EDSP

Weight-of-Evidence Process

Case Study Chemicals: Chemical A

Gregory Akerman, Ph.D.

Health Effects Division

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
Format for Case Study Presentations

 Chemical information

• Chemical class, Physical-chemical properties, etc.

 Basis for selecting case study chemical

 Tier 1 results and Other Scientifically Relevant Information 

(OSRI) considered

 Integration of data and evidence for potential interaction for:

• E Pathway

• A Pathway

• T Pathway

 Overall conclusion

3
Chemical A

Pesticidal Class: Organophosphate insecticide

Mode of pesticidal action: Cholinesterase inhibition 

leading to cholinergic toxicity

Physical-chemical Properties: 

• Relatively soluble in water

• Vapor pressure indicates compound may volatilize 

from soil and water

• Log Kow~4

• Readily metabolized and excreted

• Prone to abiotic and biotic degradation/metabolism

4
Chemical N

Basis for selecting case study chemical

• Weak/equivocal effects in some assays

• T1 responses that may be reflective of potential 

interactions with more than one endocrine modality

• Overt toxicity in fish at concentrations where potential 

endocrine effects were observed

• Challenging dose selection (overt toxicity)

5
Tier 1 In Vitro Assay Results

6

Tier 1 Assays Test Guideline

Satisfied by 

OSRI (Y/N) Study Findings

ER Binding Assay

(Rat uterine cytosol) OSCPP 890.1250 No Equivocal

ERα Transcriptional 

Activation Assay (Human 

cell line HeLa 9903)

OSCPP 890.1300;

OECD 455

Yes Negative

AR Binding Assay

(Rat prostate cytosol) OSCPP 890.1150 No Positive1

Steroidogenesis Assay 

(Human cell line H295R)

OSCPP 890.1550;

OECD 456

No Positive

Aromatase Assay (human 

recombinant microsomes) OSCPP 890.1200 No Equivocal

1 Classified as “Binder” according to Test Guideline OSCPP 870.1150
Tier 1 In Vivo Assay Results

7

Tier 1 Assays Test Guideline

Satisfied by 

OSRI (Y/N) Study Findings

Uterotrophic Assay (Rat)

OSCPP 890.1600;

OECD 440

No Negative: Estrogenic

Hershberger Assay (Rat)

OSCPP 890.1400;

OECD 441

No

Negative: Androgenic

Positive: Antiandrogenic 

Pubertal Female Assay 

(Rat) OSCPP 890.1450 No

Negative for E and

Equivocal for T pathways

Pubertal Male Assay (Rat) OSCPP 890.1500 No

Negative for A and T 

pathways

Fish Short-term 

Reproduction Assay

OSCPP 890.1350;

OECD 229

No Possible for A pathway1

Amphibian 

Metamorphosis Assay 

(Frog)

OSCPP 890.1100;

OECD 231

No Negative for T pathway

1 Based on the proposed FSTRA decision logic which considers the degree of 

complementarity among responses in the assay.
OSRI Considered

Literature study: ER transcriptional activation 

(BG1-Luc)

Guideline Mammalian Toxicity Studies

Gonadal histology and organ weights

• Rodent subchronic and chronic toxicity studies

• Dog chronic toxicity study

Two-generation reproduction study (Rat)

• PPS, VO and ovarian cycling not measured

Developmental Neurotoxicity study (Rat)

• PPS, VO were measured

8
OSRI Considered (continued)

Guideline Wildlife Studies

• Early life-cycle toxicity study with sheepshead minnow

• Chronic effects on fathead minnows and brook trout

• Partial life-cycle toxicity test with fathead minnow

• Acute toxicity testing in freshwater fish and aquaticphase amphibians

• One-generation reproduction studies in Northern 

bobwhite quail and mallard duck

• Chronic life-cycle toxicity test with the freshwater and 

estuarine/marine invertebrates

9
Chemical A: 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

Age & Weight at VO

Fertility/Fecundity

Vitellogenin

Secondary Sex 

Characteristics

Overt Toxicity 

Observed

EDSP Tier 1

ER Binding Equivocal

ERTA

No 

effect

Aromatase Equiv

Steroidogenesis

No 

effect

Uterotrophic

No 

effect

Female Pubertal Rat

No 

effect

No 

effect No effect No effect

No 

effect

No 

effect

No 

effect

FSTRA

M: 

♂ 29%

H: 

♂ 31%

♀ 42%

H: ♀

follicle 

atrophy

L,M, H: ♂

spermatog.

H: 

80% 

(fer);

94% 

(fec) 

L: 

63%

H: 

71%

♀

H: 74% 

tubercle 

score ♂

H1

1Overt toxicity at the high dose in the FSTRA included abnormal behavior and swimming; 

↓ female body wt reported at high and mid dose.
Chemical A: Lines of Evidence Indicating Potential for 

Interaction with the Estrogen Pathway

11

Study Type /

Literature 

Citation

ER Binding

ER Activation

SteroidogenesisUterine Weight

Ovarian Weight

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight

Estrous Cyclicity

Age & Weight at 

VO

Fertility/Fecundity

Vitellogenin

Overt Toxicity 

Observed

OSRI

OSRI ER 

reporter assay Positive

Subchronic 

toxicity (Rat) 

No 

effect

No 

effect

No

effect

No 

effect

Chronic toxicity 

(Rat)

No 

effect

No 

effect

No

effect

Chronic toxicity 

(Dog)

No 

effect

No 

effect

No 

effect

Two-generation 

reproduction 

(Rat)

No 

effect

No

effect

H:↓37-

59%

H

Developmental 

Neurotoxicity 

(Rat)

1.3 days 

(p<0.05)

H
Integration of Estrogen Pathway Data

ER binding and aromatase: equivocal

• occurred near the limit of solubility and did not translate into 

effects in higher levels of biological organization in mammalian T1

OSRI ER transcriptional assay: weak positive

EDSP T1 ERTA: negative

Developmental neurotoxicity study: slight delay in VO

FSTRA: E-related effects in the presence of overt 

toxicity or lack specificity in absence of overt toxicity

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Chemical A:

Estrogen Pathway Conclusions 

A chemical interacting with the E pathway would be 

anticipated to elicit more complementary and 

concordant responses in the absence of overt toxicity

WoE of T1 and OSRI: Chemical A does not 

demonstrate potential to interact with the estrogen 

pathway

13
Chemical A: Lines of Evidence Indicating Potential for 

Interaction with the Androgen Pathway

14

Study Type /

Literature 

Citation

AR Binding

Steroidogenesis

Testosterone

Testes Weight 

or Fish GSI

Gonad Staging 

and 

Histopathology

Epididymides 

Weight

Epididymides 

Histopathology 

Pituitary Weight

Accessory Sex 

Organ 

Weights/2° Sex 

Characteristics

Age and Weight 

at PPS

Fertility/

Fecundity

Vitellogenin

Overt Toxicity 

Observed

EDSP Tier 1

AR Binding Binder

Steroidogenesis Test.

Hershberger

L: SV 29% 

 VP 26%

M: SV 37% 

 VP 43%

LABC 21%

H: SV 86%

 VP 77% 

 LABC 45% 

 CG 69% 

 GP 20%

Male Pubertal 

Rat

No

effect

No

effect

No

effect

No

effect

No

effect

No

effect

No

effect

No

effect

FSTRA

M: 

♂ 29%

H: 

♂ 31%

♀ 42%

H: ♀

follicle 

atrophy

L,M, H: ♂

spermat.

H: 74% 

tubercle score 

♂

H: 

80% 

(fer)

94% 

(fec)

L: 

63%

H: 

71%

♀

H1

1Overt toxicity at the high dose included abnormal behavior and swimming; ↓ female body wt at high and mid dose.
Chemical A: Lines of Evidence Indicating Potential for 

Interaction with the Androgen Pathway

15

Study Type /

Literature Citation

AR Binding

Steroidogenesis

Testosterone

Testes Weight

Gonad Staging 

and 

Histopathology

Epididymides 

Weight

Epididymides 

Histopathology 

Pituitary Weight

Accessory Sex 

Organ 

Weights/2° Sex 

Characteristics

Age and Weight 

at PPS

Vitellogenin

Overt Toxicity 

Observed

OSRI

Subchronic toxicity 

(Rat) 

No

effect

No

effect

No

effect

No

effect

No

effect

No

effect

Chronic toxicity (Rat)

No

effect

No

effect

No

effect

No

effect

Chronic toxicity (Dog)

No

effect

No

effect

No

effect

No

effect

No

effect

Two-generation 

reproduction (Rat)

No

effect

No

effect

No

effect

No

effect

Developmental 

Neurotoxicity (Rat)

H:

1.9 

day

H
Integration of Androgen Pathway Data

AR binding: Positive (weak)

Steroidogenesis: slight reduction in testosterone 

production

• occurred at concentrations near the limit of solubility

Hershberger: Positive anti-androgenic response in sex 

accessory organ weights in castrated rats at all doses

Male Pubertal: No androgen-related effects

16
Integration of Androgen Pathway Data

Guideline mammalian studies:

• Subchronic (rat) or chronic toxicity (rat, dog):

no effects on organ weight or histopathology of the 

testes, epididymis or sex accessory organs

• 2-generation rat reproduction:

no androgen pathway-related effects observed

• Rat Developmental neurotoxicity:

a slight delay in preputial separation was observed

17
Integration of Androgen Pathway Data

FSTRA: an increasing dose trend observed in the 

number of endpoints that could be consistent with an 

anti-A response; several of these responses 

occurred at test concentrations exhibiting overt 

toxicity

• there were only two endpoints (gonadal histopathology and 

GSI) affected in the absence of overt toxicity

18
Chemical A:

Androgen Pathway Conclusions 

Chemical A appears to have the potential to initiate 

an anti-androgen AOP via androgen receptor binding 

or altered steroidogenesis

Anti-androgenic responses occurred at the tissue 

level in the Hershberger assay

In mammals with an intact HPG axis, no responses 

observed were supportive of an anti-androgen AOP

• Tier 1 male pubertal data and the OSRI (subchronic, 

chronic, and 2-generation reproductive studies)

19
Chemical A:

Androgen Pathway Conclusions 

Assuming that the MIEs observed (i.e., AR binding 

and altered steroidogenesis) are conserved across 

taxa, the Tier 1 FSTRA also indicates that an antiandrogenic effect may be expressed, but at 

concentrations that are overtly toxic

It could be that fish are more responsive than the rat 

to these MIEs/AOPs (particularly altered 

steroidogenesis)

20
Chemical A: Lines of Evidence Indicating Potential for 

Interaction with the Thyroid Pathway

21

Study Type /

Literature Citation

Hormones 

(T4 and TSH)

Pituitary Weight

Thyroid Weight

Thyroid Gross and 

Histopathology

Frog Development 

Stage

Hind Limb Length

Snout to Vent 

Length

Overt Toxicity 

Observed

EDSP Tier 1

Male Pubertal (Rat) L: ↓T4 15% 

No 

Effect

No 

Effect

No 

Effect

Female Pubertal (Rat) L: ↓T4 23%

H: ↓T4 17%

No 

Effect

No 

Effect

No 

Effect

AMA (Frog) Equiv

No 

Effect

No 

Effect

No 

Effect

OSRI

Two-generation reproduction 

(Rat)

No 

Effect

No 

Effect

No 

Effect

Subchronic toxicity (Rat) No 

Effect

No 

Effect

No 

Effect

Chronic toxicity (Rat) No 

Effect

No 

Effect

Chronic toxicity (Dog) No 

Effect

No 

Effect
Integration of Thyroid Pathway Data

Female Pubertal: Slight T4 depression was considered 

equivocal

• Also no complementary changes in TSH, thyroid weight or thyroid 

histopathology

AMA: No other noteworthy effects were reported than 

thyroid gland hypertrophy at the highest treatment level

Guideline Mammalian Studies: No changes in thyroid 

weights, pituitary weights or thyroid histopathology in the 

subchronic and chronic toxicity studies

22
Chemical A:

Thyroid Pathway Conclusions 

The lack of notable thyroid pathway-related effects 

in the AMA and mammalian toxicity studies support 

the absence of thyroid interaction

WoE evaluation: EDSP Tier 1 data and OSRI 

showed insufficient complementarity within assays 

and redundancy across assays to demonstrate a 

potential to interact with the thyroid pathway 

23
Chemical A: Overall Conclusions

24

Estrogen Pathway: Lack of 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: Lack of robust complementarity and 

redundancy to support an interaction with the HPT axis
Case Study A — Androgen Antagonism 

25

Intact HPG

Tier 1 Assays

Rat Pubertal & OSRI — anti-androgenic responses 

were not observed at a higher level of biological 

organization presumably due to compensatory 

mechanisms