EDSP

Weight-of-Evidence Process

Case Study Chemicals: Chemical J

Amy Blankinship, M.S.

Environmental Fate and 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
Chemical J

 Pesticidal Class: Triazole fungicide

 Mode of pesticidal action: Inhibits sterol biosynthesis which 

disrupts membrane function

 Physical-chemical Properties: 

• Soluble in water

• Not expected to appreciably volatilize from soil and water

• Stable under abiotic conditions (hydrolysis and photolysis)

• Slowly undergoes aerobic degradation/metabolism

• Log Kow ~ 3

• Low propensity to sorb to organic matter

3
Chemical J

Basis for selecting case study chemical

• T1 responses that may be reflective of potential 

interactions with more than one endocrine modality

• Different effects across taxa

4
Tier 1 In Vitro Assay Results

5

Tier 1 Assays Test 

Guideline

Satisfied by 

OSRI (Y/N) Study Findings

ER Binding Assay 

(Rat uterine cytosol)

OSCPP 

890.1250

No Negative1

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903)

OSCPP 

890.1300;

OECD 455

No Negative

AR Binding Assay 

(Rat prostate cytosol)

OSCPP 

890.1150

No Positive2

Steroidogenesis Assay 

(Human cell line H295R)

OSCPP 

890.1550;

OECD 456

No Positive

Aromatase Assay 

(human recombinant microsomes)

OSCPP 

890.1200

No Positive3

1 Classified as “not interactive” according to Test Guideline (TG) OSCPP 890.1250

2 Classified as “binder” according to TG OSCPP 870.1150

3 Classified as “inhibitor” according to TG OSCPP 870.1150
Tier 1 In Vivo Assay Results

6

Tier 1 Assays Test Guideline

Study Source 

(guideline / 

open lit) Study Findings

Uterotrophic Assay (Rat)

OSCPP 890.1600;

OECD 440

Guideline Negative: Estrogenic

Hershberger Assay (Rat)

OSCPP 890.1400;

OECD 441

Guideline

Negative: Androgenic

Negative: Anti-androgenic 

Pubertal Female Assay (Rat) OSCPP 890.1450 Guideline Negative for Estrogen and 

Thyroid pathways

Pubertal Male Assay (Rat) OSCPP 890.1500 Guideline

Positive for Androgen pathway;

Negative for Thyroid pathway

Fish Short-term 

Reproduction Assay 

OSCPP 890.1350;

OECD 229

Guideline Positive for Estrogen and 

Androgen pathway1

Amphibian Metamorphosis 

Assay (Frog)

OSCPP 890.1100;

OECD 231

Guideline Negative for Thyroid pathway

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

of complementarity among responses in the assay.
OSRI Considered

Guideline Mammalian Studies

Rodent subchronic and chronic toxicity studies 

and a dog chronic study

• Gonadal histology and organ weights

Two-generation reproduction study (Rat)

• PPS, VO and ovarian cycling not measured

Rodent developmental neurotoxicity studies

• PPS, VO were measured

7
OSRI Considered (continued)

Guideline Wildlife Studies

• Acute toxicity testing in freshwater and 

estuarine/marine fish

• Early life-cycle toxicity study with fathead minnow

• Chronic life-cycle toxicity test with estuarine/marine 

invertebrates (mysid shrimp)

• One-generation reproduction studies in Northern 

bobwhite quail and mallard duck

8
Chemical J: Lines of Evidence Indicating Potential for 

Interaction with the Estrogen Pathway

9

Study Type 

ER Binding

ER Activation

SteroidogenesisUterine Weight

Ovarian Weight or 

Fish GSI

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight

Estrous Cyclicity

Age & Weight at VO

Fertility/Fecundity

Vitellogenin

Estradiol

Overt Toxicity 

Observed

EDSP Tier 1

ER Binding No Effect

(N)

ERTA N

Aromatase Positive

Steroidogenesis ↓Estrogen

Uterotrophic N

Female Pubertal 

Rat

N No Effect No Effect N N N

FSTRA

M: ↑41% 

H: ↑46% ♀;

H:↑80% ♂

H: ↑ oocyte atresia; 

H: ↑ % mature

sperm

H: 98% 

(fecund)

H: 88%♀ H: 85%♀
Chemical J: 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

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight

Estrous Cyclicity

Age & Weight at 

VO

Fertility/Fecundity

Vitellogenin

Overt Toxicity 

Observed

OSRI

Subchronic 

toxicity (Rat) 

No 

effect

No

effect

Chronic toxicity 

(Dog)

No 

effect

No 

effect

No

effect

Chronic toxicity 

(Rat)

No 

effect

No 

effect

No 

effect

Carcinogenicity 

(Mouse)

No 

effect

No 

effect

Two-generation 

reproduction 

(Rat)

No 

effect

No

effect

No 

effect

Developmental 

(Rabbit/Rat) No effect

Avian/Aquatic 

Invertebrate 

Reproduction

No 

effect
Integration of Estrogen Pathway Data

 (-) in vitro ER and ERTA and in vivo uterotrophic support lack 

of direct interaction with ER

 (+) in vitro steroidogenesis (↓E) and aromatase

 Complementary responses in FSTRA:

• Females: ↓E, ↓VTG, ↑GSI, ↑ oocyte atresia, and ↓fecundity

• Males: ↑GSI; ↑ % mature sperm

 No estrogen-related effects reported in the female pubertal 

assay and mammalian or wildlife OSRI

• Although negative, differences in taxa sensitivities have been 

reported in regards to altered sterol synthesis (Ankley and Grey 

2013)

11
Chemical J:

Estrogen Pathway Conclusions 

Based upon WoE evaluation of EDSP Tier 1 data, 

Chemical J appears to alter steroidogenesis which 

then interferes with the estrogen signaling 

pathway and the HPG axis

12
Chemical J: Lines of Evidence Indicating Potential for 

Interaction with the Androgen Pathway

13

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

Fecundity/fertility

Vitellogenin

Overt Toxicity 

Observed

EDSP Tier 1

AR Binding Binder

Steroidogenesis Test.

Hershberger No effect

Male Pubertal Rat

M: 

↓49%

H: 

↓85%

No

effect

No

effect

No

effect

No

effect

L: ↓8%

M:↓10%

H:↓10%

M: 

SV↓19%;

VP↓16%

H:

SV↓40&33%

VP↓27%

DLP↓25%

LABC↓16%

H: 

↑1.7d

FSTRA

M: 

↑200%

♀

M: ↑41% 

H:↑46%♀;

H:↑80% ♂

H: ↑ oocyte 

atresia; 

H: ↑ % mature

sperm

No effect H: 88% 

(fecund)

H: 

85%

♀
Chemical J: Lines of Evidence Indicating Potential for 

Interaction with the Androgen Pathway

14

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

Fecundity/ 

fertility

Overt Toxicity 

Observed

OSRI

Subchronic toxicity 

(Rat) 

No

effect

No

effect

No

effect

No

effect

No

effect

No

effect

Chronic toxicity (Rat)

M: 23%

H: 19-

42%

Testicular 

atrophy

No 

effect

affected 

spermatogenesis

Chronic toxicity (Dog) No

effect

No

effect

No

effect

No

effect

No

effect

Two-generation 

reproduction (Rat)

No

effect

Testicular 

atrophy

No

effect

Prostate 

atrophy

affected 

spermatogenesis

Developmental (Rat)

No 

effect

Avian/Aquatic 

Invertebrate 

Reproduction

No effect
Integration of Androgen Pathway Data

 (+) in vitro AR binding (weak) but (-) in vivo Hershberger 

suggest not AR-mediated

 (+) responses in vitro steroidogenesis assay (↓T)

Complementary anti-androgenic responses in male pubertal

• ↓testosterone

• ↓sex accessory organ weights (2 organs at mid conc.; 4 at high)

• ↑ in PPS

Complementary responses in FSTRA

• Males: ↑GSI; ↑ % mature sperm

• Females: effects on enzyme activity (E, T) up through fecundity

15
Integration of Androgen Pathway Data

Guideline mammalian studies

Rat chronic/carcinogenicity:

• ↓ testes weight

• ↑ testicular atrophy

• Effects on spermatogenesis 

Rat 2-gen reproduction:

• ↑ testicular atrophy

• ↑ prostrate atrophy

• Effects on spermatogenesis

16
Chemical J:

Androgen Pathway Conclusions 

Based upon WoE evaluation of EDSP Tier 1 

data and OSRI, Chemical J appears to alter 

steroidogenesis which then interferes with the 

androgen signaling pathway and the HPG axis

17
Chemical J: Lines of Evidence Indicating Potential for 

Interaction with the Thyroid Pathway

18

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) No effect

L: ↓8%

M:↓10%

H:↓10%

No 

Effect

H: ↑ follicular

cell heights & ↓ 

colloid area1

Female Pubertal (Rat) No effect No Effect

No 

Effect

No Effect

AMA (Frog) No Effect

No 

Effect

No 

Effect

No 

Effect H, M2

OSRI

Two-generation reproduction (Rat)

No Effect No 

Effect

No Effect

Subchronic toxicity (Rat) 

No Effect No 

Effect

↑ small follicles 

♂1

Chronic toxicity (Rat)

No 

Effect

No Effect

Chronic toxicity (Dog)

No 

Effect

No Effect

1 = Effects considered to be due to normal homeostatic response of the thyroid hormone system

2 = Overt toxicity observed at two highest treatment groups (abnormal swimming, lethargy), but not at lowest which also had no indications of 

thyroid effects
Integration of Thyroid Pathway Data

No effects on thyroid hormones in male or 

female pubertal or mammalian guideline data

Alterations in thyroid organ in male pubertal and 

mammalian (chronic rat) considered normal 

thyroid response

No indications of thyroid mediated effects for all 

concentrations in AMA

19
Chemical J:

Thyroid Pathway Conclusions 

Based upon WoE evaluation of EDSP Tier 1 

data and OSRI, Chemical J does not 

demonstrate a potential to interact with the 

HPT axis

20
Chemical J: Overall Conclusions

21

Estrogen Pathway: Potential to initiate an antiestrogen AOP via altered steroidogenesis with 

complementary responses observed in fish

Androgen Pathway: Potential to initiate an antiandrogen AOP via altered steroidogenesis with 

complementarity observed in fish and rats with intact 

HPG axis

Thyroid Pathway: Lack of robust complementarity 

and redundancy to support an interaction with the 

HPT axis with alterations in thyroid tissue likely due 

to maintaining homeostasis
Case Study J — Estrogen Signaling Pathway 

(altered steroidogenesis) 

22

Tier 1 Assays

OSRI- no effects 

observed

(-) ER Binding, ERTA and Uterotrophic does not support ER binding as the MIE
Case Study J — Androgen Signaling Pathway 

(altered steroidogenesis) 

23

Tier 1 Assays

Weak AR binding and Negative Hershberger does not support AR binding as the MIE

OSRI- effects consistent 

with Tier 1 assays