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 Kow 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 BindingER ActivationSteroidogenesisUterine WeightOvarian Weight or Fish GSIOvarian/Gonad Staging and Histopathology Pituitary WeightEstrous Cyclicity

b

Age & Weight at VOFertility/FecundityVitellogeninEstradiolOvert 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 BindingSteroidogenesisTestosteroneTestes Weight or Male Fish GSIGonad Staging and HistopathologyEpididymides WeightEpididymides Histopathology Pituitary WeightAccessory Sex Organ Weights/2°Sex CharacteristicsAge and Weight at PPSFecundity/fertilityVitellogeninOvert 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 No effect

2/2 ↑1/1 ↑2/2 ↓1/2 ↑1/1 Ymedaka

*F0 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 mammals

• 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

T4TSHPituitary WeightThyroid WeightThyroid Gross and HistopathologyFrog Development StageHind Limb LengthSnout to Vent LengthOvert 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