# EDSP

Weight

- of

- Evidence Process Case Study Chemicals: Chemical N

Catherine B. Aubee, MPA 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 N

 

# Chemical Class: Ketone

 

# Mode of pesticidal action: None (inert)

 

# Physical

- chemical Properties:

• Relatively soluble in water

• Log K ow 

# ~2

• Can persist but undergoes aerobic degradation

• Moderately mobile to surface water (runoff or erosion)

3

# Chemical N

 

# Basis for selecting case study chemical

• Pesticide inert

• Decreased fish VTG but no complementary effects at that treatment level

• Additional effects at higher doses with overt toxicity

• Responses only at high dose in male rat

• Fewer additional lines of evidence (less OSRI)

4

# Assessment Tools for Evaluation of Risk

(ASTER)

 

# Predicts toxicity profile

 

# Toxicity data

• 

# Fish (ECOTOX & EPA data)

— 4 studies

• 

# Alga (ECOTOX)

— 1 study

• 

# Freshwater invertebrate (ECOTOX)

— 1 study

 

# Bioconcentration data

• 

# Fish (ECOTOX)

— 1 study

 

# Quantitative Structure

- Activity Relationships (QSARs)

• 

# Fish chronic toxicity

• 

# Fish bioconcentration

5

# Assessment Tools for Evaluation of Risk

(ASTER)

Chemical N: nonpolar narcotic (predicted absence of specific cellular interactions)

6 

## More information on ASTER: http://www.epa.gov/med/Prods_Pubs/aster.htm

# Tier 1 In Vitro Assay Results

Tier 1 Assays Test Guideline(s) 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
Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550;
OECD 456 No Negative
Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 No Negative

1 Classified as “Not Interactive” according to the Test Guidelines 890.1250

7

# Tier 1 In Vivo Assay Results

Tier 1 Assays Test Guideline(s) Satisfied by OSRI (Y/N) Study Findings
Uterotrophic Assay (Rat) OSCPP 890.1600;
OECD 440 No Negative for Estrogen Pathway

Hershberger Assay (Rat) OSCPP 890.1400;
OECD 441 No Negative for Androgen 2 Pathway
Pubertal Female Assay (Rat) OSCPP 890.1450 No Negative for Estrogen and Thyroid Pathways
Pubertal Male Assay (Rat) OSCPP 890.1500 No Positive for Androgen Pathway Negative for Thyroid Pathway Fish Short

- term Reproduction Assay OSCPP 890.1350;
OECD 229 No Possible 3 for Estrogen and Androgen Pathways Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100;
OECD 231 No Negative for Thyroid Pathway

2 Negative for both androgenic and anti

- androgenic effects.
3 Based on the proposed FSTRA decision logic which considers the degree of complementarity among responses in the assay.

8

# OSRI Considered: In Vitro (Literature)

 

# ER α yeast two

- hybrid system (Y2H)

• REC 10 

# > 10

- 4 

# M



#  

# ER α chloramphenicol acetyltransferase transcriptional activation assay

• Negative up to 10

- 5 

# M (highest test concentration)

9

# OSRI Considered: In Vivo (Guideline)

 

# Mammalian studies only

 90

- day subchronic oral toxicity

• 

# No effects on gonadal or thyroid weight or histology in rat or dog up to 3000 ppm ( ♂ 233.8 mg/kg bw /day, 

# ♀ 311.8 mg/kg bw

/day)

 103

- week carcinogenicity

• 

# No effects on gonadal or thyroid weight or histology 

# in rat or mouse up to 500 mg/kg bw /day

10Study Type / Literature Citation

ER Binding

ER Activation

Steroidogenesis

Uterine Weight Ovarian Weight or GSI Gonadal Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at
VO

Fecundity

Fertilization Success Vitellogenin Overt Toxicity Observed

EDSP Tier 1
ER Binding Not

Interactive

ERTA Negative

Aromatase Non

Inhibitor Steroidogenesis Negative

Uterotrophic No effect

Female Pubertal Rat No effect No effect No effect No

effect No effect No effect

FSTRA No effect ↑Oocyte atresia H ↓94% H ↓70% H ↓44% ♀ M ↓77% ♀ H H 

# Chemical N: Lines of Evidence Indicating Potential for 

# Interaction with the Estrogen Pathway

GSI: Gonado

- somatic index L: Low treatment, M: Medium treatment, H: High treatment

11Study Type / Literature Citation

ER Binding

ER Activation

Steroidogenesis

Uterine Weight Ovarian Weight or GSI Gonadal Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at
VO

Fecundity

Fertilization Success Vitellogenin Overt Toxicity Observed

OSRI

Yeast 2

- hybrid ER Negative

ERα transactivation Negative

Subchronic ( Rat) No effect

Subchronic ( Dog) No effect

Carcinogenicity (Rat) No effect No effect

Carcinogenicity (Mouse) No effect No effect 

# Chemical N: Lines of Evidence Indicating Potential for Interaction with the Estrogen Pathway

12

# Integration of Estrogen Pathway Data

 

# No effects in in vitro assays or HPG

- interrupted assay 

# (rat uterotrophic)

 

# 44% reduction of female fish VTG with no complementary effects (M treatment)

 

# Shutdown of fish reproduction in presence of overt toxicity (H treatment)

• Reduced feeding in males

• Reduced body weight in males and females

13

# Chemical N: Estrogen Pathway Conclusions

 

# In vitro + uterotrophic results suggest no interaction with ER or steroidogenesis

 

# Shutdown of reproduction in fish at high treatment level could be explained by reduced feeding (nonpolar narcosis)

 

# Biological significance of ↓ ♀ VTG at medium concentration with no complementary effects is uncertain

14

# Chemical N: Lines of Evidence Indicating Potential for



# Interaction with the Androgen Pathway

15 Study Type / Literature Citation

AR Binding

Steroidogenesis Testosterone Testes Weight or GSI Gonad Staging and Histopathology Epididymides Weight or Histopathology Pituitary Weight Accessory Sex Organ Weights Age and Weight at PPS Secondary Sex Characteristics Fecundity Fertilization Success Vitellogenin Overt Toxicity Observed

EDSP Tier 1

AR Binding Not

Interactive

Steroidogenesis Negative

Hershberger No effect

Male Pubertal Rat ↓58% H No effect No effect No effect No effect ↓24% H

SV ↑1.8 H

FSTRA No effect ↑ Oocyte atresia H No effect ↓94% H ↓70% H ↓44% ♀ M ↓77% ♀ H HStudy Type /

Literature Citation AR Binding Steroidogenesis Testosterone Testes Weight or GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights Age and Weight at PPS Secondary Sex Characteristics Vitellogenin Overt Toxicity Observed

OSRI

Subchronic ( Rat) No effect No effect No effect

Subchronic ( Dog) No effect No effect No effect

Carcinogenicity (Rat) No effect No effect No effect

Carcinogenicity (Mouse) No effect No effect No effect 

# Chemical N: Lines of Evidence Indicating Potential for Interaction with the Androgen Pathway

16

# Integration of Androgen Pathway Data

 

# No effects in in vitro assays

 

# No effects in HPG

- interrupted assay (rat Hershberger)

 

# Some complementarity in potential anti

- A response

• ↓ testosterone, ↓ seminal vesicle weight, and slight ↑ in age at PPS

• Only at high dose

17

# Integration of Androgen Pathway Data

 

# ↓in female fish VTG could reflect A or anti

- A response

hepatocyte vitellogenin

Vitellogenin illustration courtesy of http://opm.phar.umich.edu/protein.php?pdbid=1lsh 

# Anti

- A: ↓testosterone ↓estradiol ↓VTG

18

# Chemical N: Androgen Pathway Conclusions

 

# In vitro + Hershberger results show no AR

- mediated androgenic or anti

- A effects

 

# Some tissue level responses in rat and fish could 

# reflect an anti

- A AOP

 

# Molecular initiating event (MIE) undetermined

 

# Questions of biological relevance:

• Effects only at high dose (rat)

• At lower concentrations with normal feeding behavior, 

# no impact on fish reproduction

— only VTG

19

# Chemical N: Lines of Evidence Indicating Potential for Interaction with the Thyroid Pathway

Study Type / Literature Citation Hormones

(T 4 and TSH)

Pituitary Weight

Thyroid Weight Thyroid Gross and Histopathology Frog Developmental Stage Hind Limb Length Body Weight or Amphibian SVL Overt Toxicity Observed

EDSP Tier 1
Male Pubertal (Rat) No effect No effect No effect No effect
Female Pubertal (Rat) No effect No effect No effect No effect

AMA (Frog) No effect No effect No effect ↓15%

BW H ↓5% SVL H OSRI
Subchronic toxicity (Rat) No effect No effect
Subchronic toxicity (Dog) No effect No effect
Carcinogenicity (Rat) No effect No effect
Carcinogenicity (Mouse) No effect No effect

20

# Integration of Thyroid Pathway Data

No relevant effects

21

# Chemical N: Thyroid Pathway Conclusions

 

# Absence of complementary and redundant responses

 

# Multiple lines of evidence do not indicate interaction with the HPT axis or T pathway

22

# Chemical N: Overall Conclusions

23 

#  

# Estrogen Pathway: No evidence of receptor



# mediated interaction, possible downstream anti

- E effects in fish in vivo

 

# Androgen Pathway: No evidence of receptor

mediated interaction, possible downstream effects in male rat (anti

- A) and fish (A/anti

- A)

 

# Thyroid Pathway: No effects

# Case Study N

— Androgen Antagonism
Tier 1 Assays

24 

## * No effect on receptor mediated signaling 

## OSRI

- no relevant effects