# 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 K ow 

# ~ 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 

## 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 

## Positive 2



## Steroidogenesis Assay ( Human cell line H295R) OSCPP 890.1550;

OECD 456 

## No 

## Positive

Aromatase Assay ( human recombinant microsomes) OSCPP
890.1200 

## No 

## Positive 3

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 pathway 1 

## 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 Steroidogenesis Uterine 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 spermato

genesis

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 spermato

genesis

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

(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 L: ↓8% M:↓10% H:↓10% No

Effect H: ↑ follicular

cell heights & ↓
colloid area 1

Female Pubertal (Rat) No effect No Effect No
Effect No Effect

AMA (Frog) No Effect No
Effect No

Effect No

Effect H, M 2



### 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 ind ications 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 anti

estrogen AOP via altered steroidogenesis with 

# complementary responses observed in fish

 

# Androgen Pathway: Potential to initiate an anti

androgen 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