected with ER /luciferase ( Balaguer et al . 1999)

• MCF
- 7 ER /luciferase ( Balaguer et al . 1999; Bonefeld
- Jorgensen et al . 2007)

• T47D transfected ER/luciferase (Legler et al . 1999)

• Rainbow trout hepatocytes/vitellogenin (White et al . 1994)



###  

## AR Binding

• Human AR (Satoh et al . 2001)

• Yeast transformed with mammalian AR/luciferase (Lee et al . 2003)

• CHO cells transfected with human AR/luciferase ( Bonefeld
- Jorgensen et al . 2007)

5

# OSRI Considered (continued)

 

# Aromatase

• OCSPP 890.1200; EDSP T1 validation study



## • Human choriocarcinoma cell line ( Bonefeld
- Jorgensen et al . 2007)



##  

# Steroidogenesis

• Rat Leydig cell isolates/testosterone production (Gong and Han 2006)



## • Atlantic salmon ovarian tissue/steroidogenic enzyme transcript ( Kortner et al . 2009)



##  

# Uterotrophic

• OCSPP 890.1600; EDSP T1 interlab validation study (4 labs)

• Prepubertal rat (Laws et al . 2000)

• Ovariectomized adult rat (Laws et al . 2000)



##  

# Hershberger

• OCSPP 890.1400; EDSP T1 interlab validation study (10 labs)

• Male castrates ( Freyberger et al . 2007)

6

# OSRI Considered (F Rat)

 Prepubertal rats dosed by oral gavage PND 21 to 41 (Kim et al . 2002)

• Age at VO

• Estrous cyclicity

• Organ weights (uterine, ovary, pituitary, thyroid, liver)

• T4 + TSH

 Neonatal rats dosed PND 1 to 5; examined through puberty (Nagao et al . 2000)

• Age at VO

• Estrous cyclicity

• Copulation and fertilization rates

• Histopathology (reproductive)

 Prepubertal rats dosed PND 21 to 35 (Laws et al . 2000)

• Age at VO

• Uterine weight

• Estrous cyclicity

 Adult OVX rats dosed 11 days (Laws et al . 2000)

• Uterine weight

• Estrous cyclicity (vaginal epithelium)

 2
- gen rat (Nagao et al . 2001)

• Organ weight (uterine, ovary, thyroid, pituitary)

• Histopathology

• Age at VO (F1)

• Biochemistry (thyroid + reproductive hormones; F1)

 3
- gen rat (Chapin et al . 1999)

• Age at VO

• Organ weight (uterine, ovarian)

• Estrous cyclicity
7

# OSRI Considered (M Rat)

 Prepubertal rats dosed by oral gavage PND23 to 52/53 (Benjamin et al . 2003)

• Age at PPS

• Histopathology

• Organ weights (reproductive, thyroid, liver, kidney, adrenal)

• Spermatogenesis

 Neonatal rats dosed PND 1 to 5; examined through puberty (Nagao et al . 2000)

• Testosterone

• Age at PPS

• Testis weight

• Histopathology (reproductive)

• Behavior

• Fertility/sperm characteristics

 Adult rats dosed by oral gavage PND 35
- 37 for 50 days (Gong and Han 2006)

• Testosterone + LH

 OECD Guideline 415; ( DeJager et al . 1999a adult exposure, 1999b developmental exposure)

• Organ weights (testis, epididymis)

• Histopathology

• Fertility/ sperm characteristics

 2
- gen rat (Nagao et al . 2001)

• Age at PPS

• Organ weights (reproductive, thyroid, pituitary, liver, kidney , adrenal)

• Behavior

• Histopathology

• Biochemistry (thyroid + reproductive hormones)

8

# OSRI Considered (Fish)

 

# Fish studies

 Short
- term reproductive performance fathead minnow (Harries et al . 2000)

• 

## GSI

• 

## Secondary sex characteristics

• 

## Plasma VTG

• 

## Fecundity



#  Two generation medaka (Yokota et al . 2001)

• 

## GSI

• 

## Secondary sex characteristics

• 

## Histopathology

• 

## Fecundity and fertility

9

# Chemical X: Lines of Evidence Indicating Potential for



# Interaction with the Estrogen Pathway

10 Study Type / Literature Citation ER Binding

ER Activation

Steroidogenesis

Uterine Weight

Ovarian Weight or Fish GSI

Ovarian/Gonad Staging and Histopathology

Pituitary Weight

Estrous Cyclicity b

Age & Weight at VO

Fertility/Fecundity

Vitellogenin

Estradiol

Overt Toxicity Observed C

ER Binding P 4/4 ERTA P 5/5 Aromatase ↓ 2/2 Steroidogenesis Altered
Uterotrophic ↑ 7/7 N

Female Rat ↑ 4/5 ↓1/5 ↓ 3/3* Noted not

quantified 1/2 ↓

1/2 Altered 4/5 ↓ 4/5* N
Fish ↑FHM ↓Med* ♀ NE 1/1

♂ 1/1 ↓ 1/2 ↑

1/1 Y

Medaka *only F0 generation ovarian wt/GSI and F1 VO of multi
- gen study

# Integration of Estrogen Pathway Data

 

### Positive in vitro estrogenic activity

• ER binding

• Increased gene activation



###  

### Inhibition of aromatase activity

 

### Alteration of in vitro steroidogenesis

• Alteration of testosterone synthesis

• Altered enzyme transcripts



###  

### Positive uterotrophic assay

 

### Estrogenic effects in mammals

• Decreased age at VO

• Increased uterine weight

• Altered estrous cyclicity



###  

### Estrogenic effects in fish

• Increased VTG in fathead minnows

• Reduced fecundity

• Increased incidence of phenotypic/gonadal sex mismatch

11

# Chemical X: Estrogen Pathway Conclusions

 

# Clear estrogenic activity was observed at several levels of biological organization 

# ( in vitro , tissue, organ, organism)

 

# Based on WoE evaluation of OSRI, Chemical X demonstrated interactions with the estrogen 

# pathway

12

# Chemical X: Lines of Evidence Indicating Potential for

Interaction with the Androgen Pathway

13 Study Type / Literature Citation

AR Binding Steroidogenesis

Testosterone

Testes Weight or Male Fish GSI

Gonad Staging and Histopathology

Epididymides Weight

Epididymides Histopathology

Pituitary Weight

Accessory Sex Organ Weights/2 ° Sex Characteristics

Age and Weight at PPS Fecundity/fertility

Vitellogenin Overt Toxicity Observed b

AR Binding P

3/3
Steroidogenesis P 2/2
Hershberger No effect 11/11 N
Male Rat ↑1/3 ↓1/3 ↓2/5 ↑3/5 ↓2/5 ↑1/1* Altered ↓1/3 ↑ 1/2 Y Fish N o effect 2/2 ↑1/1 ↑2/2 ↓1/2 ↑1/1 Y medaka *F 0 only

# Integration of Androgen Pathway Data

 

## Positive in vitro androgen interaction

• Positive AR binding



### • Decreased AR
- dependent gene activation



###  

## Alteration of in vitro steroidogenesis

• Alteration of testosterone synthesis

• Altered enzyme transcripts



###  

## Negative Hershberger results

 

## Interaction with androgen pathway in mammal s

• Altered copulatory behaviors

• Altered germ cell production



### • Reduced testis weight

• Increased testosterone and LH



###  

## Interaction with androgen pathway in fish

• Decreased tubercles



### • Reduced thickness of dorsal fat pad

14

# Chemical X: Androgen Pathway Conclusions

 

# Chemical X may have the potential to initiate an anti
- androgen AOP via androgen receptor binding, decreased ARTA, and/or altered steroidogenesis

 

# Anti
- androgenic responses were absent in the Hershberger assay

 

# In mammals with an intact HPG axis, responses 

# observed varied with age of animals at treatment

 

# Distinguishing between estrogenic and antiandrogenic responses can be difficult

15

# Chemical X: Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway

16 Study Type /

Literature Citation

T 4

TSH

Pituitary Weight Thyroid Weight

Thyroid Gross and Histopathology

Frog Development Stage

Hind Limb Length

Snout to Vent Length

Overt Toxicity Observed b

Male Rat ↓ adult L 1/1 ↓ juv L 1/1 ↑ adult 1/1*

↓ juv L 1/1 ↑

1/1 ↑ 1/2* Altered

1/2 N Female Rat ↓ adult 1/2 ↓ juv L 1/1 NE adult 2/2

↓ juv 1/1 ↓

1/2 NE 2/2 NE

1/1 N *F0 only

# Integration of Thyroid Pathway Data

 

# Some indication of potential thyroid interactions in mammals

• Increased thyroid weight

• Altered pituitary weight

• Altered plasma T4 and TSH



#  

# No amphibian assay

17

# Chemical X: Thyroid Pathway Conclusions

 

# Appears to be some thyroid effects in two generation rat study, though not consistent across sex or generation

 

# Data from assays validated to detect thyroid pathway
- related effects are lacking

 

# Thyroid effects are not clearly delineated, but the potential of Chemical X to interact with the thyroid hormone pathway cannot be excluded

18

# Chemical X: Overall Conclusions

19 

##  

# Estrogen Pathway: Complementarity and redundancy in responses to support an interaction with the E 

# pathway

 

# Androgen Pathway: Potential to initiate an antiandrogen AOP via androgen receptor binding or altered steroidogenesis; however, the interaction is not robustly expressed in organisms with intact HPG axis

 

# Thyroid Pathway: Some indication of complementarity but absence of assay results that provide redundancy for delineation of interaction with the HPT axis

# Case Study X
— Estrogen Agonism

20 

# Tier 1 Assays

# Case Study X
— Androgen Agonism /Antagonism Tier 1 Assays

No +/
- A effect in Hershberger

21

# Case Study X
— Perturbation of Hypothalamic Pituitary Thyroid Axis

22