# 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

• H igh -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.

Page 2

# 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.

Page 3 

# 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

Page 4 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 m ultigeneration 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)

Page 5 

# 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.

Page 6 

## 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

Page 7 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

Page 8 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. Page 9

# Prioritization Based

On Bioactivity and Exposure

Page 10 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.

Page 11 

## All bioactivity hits (

not limited to endocrine

) as a 

## frequency distribution in oral equivalent units ToxCast 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

Page 12 Pesticide active HT Activity and HT Exposure Information Part 158/OSRI and other exposure information Prioritization and any additional EDSP related 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. Page 13

# 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

Page 15 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/text idx?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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