# 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 K ow 

# > 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 K ow 

# and K oc 

# )

 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 Negative 1



### 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 Negative 2



### 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 pathway 1



### 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

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)

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 Non

binder

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

(T 4 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 H b

Female Pubertal (Rat) No
Effect ↓ 35%

H ↓ 15%

H No
Effect H b

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