Anna B. Lowit, Ph.D. Senior Scientist, Health Effects Division

Office of Pesticide Programs, USEPA Co -Chair, Interagency Coordinating Committee on the Validation of Alternative Methods l owit.anna@epa.gov , 703- 308- 4135 

# Implementing 21st Century Toxicity Testing at EPA’s Office of Pesticide Programs

###  Introduction  Background  Guiding Principles for Data Needs  Mode of Action/Adverse Outcome Pathway (MOA/AOP)  Toxicokinetic Data  Read Across 

# Outline

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##  EPA’s Strategic Plan for Evaluating the Toxicity of Chemicals  http://www.epa.gov/osa/spc/toxicitytesting/  Characterizing or predicting potential human exposures;  Estimating the resulting chemical dosimetry (magnitude, frequency, and duration) for target pathways, tissues or organs;  Measuring toxicity pathway response at doses consistent with human exposures;  Predicting the in vivo human response resulting from pathway perturbations;  Quantifying the range of human variability and susceptibility; and  Validating predictions utilizing in vivo systems (e.g., laboratory animals, human data). 

# Introduction

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###  Office of Pesticide Programs is a licensing program regulating pesticide products in the U.S.  One of the major roles is to review effects of pesticides on human and ecological health 

###  The types of information typically required to allow the agency to make the appropriate statutory safety findings under both of these acts are identified under 40 Code of Federal Regulations (CFR) Part 158.  Flexibility in implementing Part 158 data requirements (§ 158.30):  Waivers may be granted as permitted by 40 CFR Part 158.45;  Additional data beyond the 158 data requirements may be important to the risk assessment (§ 158.75), alternative approaches can be accepted, and other data can be used (158.70).  E.g., database can include non -guideline or special studies. 

# Part 158 Toxicology Data Requirements

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# Data sources
 OPP considers multiple sources of information when conducting risk assessments for pesticides  Registrant submitted guideline studies conducted according to 40CFR Part160 Good Laboratory Practice Standards (GLP)  Alternative non -guideline studies  QSAR/SAR  Data in the open literature  submitted by registrants  identified through a literature search by OPP  posted by the public to a regulatory docket for a pesticide evaluation. 5

###  EPA’s Office of Pesticide Programs has developed a Strategic Direction for New Pesticide Testing and Assessment Approaches  http://www.epa.gov/pesticides/science/testing - assessment.html .  A broader suite of computer -aided methods to better predict potential hazards and exposures, and to focus testing on likely risks of concern;  Improved approaches to more traditional toxicity tests to minimize the number of animals used while expanding the amount of information obtained;  Improved understanding of toxicity pathways to allow development of non -animal tests that better predict how exposures relate to adverse effects; 

# Background

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###  Guiding Principles for Data Requirements  Purpose: provide consistency in the identification of data needs, promote and optimize full use of existing knowledge, and focus on the critical data needed for risk assessment.  http://www.epa.gov/pesticides/regulating/data -require - guide -principle.pdf 

###  Part 158 Toxicology Data Requirements: Guidance for Neurotoxicity Battery, Subchronic Inhalation, Subchronic Dermal and Immunotoxicity Studies  Purpose: use a weight of evidence evaluation to determine data needs or to review a waiver justification  http://www.epa.gov/pesticides/regulating/part158-tox-data - requirement.pdf 

# Guiding Principles for Data Needs

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###  “...ensure there is sufficient information to reliably support registration decisions that are protective of public health and the environment while avoiding the generation and evaluation of data that does not materially influence the scientific certainty of a regulatory decision....”  “It is important to only require data that adequately inform regulatory decision making and thereby avoid unnecessary use of time and resources, data generation costs, and animal testing.” 

# Guiding Principles for Data Needs

8Chemical Properties Receptor/Ligand Interaction DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling

Protein Depletion Altered Physiology

Disrupted Homeostasis Altered Tissue Development

or Function Lethality Impaired Development Impaired Reproduction Cancer Structure Recruitment

Extinction Toxicant Macro - Molecular Interactions Cellular Responses Organ Responses Organism Responses Population Responses

Toxicity Pathway Anchor 1 (initiating

event) Anchor 2 (adverse outcome at the organism or population level) 9

## Adverse Outcome Pathway

Chemical s Molecular Target Cellular Response Tissue/ Organ Individual Population Pharmaco

- kinetics In vitro studies Biomonitoring & Exposure data Epidemiology Structure Activity Relationships
Toxicity Pathways Human Incidents In vivo studies

Molecular initiating event Key events or predictive relationships spanning

levels of biological organization Adverse outcome relevant to risk assessment Greater Toxicological Understanding Greater Risk Relevance 10

###  TK data support smarter testing strategies & implementation of the 3R’s (replacement, reduction, and refinement)  By better understanding the TK properties, we can tailor the tox data for a particular pesticide or group. 

###  Many potential uses:  Dose selection: avoid excessively toxic doses & characterization of doses closer to human exposures  Lifestage sensitivity  Incorporation in mode of action/adverse outcome pathway (MOA/AOP) evaluation  Animal to human extrapolation  Human relevance of animal outcomes  Read across 

# Toxicokinetic Data

11 Reduce : Evaluating potential to waive studies is primary role of HASPOC  Prescreens for new active ingredients  Scoping phase of registration review  Although not specifically covered by the guidance, we still consider other guideline studies using the same principle  Waivers for developmental, reproductive, DNT, chronic/carcinogenicity toxicity  Consider waiver submissions from registrants  Read across  Refine : Special protocol studies (e.g., acute inhalation for fumigants, comparative cholinesterase assay studies, shorter duration) instead of standard guideline protocols  Replace : Pharmacokinetic studies in lieu of toxicity study  Since December, 2011, HASPOC has recommended waivers for over 550 in vivo studies 

## Hazard and Science Policy Council (HASPOC)

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###  http://www.oecd.org/env/ehs/risk- assessment/groupingofchemicalschemicalcategoriesandre ad -across.htm  In the read -across approach, toxicity information for one chemical is used to predict for another chemical, which is considered to be "similar" in some way (usually on the basis of structural similarity or on the basis of the same mode or mechanisms of action)  In principle, read -across can be used to assess physicochemical properties, toxicity, environmental fate and ecotoxicity. For any of these endpoints, it may be performed in a qualitative or quantitative manner. 

# Read Across Approaches

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###  Move away from apical endpoints towards hypothesisbased testing using targeted in vivo studies, in vitro studies, and predictive modeling
 Focus on toxicokinetics & internal dosimetry 

# Case Example: Synthetic pyrethroids & naturally occurring pyrethrins

14May 19, 2015 Monique Perron, Jaime D’Agostino , William Irwin, Anna Lowit U.S. Environmental Protection Agency Office of Pesticide Programs Health Effects Division 

# Evaluation of Potential Juvenile Sensitivity from Pyrethroid Exposure

###  Registered Pyrethroids and OPP Risk Assessments  Evidence of Potential Juvenile Susceptibility  Mode of Action/Adverse Outcome Pathway (MOA/AOP)  CAPHRA Research  Pharmacodynamics (PD)  Pharmacokinetics (PK)  Pharmacologically -based pharmacokinetic (PBPK) model 

###  Path Forward 

# Outline

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###  >20 pyrethroids currently registered as pesticides
 Target sodium ion channels resulting in neurotoxic effects
 Increased use over the past decade
 Replacements for organophosphates (OPs) and n -methyl carbamates (NMCs)

# Registered Chemicals

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###  New uses under Pesticide Registration Improvement Act (PRIA) – all actions on hold
 Registration Review – draft risk assessments for public comment by September 2016 (http :// www.epa.gov/oppsrrd1/reevaluation/pyrethroids -pyrethrins.html)


###  Cumulative – 2011 assessment for registered pyrethroids/pyrethrins; reexamined with all actions 

# Pyrethroid Risk Assessments

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###  Previously, developmental neurotoxicity studies (DNTs) were required to evaluate potential juvenile susceptibility
 DNT not particularly sensitive for evaluating comparative sensitivity of young animals to adults for pyrethroids
 In contrast, literature data indicated susceptibility, albeit at relatively high doses 

# Potential Juvenile Susceptibility

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###  Conducting additional DNTs on pyrethroids will not contribute new information that is relevant for risk assessment  Agency concluded that they did not have adequate data to characterize post-natal juvenile susceptibility
 Protocol request (Feb 2010 )
“ In order to make the best use of available experimental techniques appropriate for the toxicological properties of pyrethroids, the Agency will consider protocols which include data from in vivo, in vitro, and/or in silico studies (or any combinations thereof) ” 

# Alternative to DNT Submission

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###  Current toxicity testing approach is at intersection of toxicity and exposure
 2011 review of existing developmental, reproduction, and DNT studies  76 guideline studies for 24 pyrethroids  No sensitivity observed following in utero exposure  No consistent or compelling evidence of sensitivity to offspring at doses relevant for risk assessment 

# Population of Concern

21 The population of greatest concern to the Agency for pyrethroid exposure is children from birth to <6 years of age  Post- natal issue given in utero data  Ontogeny of metabolic enzymes  Early infants  Drinking water not an issue; therefore, bottle feeding not a concern  N ot mobile  Early lifestage behavior contributes to higher exposures  Young children playing on lawns or floors  Greater amounts of time outside compared to adults  Inadvertent and/or intentional ingestion (hand -to - mouth ) Based on a combination of exposure and behavior pattern, toddlers (3- 6 yrs old) are considered most highly exposed 

# Population of Concern

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###  Research proposal submitted by the Pyrethroid and Pyrethrin Technical Working Group (PPTWG) reviewed by July 2010 SAP
 Dec 2010: three major components of work  i n vitro studies: high -throughput human sodium channels and neurolemma  in vivo acute, behavioral studies: acoustic startle response (ASR)  Pharmacologically -based pharmacokinetic (PBPK) modeling 

###  Mode of Action/Adverse Outcome Pathway (MOA/AOP) has been used as guiding principle for CAPHRA research 

# Research Proposal

23Chemical Properties Receptor/Ligand Interaction DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling

Protein Depletion Altered Physiology

Disrupted Homeostasis Altered Tissue Development

or Function Lethality Impaired Development Impaired Reproduction Cancer Structure Recruitment

Extinction Toxicant Macro - Molecular Interactions Cellular Responses Organ Responses Organism Responses Population Responses

Toxicity Pathway Anchor 1 (initiating

event) Anchor 2 (adverse outcome at the organism or population level) 24Slide adapted from CAPHRA, 7 - 10-2012 Apical outcome Development of PBPK models for human children Tremor & Acoustic Startle ChanTest  Showed a weak response by human sodium channels  Could not evaluate sensitivity  Neurolemma  No lifestage sensitivity using adult and juvenile rat membranes  Meacham et al. 2008 demonstrated that an embryonic sodium channel isoform (Nav 1.3) is more sensitive to type II pyrethroids than an adult isoform (Nav 1.2). However, expression of the Nav 1.3 isoform peaks around birth and falls quickly afterward.  Literature data (Choi and Soderlund 2006, Tan and Soderlund 2009) suggest the rat sodium channels are highly sensitive in comparison to human isoforms .  Overall, data from CAPHRA and the literature do not support a PD - related sensitivity for juveniles . 

# CAPHRA Research - Pharmacodynamics (PD)

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###  In vivo acute behavioral studies  Acoustic startle response (ASR)  Detailed clinical observations (DCO) tailored for pyrethroid toxic signs  2010 SAP cautioned challenges with ASR  Juvenile sensitivity observed with both measurements for deltamethrin, but not for permethrin  Observed primarily at doses higher than currently regulating  Consistent with submitted guideline and literature studies  Utility in conducting additional in vivo apical outcome research? 

# CAPHRA Research – Pharmacokinetics (PK)

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###  Modeling follows the OPP strategic direction for toxicity testing
 Chlorpyrifos PBPK Model  SAP and peer - reviewed (2011)  Chlorpyrifos PBPK model took >10 years to develop


###  CAPHRA is taking a more streamlined, efficient approach  Read across - consistent with EPA proposal of “family modeling approach ”  Relying on i n vitro - in vivo extrapolation (IVIVE) 

# PBPK Model

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###  Rat PBPK model demonstrated juveniles and adults are pharmacokinetically different  Higher levels of deltamethrin in plasma and brain of juveniles  Validation of internal dosimetry measurements 

###  In contrast, human PBPK model showed juveniles had lower values
 Charge Question 8a 

# CAPHRA Research – PBPK Model

29Measured Brain Deltamethrin in Juvenile (PND 15) Rats

Measured Brain Deltamethrin in Adult Rats Model Predictions of Brain Deltamethrin in Humans 30

###  Models incorporate CAPHRA generated and literature data for chemical and non -chemical specific parameters  Utility of SimCyp database to provide enzyme ontogeny patterns  Recombinant enzymes vs. directly measured tissue data  Addition of intestinal absorption and potential to further refine


###  Additional research identified by CAPHRA for read -across 

# CAPHRA Research – PBPK Model

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###  Current data generated by CAPHRA fits well with overall pyrethroid knowledge base
 Utilize available data and models to address potential juvenile susceptibility for regulatory actions
 Further research needs to be considered within the context of regulatory deadlines  Registration Review – September 2016 for public comment  Science work ~1 year prior 

# Moving Forward

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