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

lowit.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. 

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 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

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Chemical 

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)

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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

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 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

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 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

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May 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

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 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

 Not 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

 in 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

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Chemical 

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

Anchor 1 Toxicity Pathway

(initiating 

event)

Anchor 2

(adverse outcome at the organism 

or population level)

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Slide 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 in 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

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Measured Brain Deltamethrin 

in Juvenile (PND 15) Rats

Measured Brain 

Deltamethrin in Adult Rats

Model Predictions of Brain 

Deltamethrin in Humans

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 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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