EDSP

Weight-of-Evidence Process

Case Study Chemicals: Chemical S

John Liccione, 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
Chemical S

Pesticidal Class: Organometal miticide

Mode of pesticidal action: Inhibition of oxidative 

phosphorylation

Physical-chemical Properties: 

• Relatively insoluble in water

• Stable to abiotic routes of degradation and to aerobic 

metabolism

• Log Kow > 5; Koc ≈ 321,000 L/kg

• Difficult to test in aquatic systems and in vitro assays

3
Chemical S

Basis for selecting case study chemical

Difficult-to-test substance (low solubility; high Kow and Koc)

Overt toxicity in rat and fish assays coincident with 

potential endocrine effects

• severe gastric irritant

• poor palatability of the diet

Lack of molecular initiating event based on negative 

results in the Tier 1 in vitro assays

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 Negative2

Steroidogenesis Assay 

(Human cell line H295R)

OSCPP 890.1550;

OECD 456

No Negative

Aromatase Assay (human 

recombinant microsomes) OSCPP 890.1200 No Equivocal

1 Classified as “Not Interactive” according to Test Guideline OSCPP 870.1250

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

6

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

Positive for E and

Negative 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 Limited exposure

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

among responses in the assay.
OSRI Considered

Guideline Mammalian Studies

Chronic toxicity studies (Rat, Mouse, Dog)

• Gonadal histology and organ weights

Two-generation reproduction study (Rat)

• Gonadal histology and organ weights — evaluated

• PPS, VO and ovarian cycling — not evaluated

7
OSRI Considered (continued)

Guideline Wildlife Studies

• Early life stage test with rainbow trout

• Acute toxicity study in rainbow trout

• One-generation reproduction study with bobwhite quail

• Chronic toxicity study with Daphnia magna

8
Chemical S: Lines of Evidence Indicating Potential for 

Interaction with the Estrogen Pathway

9

Study Type /

Literature 

Citation

ER Binding

ER Activation

Steroidogenesis

Uterine Weight or 

Fish GSI

Ovarian Weight

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

Not

interactive

ERTA Negative

Aromatase Equivocal

Steroidogenesis Negative

Uterotrophic No effect

Female Pubertal Rat

↓24%

H

↓43%

H

No effect ↓35%

H

No 

effect

VO: ↑ 

2days (L); 

7days (H) 

H

FSTRA

H: 

↑(39%) 

♂ No effect

H: ↓(13%) 

(fer)

No 

Effect

H: ↓(34%) 

tubercle 

score ♂

H

Overt toxicity in FSTRA was a 50% decrease in survival at high concentration. 

In the FPUB assay, salivation, ↓BW and FC were noted. 
Chemical S: Lines of Evidence Indicating Potential for 

Interaction with the Estrogen Pathway

10

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

Subchronic 

toxicity (Rat) 

Chronic toxicity 

(Rat)

No 

effect

No 

effect

No

effect

No

effect

Chronic toxicity 

(Mouse)

No

effect

Two-generation 

reproduction 

(Rat)

No

effect

No 

effect

No

effect

No

effect

No 

effect
Integration of Estrogen Pathway Data

 In vitro assays: Negative in ER binding, ERTA and 

steroidogenesis assays. Equivocal in the aromatase 

assay.

 In vivo assay: Delayed VO in the Female Pubertal 

Assay at low dose in absence of overt toxicity

No estrogen-related effects in FSTRA in the absence of 

overt toxicity

No estrogen-related effects in the OSRI

11
Chemical S:

Estrogen Pathway Conclusions 

WoE evaluation: T1 and OSRI demonstrated a 

potential to interact with the estrogen pathway. 

Based on:

• Delayed VO in female pubertal assay in the absence 

of overt toxicity

• Estrogen-related effects in the female pubertal and 

FSTRA assays in presence of overt toxicity

12
Chemical S: Lines of Evidence Indicating Potential for 

Interaction with the Androgen Pathway

13

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

Fertility/

Fecundity

Vitellogenin

Fish Gonadal Wt 

(GSI)

Overt Toxicity 

Observed

EDSP Tier 1

AR Binding

Nonbinder

Steroidogenesis Negative

Hershberger

L: VP

 GP

M & 

H

M & H: 

 SV

 VP

 LABC

 CG

 GP

Male Pubertal 

Rat

56%

H

17%

H

No

effect

24%

H

No

effect

No

effect

SV, VP, DP, 

LABC

H

PPS:

↑ 6 days

H

H

FSTRA No Effect

H: ↓(34%) 

tubercle score ♂

H: ↓ 

(13%) 

(fer)

No 

effect

H: 

↑(39%) 

♂

H

Overt toxicity: Salivation and body weight in the Hershberger and Male Pubertal.

50% decrease in survival at high dose in the FSTRA. 
Chemical S: 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

Vitellogenin

Overt Toxicity 

Observed

OSRI

Subchronic toxicity (Rat) No

effect

No

effect

Chronic toxicity (Rat) ↑27% No

effect

No

effect

Chronic toxicity (Mouse) No

effect

No

effect

Chronic toxicity (Dog)

No

effect

Two-generation 

reproduction (Rat)

No

effect

No

effect

No

effect

No

effect

No

effect
Integration of Androgen Pathway Data

In vitro assays: Negative in AR binding and 

steroidogenesis assays

In vivo assay:

• Anti-androgenic response in sex accessory organ weights in 

Hershberger at all doses. Response at low dose occurred in 

the absence of overt toxicity.

• Decreased SV, VP, DP and LABC weights in the pubertal male 

at high dose only in presence of overt toxicity

15
Integration of Androgen Pathway Data

Guideline mammalian chronic toxicity study in 

the rat showed ↑ testes weight but no 

corresponding histological effects 

No other androgen-related effects observed in 

other mammalian studies, including the

2-generation rat reproduction study

16
Integration of Androgen Pathway Data

In the FSTRA, potential endocrine effects (e.g., 

alterations in fertility, male GSI, male tubercles) 

were observed only in the presence of overt toxicity 

(increased mortality)

• Uncertainty if these responses are due to potential 

endocrine interaction or from a response in decreased 

energy output

17
Chemical S:

Androgen Pathway Conclusions 

WoE evaluation: T1 and OSRI demonstrated a 

potential to interact with the androgen pathway

• Decreased VP and GP weights in the Hershberger 

assay in the absence of overt toxicity

• Androgen-related effects in male pubertal and

FSTRA assays in presence of overt toxicity

18
Chemical S: Lines of Evidence Indicating Potential for 

Interaction with the Thyroid Pathway

19

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

No 

Effect

No 

Effect

No 

Effect

Hb

Female Pubertal (Rat) No

Effect

↓ 35% 

H

↓ 15% 

H

No 

Effect

Hb

AMA (Frog)a No 

Effect

No 

Effect

No 

Effect

No 

Effect

OSRI

Two-generation reproduction (Rat)

No 

Effect

Subchronic toxicity (Rat) 

Chronic toxicity (Rat) No

Effect

No 

Effect

No 

Effect

Chronic toxicity (Mouse) No

Effect

No 

Effect

No 

Effect

Chronic toxicity (Dog)

No 

Effect

a Limited exposure.

b Decreased BWs and FC, and increased salivation in the male and female pubertal assays. 
Integration of Thyroid Pathway Data

No evidence that Chemical S has the potential to interact 

with the T pathway

• ↓pituitary and thyroid weights at high dose in the female pubertal 

assay occurred in presence of overt toxicity

• No changes in thyroid weights, pituitary weights or thyroid 

histopathology in the mammalian chronic toxicity studies

Limited exposure in the AMA assay — restricted by 

physico-chemical properties of the chemical.

No thyroid effects at concentrations tested.

20
Chemical S:

Thyroid Pathway Conclusions 

Based upon WoE evaluation of EDSP Tier 1 

data and OSRI, Chemical S does not 

demonstrate a potential to interact with the 

thyroid pathway

21
Chemical S: Overall Conclusions

22

 Estrogen Pathway:

• Chemical S has the potential to interact with the E pathway 

based on delayed VO in the Female Pubertal assay

• Negative in vitro data may indicate a different MIE or an active 

metabolite is responsible for in vivo responses

 Androgen Pathway:

• Chemical S has the potential to interact with the A pathway 

(Hershberger assay)

• Negative in vitro data may indicate a different MIE or an active 

metabolite is responsible for in vivo responses

 Thyroid Pathway: Chemical S did not show the potential to 

interact with the HPT axis
Case Study S — E and A Pathway Interactions 

23

Tier 1 Assays

OSRI- no effects 

found

Estrogen Pathway

Androgen Pathway