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 Kow~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 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 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 Androgen2 

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

Possible3 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)

ERα yeast two-hybrid system (Y2H)

• REC10 > 10-4 M

ERα 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

10
Study 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 NonInhibitor

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

11
Study 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

H
Study 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 

(T4 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 receptormediated interaction, possible downstream 

anti-E effects in fish in vivo

Androgen Pathway: No evidence of receptormediated 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