Pesticides and the EDSP: 

A “Lines of Evidence” Evaluation 

Colleagues from Bayer CropScience, 

Dow AgroSciences, and Syngenta

Presentation to the SAP, “Integrated Bioactivity and Exposure Ranking: A 

Computational Approach for Prioritization and Screening of Chemicals in the 

Endocrine Disruptor Screening Program”

December 2014

Katie Paul Friedman; katie.paul@bayer.com
Objectives of our collaboration

• High-throughput screening (HTS) information as a first line of 

evidence for determining the priority to identify endocrine activity.

• Goal: Demonstrate a “roadmap” for data-rich chemicals through 

the EDSP.

– Transparency

– Hazard and Exposure

– Organize information based on the OECD Conceptual Framework for 

Endocrine Information.

– Structured, systematic data review that considers the level of 

biological relevance of the data.

• Share our approach via a manuscript.

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HT hazard and exposure information should be used as a first step in 

prioritization, followed by consideration of data available for pesticides or 

other data-rich chemicals.

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Pesticide active

HT Activity and HT Exposure 

Information

Part 158/OSRI and other 

exposure information

Proposed Roadmap for EDSP Prioritization
Using the OECD Conceptual Framework to 

organize data by biological relevance

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OECD Level General Information EDSP Tier 1 specific information Data from screening, pesticide 

registration studies, and OSRI

Level 1 • Prioritization information • HTS bioactivity data (ToxCast)

• HT exposure predictions (ExpoCast)

Level 2 • In vitro indicators of 

potential endocrine 

perturbation

• ER and AR binding 

• ER transcriptional activation 

assay

• Steroidogenesis assay

• Aromatase assay

• Validated and/or low-throughput in 

vitro assays

Level 3 • In vivo endocrine measures 

that indicate the potential 

for adversity

• Uterotrophic assay 

• Hershberger assay 

• Amphibian metamorphosis 

• Pubertal assays, by endpoint

• Short-term animal exposures, repeat 

dose studies, or other studies that do 

not include chronic or reproductive 

information

Level 4 • In vivo adverse endocrine 

outcomes

• Pubertal assays, by endpoint

• Fish short-term reproduction 

assay

• 1-generation reproduction

• Prenatal developmental toxicity

• Old multigeneration studies 

• Chronic toxicity and carcinogenicity

• Endocrine histopathology findings

Level 5* • In vivo endocrine adversity 

over the life cycle

• EDSP Tier 2 studies • Extended 1-generation reproduction

• Modern two-generation reproduction

• Life cycle studies

* No Level 5 data were available for our case study.
Line of evidence: ToxCast Data (Level 1)

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Ex. Assays considered for androgen, anti-androgen, steroidogenesis hypotheses

Assay Assay activity

ATG_AR_TRANS Inducible exogenous androgen receptor (NR3C4) transcription factor activity in HepG2 cells.

NVS_NR_hAR Cell-free radioligand binding assay using LnCAP-cell derived human AR.

NVS_NR_rAR Cell-free radioligand binding assay using rat prostate-derived AR.

NVS_NR_cAR Cell-free radioligand binding assay using recombinant Sf9-derived chimpanzee AR.

Tox21_AR_BLA_Agonist_ratio Inducible AR transcription factor activity detected by a beta-lactamase-androgen response element 

reporter in agonist mode using HEK293T cells.

Tox21_AR_BLA_Antagonist_ratio Inducible AR transcription factor activity detected by a beta-lactamase-androgen response element 

reporter in antagonist mode using HEK293T cells.

Tox21_AR_BLA_Antagonist_viability (for 

context)

Cell viability assay for HEK293T cells with the AR-BLA reporter system.

Tox21_AR_LUC_MDAKB2_Agonist Inducible AR transcription factor activity detected by a luciferase-androgen response element fusion 

reporter in agonist mode using MDA cells.

Tox21_AR_LUC_MDAKB2_Antagonist Inducible AR transcription factor activity detected by a luciferase-androgen response element fusion 

reporter in antagonist mode using MDA cells.

Tox21_AR_LUC_MDAKB2_Antagonist_viab

ility (for context)

Cell viability assay for MDA cells with the luciferase-androgen response element reporter.

OT_AR_ARE_LUC_AG_1440 Luciferase protein-fragment complementation assay measuring ERα binding to the estrogen response 

element detected by microscopy in CHO-K1 cells (24 hr).

OT_AR_ARSRC1_0480 Fluorescent protein-fragment complementation assay measuring ERα binding to the estrogen response 

element detected by microscopy in HeLa cells (8 hr).

OT_AR_ARSRC1_0960 Fluorescent protein-fragment complementation assay measuring ERα binding to the estrogen response 

element detected by microscopy in HeLa cells (16 hr).

Tox21_Aromatase_Inhibition Assay indicates inhibition of aromatase using human breast carcinoma MCF-7 aro ERE cells stablytransfected with a testosterone receptor-responsive luciferase reporter. 

Tox21_Aromatase_Inhibition_viability Cell viability assay for the MCF-7 aro-ERE reporter system.

NVS_ADME_hCYP19A1 Cell-free, biochemical assay of human CYP19A1 activity inhibition.

NVS_ADME_hCYP19A1_Activator Cell-free, biochemical assay of human CYP19A1 activity induction.

Additional assays 

considered for 

steroidogenesis

All 3 ToxCast

Phase II AR 

binding assays 

were considered

Different from 

AUC approach:
A qualitative scoring system was developed to evaluate the 

endocrine relevance of each level of evidence.

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Qualitative 

Score

Description Assessment Criteria

0 Negative There is no evidence that the endocrine pathway is perturbed.

1 Marginal 

Evidence

 There is limited evidence that the endocrine pathway is 

perturbed;

 There is conflicting evidence;

 The effect occurs inconsistently;

 The effect occurs concomitantly with cell-based or systemic 

toxicity;

 There is other information that may discount this finding.

2 Weak Evidence  There is limited evidence that the endocrine pathway is 

perturbed;

 The observation has been made occasionally;

 There is no information to discount the effect;

 The magnitude of the effect is low;

 The relevance to specific endocrine toxicity is unknown.

3 Moderate 

Evidence

 There is some evidence that the endpoint(s) is perturbed;

 The observation has been made consistently;

 The magnitude of the effect is low to moderate;

 The relevance to specific endocrine toxicity is low or uncertain. 

4 Strong Evidence  There is evidence that the endpoint(s) is perturbed;

 The observation has been made consistently;

 The magnitude of the effect is moderate to substantial;

 The relationship to specific endocrine toxicity is understood, 

and of moderate to high relevance.

5 Very Strong 

Evidence

 Adversity in the endocrine pathway is evident.
Androgen/Anti-Androgen/Steroidogenesis: 

3 “lines of evidence” by OECD Level

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Increasing level 

indicates increasing 

confidence in the 

ability to observe 

endocrine activity

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Myclobutanil

Propiconazole

Triadimefon

Androgenicity Anti-Androgenicity
Estrogen/Anti-Estrogen/Steroidogenesis: 

3 “lines of evidence” by OECD Level

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Increasing level 

indicates 

increasing 

confidence in the 

ability to observe 

endocrine activity

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Very Strong

Strong

Moderate

Weak

Marginal

Negative

Myclobutanil

Propiconazole

Triadimefon

Estrogenicity Anti-Estrogenicity
HT bioactivity data is a useful line of 

evidence for prioritizing the need for 

endocrine evaluation.

• HTS information was useful as Level 1 evidence in determining 

potential endocrine hazard by identifying putative molecularinitiating events in vitro. 

• With increasing levels of information for these fungicides, it is 

clear that endocrine hazard observations were coincident 

with other toxicities, i.e. endocrine toxicity was likely not the 

primary effect for the triazoles.

• As demonstrated by the IBER approach, combining HT 

bioactivity and HT exposure information may indicate priority 

for obtaining further endocrine information.

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Prioritization Based

On Bioactivity and Exposure

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Exposure

mg/kg BW/day

Bioactivity

Low

Priority

Med

Priority

Higher

Priority

No

Priority (Hold)

No

Priority (Hold)

Agree with this conceptually, with the caveats that:

• Potency may be low enough that exposure at that level may have no biological result;

• Conservative thresholds to distinguish levels of priority for further testing are not yet 

clear.

From Page 12 of SAP White Paper
HT activity and exposure prediction demonstrate no priority for further 

human health hazard screening for pesticide actives in our case study.

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All bioactivity hits (not limited to endocrine) as a 

frequency distribution in oral equivalent unitsToxCast Assay Hits, Frequency

Similar to IBER approach, and concordant.

IBER for triadimefon was 1000000.

Wambaugh et al. 2013 = source of ExpoCast range; Wetmore et al. 2012 = source of oral equivalents for bioactivity
A roadmap for pesticide actives 

(or other data-rich chemicals) in EDSP

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Pesticide active

HT Activity and HT 

Exposure 

Information

Part 158/OSRI and 

other exposure 

information

Prioritization and any 

additional EDSPrelated RA decision 

can be made

No further EDSP 

testing

Priority and information 

gaps require more hazard 

data for RA decision

Select assays 

from EDSP Tier 1 

battery

EDSP Tier 1 

battery

EDSP Tier 2

Alternative 

mechanistic 

endpoints

Priority and information 

gaps require more 

exposure data for RA 

decision?

Exposure 

refinements

This avenue needed for 

when endocrine activity is 

expected to change the 

POD.

This avenue needed for when 

endocrine activity is understood 

without EDSP Tier 1, but exposure 

information needs refinement.

Direct path indicates endocrine activity is already identified or demonstrated 

appropriate margin-of-exposure.
Conclusions

• Combined HT hazard and exposure is adequate for 

determining priority for EDSP Tier 1 evaluation.

• The standard test guideline studies (OECD and/or 

OCSPP) used for evaluating the hazard potential of 

chemicals are sufficient for informing the potential 

endocrine activity of tested molecules.

• The combined evaluation of HT exposure and hazard 

data for these 3 triazoles suggests a low priority for 

testing through the EDSP Tier 1 battery.

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Acknowledgments

BCS contributors: Katie Paul Friedman, Ann Blacker, Pat Kwiatkowski;

DAS contributors: Daland Juberg, Sabitha Papineni, Sue Marty, Kerry Hastings, Reza 

Rasoulpour, Ray Brinkmeyer;

Syngenta contributors: Doug Wolf, Richard Peffer, Amber Goetz, Sue Yi, and Arpad 

Szarka
Example from Lines of Evidence Comparison

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Review Table for Triadimefon and Androgen/Anti-Androgen/Steroidogensis Pathway

Information 

Level

Endocrine 

Pathway

HTS Tier 1 Part 158/OSRI

Level 1

A

Weak evidence; 

aromatase inhibition at 

high concentration

Anti-A

Marginal evidence; 

hAR binding without 

agreement across 

assays

Level 2

A

Moderate evidence; Inhibits aromatase with 

IC50 = 1.71 μM; part of US EPA validation study 

for the aromatase assay; Negative binding to AR

Weak evidence; Aromatase inhibition at high 

concentrations.

Anti-A Negative; no binding to AR

Moderate evidence; ↓ testosterone 

production in vitro in two assays, suggests 

possible inhibition of steroidogenesis

(CYP17A1).

Level 3

A Negative

Weak evidence in rats, ↑ abs testes weight, ↑ 

rel ventral prostate, and ↑ testosterone in 

vivo in two studies (at 500 & 1800 ppm, with 

toxicity). 

Anti-A Negative

Marginal evidence in rats; slight delay in PPS 

with BW ↓ and conflicting with ↑ anogenital

distance in males at 1800 ppm.

Level 4

A Negative Negative

Anti-A

Marginal evidence in fish; ↑ VTG in males but 

within historical control, ↓ fecundity and ↓ male 

BW, increased GSI in males in FSTRA; There were 

no effects on fertilization, gonad histology, or 

secondary sex characteristics in the FSTRA. 

Weak evidence in rat; Reduced mating by F1 

males at 1,800 ppm coincident with other 

toxicity

Apical effects occur concomitantly with other toxicities and/or resultant to effects on 

steroidogenesis.
Definitions

• EDSP: Endocrine Disruptor Screening Program

• HT: high-throughput

• HTS: high-throughput screening

• IBER: integrated bioactivity and exposure ranking

• POD: point of departure

• OECD Conceptual Framework for Testing and Assessment of Endocrine Disruptors 

(rev. 2012): 

(http://www.oecd.org/env/ehs/testing/OECD%20Conceptual%20Framework%20for%20Testing%20and%2

0Assessment%20of%20Endocrine%20Disrupters%20for%20the%20public%20website.pdf)

• OSRI: Other scientifically-relevant information, with reference on its use in EDSP: 

http://www.ncbi.nlm.nih.gov/pubmed/24151143

• “Part 158” is part of Code of Federal Regulations, Title 40, Part 158, describing test 

guidelines for the registration of pesticides: 

– eCFR source: http://www.ecfr.gov/cgi-bin/textidx?SID=eb9e4de396d2dafe53d71367e904e06f&node=se40.24.158_1500&rgn=div8

– EPA source on test guidelines: 

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series870.htm

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