# 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 K ow 

# ~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 Positive 1



### 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 pathway 1



### 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 aquatic

phase 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 ♂ H 1

1 

### Overt 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

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

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

12

# 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% ♀ H 1

1 Overt 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 anti



# androgenic 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

(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) 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 anti

androgen 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