cc:

Jim Jones

Louise Wise

Jack Housenger

William Jordan

Yu-Ting Guilaran 

Robert McNally

Donald Brady

Jacqueline Mosby

Jennifer McLain

Dana Vogel

Susan Lewis 

Richard Keigwin 

Laura Bailey

Anna B. Lowit

Monique Perron 

Jaime D’Agostino

William Irwin 

Cathy Milbourn

Linda Strauss 

OPP Docket

FIFRA Scientific Advisory Panel Members

Stephen Klaine

Kenneth Delclos

Marion Ehrich

David A. Jett

James McManaman

FQPA Science Review Board Members

Russell Carr, Ph.D.

Veronica Berrocal, Ph.D.

Muhammad Hossain, Ph.D.

Allison Jenkins, MPH

Teresa Leavens, Ph.D.

Igor Linkov, Ph.D.

Thomas Murray, Ph.D.

Isaac Pessah, Ph.D.

Bingfang Yan, Ph.D.

 
1

FIFRA Scientific Advisory Panel Minutes No. 2015-02

A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding

Research to

Evaluate the Potential for Juvenile Sensitivity to Pyrethroids

May 19-21, 2015

FIFRA Scientific Advisory Panel Meeting

Held at the

EPA Conference Center

Arlington, VA

The meeting minutes represent the views and recommendations of the FIFRA SAP and do not 

necessarily represent the views and policies of the EPA or of other agencies in the Executive 

Branch of the Federal government. Mention of trade names or commercial products does not 

constitute an endorsement or recommendation for use. The meeting minutes do not create or 

confer legal rights or impose any legally binding requirements on the EPA or any party. 
2

TABLE OF CONTENTS 

PANEL ROSTER .........................................................................................................5

LIST OF COMMON ACRONYMS USED ..................................................................8

INTRODUCTION .........................................................................................................9

PUBLIC COMMENTS .................................................................................................10

OVERALL 

SUMMARY.........................................................................................11

EXECUTIVE SUMMARY OF PANEL DISCUSSION AND 

RECOMMENDATIONS.................................................................................................13 

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE ..............26

LITERATURE CITED.....................................................................................................56
3

NOTICE

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel 

(SAP) is a Federal advisory committee operating in accordance with the Federal Advisory 

Committee Act and established under the provisions of FIFRA as amended by the Food Quality 

Protection Act (FQPA) of 1996. The FIFRA SAP provides scientific advice, information, and 

recommendations to the EPA Administrator on pesticides and pesticide-related issues regarding 

the impact of regulatory actions on health and the environment. The Panel serves as the primary 

scientific peer review mechanism of the Environmental Protection Agency (EPA), Office of 

Pesticide Programs (OPP), and is structured to provide balanced expert assessment of pesticide 

and pesticide-related matters facing the Agency. FQPA Science Review Board members serve 

the FIFRA SAP on an ad hoc basis to assist in reviews conducted by the FIFRA SAP. The 

meeting minutes have been written as part of the activities of the FIFRA SAP. 

In preparing the meeting minutes, the FIFRA SAP carefully considered all information provided 

and presented by EPA and the Council for the Advancement of Pyrethroid Human Risk 

Assessment, LLC (CAPHRA). The minutes represent the views and recommendations of the 

FIFRA SAP and do not necessarily represent the views and policies of the EPA, nor of other 

agencies in the Executive Branch of the Federal government. Mention of trade names or 

commercial products does not constitute an endorsement or recommendation for use. The 

meeting minutes do not create or confer legal rights or impose any legally binding requirements 

on EPA or any party. 

The meeting minutes of the May 19-21, 2015 FIFRA SAP meeting held to consider and review 

scientific issues associated with “Research to Evaluate the Potential for Juvenile Sensitivity to 

Pyrethroids” were certified by Stephen Klaine, Ph.D., FIFRA SAP Chair, and Fred Jenkins, 

Ph.D., FIFRA SAP Designated Federal Official, on August 18, 2015. The minutes were 

reviewed by Laura E. Bailey, M.S., FIFRA SAP Executive Secretary. The minutes are publicly 

available on the SAP website (http://www.epa.gov/scipoly/sap/) under the heading of 

“Meetings” and in the public e-docket, Docket No. EPA-HQ-OPP-2015-0130, accessible 

through the docket portal: http://www.regulations.gov. Further information about FIFRA SAP 

reports and activities can be obtained from its website at http://www.epa.gov/scipoly/sap/. 

5

PANEL ROSTER

FIFRA SAP Chair

Stephen Klaine, Ph.D. 

Professor, Department of Biological Sciences 

Director, Institute of Environmental Toxicology

Clemson University

Clemson, SC

Designated Federal Official

Fred Jenkins, Jr., Ph.D.

U.S. Environmental Protection Agency

Office of Science Coordination and Policy

FIFRA Scientific Advisory Panel

EPA East Building, MC 7201M

1200 Pennsylvania Avenue, NW 

Washington, DC 20460

Phone: (202) 564-3327

jenkins.fred@epa.gov

FIFRA Scientific Advisory Panel Members

Kenneth Delclos, Ph.D.

Research Pharmacologist

Division of Biochemical Toxicology

National Center for Toxicological Research

U.S. Food and Drug Administration

Jefferson, AR

Marion Ehrich, Ph.D., D.A.B.T., A.T.S.

Professor of Pharmacology and Toxicology

Department of Biomedical Science and Pathobiology

Virginia-Maryland Regional College of Veterinary Medicine

Virginia Polytechnic Institute and State University

Blacksburg, VA

David A. Jett, Ph.D.

Director, National Institute of Health CounterACT Program

National Institute of Neurological Disorders and Stroke

National Institutes of Health

Bethesda, MD
6

James McManaman, Ph.D.

Professor and Chief

Section of Basic Reproductive Sciences

Department of Obstetrics and Gynecology, Physiology

and Biophysics

University of Colorado Denver

Aurora, CO

FQPA Science Review Board Members

Russell Carr, Ph.D.

Associate Professor 

College of Veterinary Medicine

Mississippi State University

Mississippi State, MS

Veronica Berrocal, Ph.D.

Assistant Professor of Biostatistics 

Department of Biostatistics

School of Public Health

University of Michigan

Ann Arbor, MI

Muhammad Hossain, DVM, MS, Ph.D.

Neurotoxicologist 

Department of Environmental and Occupational Medicine 

Robert Wood Johnson Medical School 

Rutgers University 

Piscataway, NJ

Allison Jenkins, MPH

Senior Toxicologist 

Toxicology Division of the Texas Commission on Environmental Quality (TCEQ)

Austin, TX

Teresa Leavens, Ph.D.

Independent Pharmacokinetic Consultant

Cary, NC 

Igor Linkov, Ph.D.

Team Leader

Risk and Decision Science Focus Area Lead

US Army Engineer Research and Development Center

Concord, MA 
7

Thomas Murray, Ph.D.

Associate Vice Provost for Research and Scholarship

Professor and Chair

Department of Pharmacology

Creighton University School of Medicine

Omaha, NE

Isaac Pessah, Ph.D.

Associate Dean of Research and Graduate Education, Professor

Department of Molecular Biosciences 

University of California Davis School of Veterinary Medicine 

Department of Veterinary Medicine: Molecular Biosciences 

University of California 

Davis, CA 

Bingfang Yan, Ph.D.

Professor and Department Chair of Biomedical and Pharmaceutical Sciences 

Director of the Center for Integrated Drug Development 

University of Rhode Island 

Kingston, RI
8

LIST OF COMMONLY USED ACRONYMS AND ABBREVIATIONS

AOP: Adverse Outcome Pathway 

AP: Apical 

ASR: Acoustic Startle Response 

AUC: Area Under Curve 

BBB: Blood-brain barrier 

BL: Basolateral 

CAPHRA: Council for the Advancement of Pyrethroid Human Risk Assessment 

CSAF: Chemical specific adjustment factor 

CE(S): Carboxylesterase(s) 

CPPGL: Cytosolic protein per g liver 

CSPA: Consumer Specialty Products Association, Inc. 

CYP: Cytochrome P450 

DCO: Detailed Clinical Observations 

DLM: Deltamethrin 

fu: fraction unbound 

GC-MS: Gas chromatography-mass spectrometry 

GI: Gastrointestinal 

HTS: High-throughput screening (HTS) 

ISEF: Inter-system extrapolation factor 

LW: Liver weight 

MPPGL: Microsomal protein per g liver 

IVIVE: In Vitro to In Vivo Extrapolation 

PBPK: Physiologically-based pharmacokinetic modeling 

Papp: Permeability coefficient 

PC: Partition coefficients 

PD: Pharmacodynamic
9

INTRODUCTION

On May 19-21 the US EPA Federal Insecticide, Fungicide, and Rodenticide Act Scientific 

Advisory Panel (FIFRA SAP) met in Crystal City, VA to consider and review scientific issues 

associated with “Research to Evaluate the Potential for Juvenile Sensitivity to Pyrethroids.” The 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) authorizes EPA to register 

pesticides and the Federal Food, Drug, and Cosmetic Act (FFDCA) gives the Agency the 

authority to establish tolerances for residues on food and/or feed resulting from use of a 

pesticide. The studies required to allow the Agency to make the appropriate statutory safety 

findings under both of these acts are stipulated under 40 Code of Federal Regulations (CFR) Part 

158. There is flexibility, however, in implementing Part 158. Additional data can be required 

(§158.75), alternative approaches can be accepted, and studies can be waived (§158.45). The 

2007 National Research Council (NRC) report from the National Academy of Sciences (NAS) 

on Toxicity Testing in the 21st Century describes this new vision for toxicity testing. In response 

to the NRC report, EPA’s Office of Pesticide Programs (OPP) developed a Strategic Direction 

for New Pesticide Testing and Assessment Approaches 

(http://www.epa.gov/pesticides/science/testing-assessment.html) which describes OPP’s 

approach to implementing the NAS vision. One of the key components of OPP’s Strategic 

direction is improved approaches to more traditional toxicity tests to minimize the number of 

animals used while expanding the amount of information obtained. OPP also has a recent 

document, Guiding Principles for Data Requirements 

(http://www.epa.gov/pesticides/regulating/data-require-guide-principle.pdf) which describes the 

principles for requiring toxicology data for pesticides, specifically 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.” 

OPP is actively working on a reevaluation of the human health effects of the pyrethroids and 

pyrethrins under the OPP registration review program 

(http://www.epa.gov/oppsrrd1/registration_review/index.htm), required under FIFRA. Until late 

2009, OPP requested developmental neurotoxicity (DNT) studies for pyrethroids. However, the 

Agency determined that the DNT studies were not providing adequate data to evaluate the 

potential for post-natal sensitivity to pyrethroids. In July, 2010, the FIFRA Scientific Advisory 

Panel (SAP) reviewed a proposed research strategy to assess the potential for juvenile sensitivity 

consistent with the recommendations of the NAS in its report on Toxicity Testing in the 21st

Century using a combination of in vitro studies, targeted in vivo studies, and physiologicallybased pharmacokinetic (PBPK) models. 

Based on feedback from the SAP and the Agency, the industry research proposal was revised. 

Since late 2010, the Council for the Advancement of Pyrethroid Human Health Risk Assessment 

(CAPHRA) has worked with industry and academic scientists to develop assays and models to 

assess the potential for juvenile post-natal sensitivity to pyrethroids. The on-going research effort 

is organized around the adverse outcome pathway (AOP) for pyrethroids alterations with 

voltage-gated sodium channels (VGSC), leading to alterations in membrane excitability and 

firing potentials and ultimately to in vivo clinical syndromes. Specifically, the CAPHRA is 

evaluating the potency of pyrethroids to human sodium channels and transplantation of adult & 

juvenile rat synaptic membrane into oocytes. In addition, the CAPHRA is conducting targeted in 

vivo studies on behavioral metrics and developing PBPK models. The research, thus far, has 
10

focused on development of the overall approach using data for deltamethrin and permethrin 

(Type II and Type I pyrethroids, respectively). The CAPHRA research is at a point where 

feedback on extending this research to the other pyrethroids would be constructive. The 

CAPHRA proposal is to use the knowledge gained with deltamethrin and permethrin to develop 

more targeted datasets using read across and computational approaches (i.e., less data 

generation) for other pyrethroids. As such, the Agency sought the FIFRA SAP’s advice on the 

current state of the science with the CAPHRA research effort and proposals for next steps which 

include extension of data on deltamethrin and permethrin to other pyrethroids. 

PUBLIC COMMENTERS

There were no oral or written public comments provided for this meeting.
11

OVERALL SUMMARY 

The Panel was charged with advising the Agency on 8 topics areas regarding the development of 

assays and models to assess the potential for juvenile post-natal sensitivity to pyrethroids

insecticides. The following provides a summary of the Panel’s advice for each topic area. 

• Topic 1) High-throughput screening studies using human sodium channels expressed in 

mammalian cells with their regulatory beta subunits (ChanTest Data)- The Panel 

concurred that the proposed high-throughput screening approaches would not be useful in 

depicting the potential for juvenile sensitivity from pyrethroids. They noted that there 

were several factors that diminished the strength of the data (e.g. the large variability in 

protein expression and many of the electrophysiological parameters).

• Topic 2) Neurolemma studies: Transplantation of adult & juvenile rat brain synaptic 

membrane into (Xenopus) \oocytes- The Panel expressed a lack of confidence in the 

results of these studies due to various factors (e.g. the small sample sizes of the studies).

• Topic 3) Targeted in vivo studies in adult and juvenile rat: acoustic startle/detailed 

clinical observation: Generally, the Panel thought that the study was well designed. 

However, they noted that several key pieces of information regarding the studies were 

omitted. For example rationale for the use of male Sprague Dawley (SD) Rats and the 

dose selection was lacking. The Panel found that the detailed clinical observation (DCO) 

method is subjective and not a rigorous unbiased method. Relative to DCO, the 

automated acoustic startle response (ASR) or tactile startle response (TSR) may be better 

approaches, however, the Panel found that the use of the ASR for juveniles is problematic 

in some areas, and that further testing with the TSR is warranted. The Panel encouraged 

a continued investigation of the comparison of levels of neurotoxicity in young and adult 

rats with similar levels of exposure.

• Topic 4) Pharmacokinetic studies: The Panel commented that the CAPHRA’s efforts to 

develop a human PBPK model appeared to be on the right track for addressing the 

information needed. However, the Panel detailed a few deficiencies. . One deficiency 

noted was the lack of data in addressing age-related differences.

• Topic 5) Physiologically: based pharmacokinetic model in rat- The Panel had concerns 

regarding the confidence, accuracy, and uncertainty associated with this model. They also 

noted that the model appeared to be in a preliminary form. They also recommended 

further research to better validate the model.

• Topic 6) Physiologically: based pharmacokinetic model for human-The Panel noted 

several concerns regarding the structure of the model. Consequently, the Panel thought 

that there was insufficient evidence to comment on the reliability, confidence and 

uncertainty of the age-related brain Cmax predictions of the model.

• Topic 7) Physiologically: based pharmacokinetic models for humans with other 

pyrethroids- The Panel concurred that using the PBPK model to evaluate other 
12

pyrethroids was not yet warranted as the model, according to the Panel judgment was still 

under development. 

• Topic 8) Integration of lines of evidence: The Panel expressed doubt that the proposed rat 

model is a good model to learn about the response of human brains to exposure to 

deltamethrin. They recommended exploration of alternative experiments to learn about 

internal dosimetry. Also the Panel recommended the consideration of the Weight of 

Evidence (WOE) approaches that have been recently developed by the Organization for 

Economic Co-operation and Development (OECD) for developing Adverse Outcome 

Pathways (AOPs) (OECD, 2014, Becker et al., 2015).
13

EXECUTIVE SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS

1. High-throughput screening studies using human sodium channels expressed in 

mammalian cells with their regulatory Beta subunits (ChanTest Data): Human voltagegated sodium channels (NaVs 1.1, 1.2, 1.3, 1.6) were expressed in human embryonic kidney 

(HEK) cells and 9 pyrethroids were tested for their effects on channel conductance, as well as 

the effects of co-expressing the beta 1 and 2 regulatory subunits. 

a. Please comment on the ChanTest studies conducted for nine pyrethroids. Please include in 

your comments a consideration of their robustness (i.e. reproducibility, controls, statistics, 

background information, NaV selection, etc). Please comment on the confidence and 

uncertainties in the ChanTest experiments and related findings.

The FIFRA SAP Panel unanimously agreed that the ChanTest approach is unlikely to contribute 

useful information to characterize the potential for juvenile sensitivity of pyrethroids. The 

approach is fundamentally limited by the heterologous expression system where the NaV 

isoforms are over-expressed out of context of their native cell environment, and exposed to acute 

challenges to most pyrethroids above their solubility range. Experimental factors that weakened

the robustness of the data included large variability in protein expression and many of the 

electrophysiological parameters measured including peak current (Ipeak), normalized tail current, 

and persistent (late) sodium current. Presumably the variability in the ChanTest approach 

extends to all NaV isoforms and each of the electrophysiological parameters measured. Such 

variability limits the usefulness of the ChanTest approach to measure small to moderate 

differences in whole cell current parameters that are necessary before conclusion can be drawn 

about differential influences on NaV isoforms and juvenile toxicity. Importantly, many of the 

most acutely toxic pyrethroids in vivo failed to show detectable influences on NaV channel 

parameters, bringing into question whether the approach actually reflects an outcome relevant to 

the pyrethroid AOP. 

A more defensible approach for high-throughput screening would be to implement primary 

neuronal networks in cultures isolated from juvenile rodent or human iPSC derived neuronal 

cells grown in culture. Secondly measure electrical network activity or patterns of synchronous 

Ca2+ oscillations, which are tightly coupled to membrane electrical activity as an integral part of 

the AOP.
14

b. CAPHRA thinks that additional data from this line of evidence are unlikely to contribute 

useful information to characterize the potential for juvenile sensitivity. Thus, the CAPHRA 

has proposed NOT to continue this line of research. Please comment on this proposal and 

degree to which the ChanTest data inform the issue of evaluating the potential for juvenile 

sensitivity.

The Panel generally agreed with CAPHRA that the ChanTest approach using expressed NaV

subunits, with or without ß1and ß2 subunits, does not provide useful information about the 

potential of differential juvenile toxicity.

c. The ChanTest experiments focus on human sodium channels and generally show weak 

response. In contrast, in vitro studies in rodents in the literature (e.g. Choi and Soderlund, 

2006; Meacham et al., 2008; Tan and Soderlund, 20091) show stronger responses to 

pyrethroids. Please comment on the extent to which the expressed human sodium channels 

can be used in combination with these sources to infer relative pharmacodynamic sensitivity 

between rats and humans. 

The Panel unanimously concluded that the ChanTest experiments do not sufficiently 

discriminate among the electrophysiological properties of expressed NaV isoforms. Moreover, 

the ChanTest approach does not have the sensitivity to identify modest to moderate differential 

influences of pyrethroids of sodium channel gating kinetic parameters. This is a significant 

concern since the large range of acute neurotoxicological potencies of the pyrethroids examined 

seem to be largely discordant from their apparent activities towards the NaV isoforms measured 

by ChanTest. The data reporting stronger response of rat NaV to pyrethroids from the cited 

literature cannot be used in combination with the ChanTest data because the cited data were 

obtained from a non-mammalian expression system (i.e., Xenopus oocytes) which is likely to 

have differences in protein-protein interactions, have divergent membrane composition, and/or 

differences in posttranslational modifications, especially protein phosphorylation status. Any and 

all of these factors could influence interactions between pyrethroids and NaVs thereby making 

comparisons difficult and possibly misleading. The weak effects of pyrethroids in the ChanTest 

protocol and the lack of neuronal and developmental contexts are unlikely to add knowledge 

about isoform sensitivity, unless the rat and humans clones are tested under identical 

experimental conditions with a more robust sample size. 

2. Transplantation of adult & juvenile rat brain synaptic membrane into Xenopus oocytes:

Purified neurolemma membranes from adult and juvenile rats were separately micro-injected 

into Xenopus oocytes. Patch clamp testing was performed on the oocyte membranes versus 

various doses of pyrethroids to determine their EC50 values for NaV channel activation. 

Inhibitors of competing channels were added to isolate the sodium channel conductance (i.e. 

chloride and calcium channels). 

 1 Choi JS and Soderlund DM. 2006. Structure-activity relationships for the action of 11 pyrethroid insecticides on rat Na v 1.8 

sodium channels expressed in Xenopus oocytes. Toxicol Appl Pharmacol. 2006 Mar 15;211(3):233-44. Epub 2005 Jul 26 

Meacham, C.A., et al., Developmentally-regulated sodium channel subunits are differentially sensitive to [alpha]-cyano 

containing pyrethroids. Toxicology and Applied Pharmacology, 2008. 231: p. 273-81. Tan, J. and D.M. Soderlund, Human and rat 

Nav1.3 voltage-gated sodium channels differ in inactivation properties and sensitivity to the pyrethroid insecticide tefluthrin.

NeuroToxicology, 2009. 30(1): p. 81-89.
15

a. Please comment on the synaptic membrane (“neurolemma”) studies conducted for 

deltamethrin and permethrin. Please include in your comments a consideration of the 

robustness of data from the synaptic membrane transplantation into oocytes (reproducibility, 

control compounds, channel modulator reagents, statistics, use of Na blocker tricaine as an 

anesthetic, etc) and the degree to which these data inform the issue of evaluating the potential 

for juvenile sensitivity. Please comment on the confidence and uncertainties and associated 

findings in the oocyte experiments.

The Panel believes that the neurolemma studies were conducted using appropriate techniques 

and methodology. The Panel applauds the scientists for providing a detailed and accurate 

documentation of the experiments and of the results found. Nonetheless, despite the 

appropriateness of the studies performed and the accuracy in documenting the oocyte 

experiments, the Panel is hesitant in placing a great amount of confidence in the findings of these 

experiments for three main reasons: the sample sizes used were too small, the estimated 

uncertainties both in the final results and in the intermediary analyses steps (e.g. the response 

concentration curves fit) are very large, and the primary data and measurements had to undergo 

several transformations in order to be compared. 

b. In the context of your response to 2a, the CAPHRA has proposed to collect dose-response 

data in adult and juvenile rat synaptic membrane transplanted into oocytes for five more 

pyrethroids (including Type I, Type II, and mixed). CAPHRA’s proposed path forward: If the 

additional five pyrethroids show similar patterns to deltamethrin and permethrin (i.e. no 

lifestage sensitivity observed), no additional pyrethroids will likely be tested in this 

system. Alternatively, if a different pattern is observed, additional pyrethroids are likely to be 

tested. Please comment on the CAPHRA’s proposed path forward.

The Panel acknowledged that age-related differences in the sensitivity of rat brain sodium 

channels could be studied using this system. However, the only age-related difference appears to 

be the lower number of sodium channels in the juveniles as compared to the adults. The system 

also suffers from other issues that lead to large uncertainties concerning the confidence in the 

findings. The Panel concluded that the usefulness of using this system for determining agerelated differences is minimal.
16

3. Targeted in vivo studies in adult and juvenile rat: acoustic startle/detailed clinical 

observations: Preliminary experiments that measure acoustic startle and detailed clinical 

observations have been conducted by the University of Cincinnati. In these experiments, 

juvenile and adult male Sprague-Dawley rats were treated with deltamethrin and permethrin 

as model Type II and Type I pyrethroids, respectively. In addition, brain and plasma 

concentrations were measured in PND 15 and 90 rats exposed to deltamethrin. The 

preliminary results suggest that juvenile rats are more sensitive to deltamethrin based on 

changes in detailed clinical signs and, to less extent, acoustic startle. However, there was no 

greater sensitivity in juvenile rats exposed to permethrin. Preliminary data from whole brain 

tissue samples with deltamethrin indicated that PND 15 rats had higher deltamethrin 

concentrations as compared to adult rats given the same dose. The brain concentration and 

toxicity data from the CAPHRA studies for deltamethrin are consistent to those previously 

published by the Bruckner lab (Kim et al., 20102

) which showed that at a similar dose (2 

mg/kg deltamethrin) PND 10 rats had increased severity of clinical signs and higher Cmax of 

deltamethrin in the brain as compared to PND 21, PND 40, and PND 90 rats.

a. The in vivo behavior studies reported thus far are preliminary evaluations. Please comment 

on the study design in the preliminary in vivo studies for deltamethrin and permethrin 

conducted at the University of Cincinnati (Vorhees lab). 

In general, the Panel believed that the study design was carefully planned and permitted rigorous 

statistical analysis. The Panel commented that more information on the selection of the Postnatal 

Day 15 (PND15) age in rats was needed, especially since the lack of sensitivity to the Acoustic 

Startle Response (ASR) observed in PND15 rats was not observed in PND17 and PND21 rats. 

The rationale behind the use of only male Sprague Dawley (SD) rats in the CAPHRA in vivo

studies was also not discussed in the study reports and should have been included. The Panel 

commented that the rationale for the dose selection was also insufficient for several studies, and 

questioned whether the dynamic range of doses was appropriate. 

The Panel also agreed that the description of the pretesting of animals in order to improve 

performance was sufficient, and a discussion of how this could affect the overall study design 

and results would have been beneficial. The Panel noted that the effect of litter should have been 

included in the statistical analysis since the dams were required to care for the pups during transit 

and the shipping, and this could have induced some level of stress in the dams. Finally, there was 

no chemical characterization information provided for permethrin.

 2 Kim KB, Anand SS, Kim HJ, White CA, Fisher JW, Tornero-Velez R, Bruckner JV. 2010. Age, dose, and time-dependency of 

plasma and tissue distribution of deltamethrin in immature rats. Jun;115(2):354-68. doi: 10.1093/toxsci/kfq074. Epub 2010

Mar 8.
17

b. In 2010, FIFRA SAP commented on some challenges that were anticipated with the use of 

auditory startle to compare the relative sensitivity of juveniles and adults. In the studies 

conducted by the CAPHRA, both auditory startle and detailed clinical observations were 

evaluated. Please comment on the degree to which the auditory startle and detailed clinical 

observations provide useful data for evaluating the potential for juvenile sensitivity. Please 

include in your comments discussion of the dynamic range; the direction of the response 

varying between Type I and IIs; and the type of data obtained (continuous, ranked).

The Panel remarked that one study indicates that the Tactile Startle Response (TSR) test provides 

a somewhat larger dynamic range; however, more studies may need to be done across ages, 

compounds and doses, to justify including this paradigm at this late stage. The use of ordinal 

Detailed Clinical Observations (DCO) data is valuable as secondary observation, but it is 

subjective and not a rigorous unbiased method for decisions related to hazard assessment across 

many compounds. Relative to DCO, the automated ASR (or TSR) approach is a better primary 

method for assessing in vivo toxicity. However one panel member noted that the DCO 

observations, and tremors in particular, might better fit the “adverse” description as opposed to a 

compound effect. For the PM pre-weaning results, there seems to be no clear relationship 

between the magnitude of the tremor response and the magnitude of the ASR response. With 

respect to the ASR data for deltamethrin (DLM), CAPHRA concluded that DLM did not induce 

statistically significant effects on ASR in PND90 rats (Figure 2-5a) while DLM induced 

statistically significant effects on ASR in PND15 pups (Figure 2-5b). Shortcomings in the 

statistical analysis of these data decreased the confidence of the Panel concerning the absence of 

significant effects on ASR in PND90 rats exposed to DLM. 

The ASR has a history as a measure of pyrethroid neurotoxicity and data generated should be 

comparable to values in the literature. However, the predictability of the ASR model to adverse 

effects in humans, published or hypothesized, has not been established. The Panel commented 

that tremors and/or writhing in rats might be more sensitive and relevant to the human condition. 

Another primary question is whether ASR is linked to any known mechanism of action for 

pyrethroids, including their influences on sodium channels, or other ion channels. Overall it does 

appear that use of the ASR for juveniles is problematic because of the overlap with non-specific 

effects. Going forward, the focus should be on further testing with the TSR, either alone or in 

combination with the ASR, to see if the test is sensitive enough to separate out the specific 

effects from the non-specific effects. 

c.Please provide comments comparing the temporal pattern and magnitude of the brain and 

plasma concentration data from the Vorhees & Bruckner labs and utility of such data to aid in 

the interpretation of the auditory startle and detailed clinical observation data. 

For DLM, the time of the rise in plasma and brain levels does not coincide with the peak in ASR 

effects, but the trends are consistent perhaps with slight delays. It is not expected that these 

should match perfectly since there are several unknown factors involved in the potential doseand time-related mechanisms underlying the effect of DLM on ASR.

A plan to continue investigating the comparison in levels of neurotoxicity in young and adult rats 

with similar levels of exposure (concentrations) within the brain may be appropriate. If the data 
18

support the findings available to date, the Panel would concur that there would be no further in 

vivo work needed to resolve the pharmacodynamics question.

The Panel noted that it may be important to examine the effects of pyrethroids in young and old 

rats at a matched internal dose (e.g. brain/plasma concentration), but the difficulties in obtaining 

a high enough dose in adults and the temporal variability in these doses may be prohibitive. This 

difficulty must be weighed against the added value of these data to the overall question of 

whether young rats/humans are more affected by a given external dose, regardless of intrinsic 

differences. The Agency will need to consider what information will be critical for the final 

hazard assessment and regulatory activity.

d. Please comment on the use of a 5 ml/kg dosing volume in the CAPHRA studies and how any 

impact on pyrethroid kinetics affects correlations with behavioral effects. 

The Panel concluded that the dose volume does not seem to appreciably influence the DLM 

experiments in terms of the correlation to the timing of ASR effects. The Agency should 

consider the question: Is a slower release and delayed or extended period of the ASR desirable? 

For example, is it better to give a smaller volume and test at 2 hours, or larger volume and test at 

4 or 6 hours? The answer may lie in the optimal study design for comparable results across 

compounds, as well as how best to design the model in order to simulate exposure in humans. 

The Panel questioned the justification of using 5 mL/kg in the CAPHRA studies given that 

previous studies in the literature used 1 mL/kg. CAPHRA stated that the methods were 

developed to allow for the testing of additional pyrethroids with very low toxicity that would 

require very high dosages. Considering the Agency goal to modify toxicity testing not only to 

reduce animal use but also to test more human relevant doses, it may be worth considering if it 

would really be necessary to push the doses of low potency compounds to such high levels. 

Panel members agreed that there is a concern that doses administered to a pup with a full 

stomach of milk may be problematic. It was stated that 15-day old rat pups were not removed 

from the dam prior to dosing which did not allow gastric emptying. It is possible that the milk in 

the stomach altered the absorption and pharmacokinetics of the pyrethroid. 

4. Pharmacokinetic studies: A number of pharmacokinetic studies using deltamethrin were 

performed by the CAPHRA in order to further refine and validate the developing rat PBPK 

model and construct a developing human PBPK model. For refinement of the rat PBPK 

model, tissue: plasma partition coefficients (in vivo measurements in PND 21 and adult rats), 

age dependent plasma protein binding (in vivo measurements in PND 10, 15, 21, and 90 rats), 

and cytochrome P450 (CYP) and carboxylesterase (CES) metabolism (in vitro measurements 

using rat liver and plasma preparations from PND 15, 21, and 90 rats) were evaluated. 

Further in vivo pharmacokinetic studies in rats were conducted with single IV (PND 90), 

single oral (PND 90), and multiple oral (PND 15, 21, and 90) doses to generate plasma and 

tissue data to validate the developing rat PBPK model. Additional experiments with 

deltamethrin were conducted for constructing the developing human PBPK model. 

Parameters evaluated in vitro included age dependent plasma protein binding derived in 

plasma from human donors aged from birth to adults, transport across the blood-brain 
19

barrier using a human brain microvascular endothelial cell line, estimates of gastrointestinal 

absorption in caco-2 cells, and ontogeny data for CES, CYP1A2, and CYP2C8 determined in 

human liver tissue from donors (age 1-18 years old). Further in vitro experiments are 

currently underway by the CAPHRA to determine human adult and juvenile liver metabolism 

of deltamethrin by CYP and CES enzymes using human liver preparations or recombinant 

human enzymes.

a. Please comment on the in vitro experiments to support the PBPK model development in the 

rat conducted thus far for deltamethrin. 

Based on the needs of information for the development and validation of the PBPK model for 

deltamethrin, the in vitro work conducted so far by CAPHRA appears to be relevant towards the 

stated goal of constructing a developing human PBPK model. CAPHRA has made considerable 

progress with proposed kinetic studies for deltamethrin, and, overall, the Panel thought that the 

experiments completed or underway were excellent in quality and appropriate for addressing the 

age-dependence of liver and plasma metabolism and protein binding. The age-dependence of 

these kinetic factors is particularly important to be captured in the model, and the experiments 

have quantitated important issues that are critical for the In Vitro- In Vivo Extrapolation (IVIVE)

approach. The Panel recommended that the CAPHRA program should complete the propose 

metabolism work, in particular utilizing enzyme preparations from various age groups. The Panel 

identified a few deficiencies in the work including the lack of data or experiments addressing 

age-related differences in permeability and active transport at the blood-brain barrier, lack of in 

vitro studies that addressed possible age-related changes in the oral absorption of deltamethrin, 

lack of studies to address potential sex-specific differences in kinetics, and lack of studies to 

address extrahepatic metabolism.

b. Please comment on the in vivo experiments to support the PBPK model development in the rat 

conducted thus far. 

The CAPHRA program systematically carried out and collected high quality data for numerous 

toxicokinetic and tissue distribution studies that had several strengths including: (a) use of 

various age-groups to determine age-related changes in toxicokinetics; (b) administration of 

deltamethrin by intravenous and oral dosing to establish bioavailability; and (c) dosing over a 

range of deltamethrin concentrations doses to provide important information on the 

detoxification capacity (saturation). However, the Panel expressed some concerns about the 

study design and the data analysis, including the lack of repeated exposure studies, lack of 

information regarding sex selection for the study, lack of adequate discussion for results from 

studies, and lack of adequate quantitation of bioavailability. Two issues that generated significant 

discussion among Panel members were the potential influence of vehicle on absorption and 

subsequent impacts on lymphatic versus portal system distribution. This is an important issue 

because the assumption of bypassing the liver in the approach described may have influenced 

modeling results, which predicted a higher brain Cmax in the adults than in children.

c. Please comment on the in vitro experiments in the human tissue conducted thus far. Please 

include in your comments a discussion of the ongoing in vitro experiments with recombinant 
20

enzymes for use in PBPK models and associated confidence and uncertainty with the use of 

such data.

The Panel noted the approach that CAPHRA was using for the IVIVE with recombinant 

enzymes seemed to be thorough and a logical method strategy to predict metabolism in the liver 

across age groups. The Panel noted several areas of confidence for the approach including the 

following: ontogeny data on human liver carboxylesterases; ontogeny data on CYP2C8 and 

CYP1A2 in the liver; and, determination of enzyme kinetics with recombinant CYPs and CESs. 

However, the Panel had concerns about some aspects of the study that would generate 

uncertainty in the use of the data. These included lack of estimation in hepatic clearance in 

primary human hepatocytes from various age populations (cryopreserved hepatocytes), and, use 

of different laboratories to determine ontogeny expression, which adds another source of 

variability to the data.

5. Physiologically-based pharmacokinetic model in rat: Using data described in Question 4, 

the CAPHRA has developed a PBPK model for deltamethrin using age-specific metabolism 

parameters in rats to simulate plasma and brain internal exposures in young and adult rats. 

This PBPK model relies on in vitro to in vivo extrapolation (IVIVE) to use age-specific 

metabolic data collected in vitro to estimate hepatic metabolic clearance in vivo. The 

deltamethrin brain or plasma concentrations estimated by the model in rats are then 

compared to measured concentrations from in vivo rat studies to verify the model. Nonchemical specific physiological parameters for rats were obtained from the published 

literature, including body weight, cardiac output, hematocrit levels, tissue volumes, and tissue 

blood flows. As discussed in Question 4, recently generated data by the CAPHRA and 

published data were used for compound-dependent parameters, including partition 

coefficients, metabolic rate constants, absorption rates, protein binding, compartments and 

tissue permeability. 

a. Please comment on the robustness of the rat PBPK model for simulating internal exposures in 

the developing rat. In your response, please include evaluation of the structure and 

parameters used to build the model, as well as its ability to accommodate different oral 

absorption scenarios (i.e. different vehicles used for in vivo studies) and discussion of 

confidence, accuracy and uncertainties associated with the deltamethrin developing rat 

model. Please also comment on the sensitivity analyses of parameters CAPHRA has 

completed thus far.

While the Panel recognized the fluidity of the modeling process and commended the researchers 

and modelers on the iterative nature of the experimental work with model development, the 

Panel were reticent to comment on the confidence, accuracy and uncertainty of a model that did 

not appear to be in the final form. The PBPK model structure was based upon the model by 

Tornero-Velez et al. (2010) and was expanded to account for vehicle-specific absorption of 

deltamethrin, plasma metabolism age-specific plasma protein binding, and the metabolic 

contribution of CES to pyrethroid hydrolysis. The Panel felt the incorporation of this additional 

information increased the confidence and accuracy of the model, however, the Panel expressed 

concern regarding the adequacy of the model structure and parameter values due to lack of 

consistency of the model predictions with observed data and experimental data to support certain 
21

model structures (e.g. lymphatic versus portal uptake of deltamethrin). The lack of a quantitative 

assessment of the goodness of fit adequacy of fit for the absorption parameters decreased the 

confidence in the model predictions. One parameter, which appeared to be influential and for 

which no age-dependent changes were incorporated was the permeability-surface-area cross 

product in the brain. This appeared to be a parameter that was highly uncertain and the Panel 

recommended that potential age-related changes in the permeability of the blood-brain barrier 

should be investigated and incorporated in the model. The Panel was unable to provide 

comments on the sensitivity analysis because information was not provided in the overview 

documents or presentations for the rat model. The Panel did recommend that a global sensitivity 

analysis, in addition to a local analysis, be performed for the modeling work.

b. In the context of your response to 5a, please comment on the extent to which additional data 

are/are not needed to refine the developing rat model.

The Panel recommended several studies that are needed to better validate the predictions of the 

current model as well as provide further refinement with respect to predicted brain concentration 

of deltamethrin in older versus younger rats. These include repeated dosing of pyrethroids to 

look at the potential accumulation in the brain, studies to determine lymphatic versus portal 

absorption, quantitation of bioavailability, and, evaluation of age-dependent differences in the 

blood-brain barrier. Based on the discussions of the Panel during the meeting, the recommended 

studies will provide important data to have confidence in the use of the PBPK model for risk 

assessment for children.

6. Physiologically-based pharmacokinetic model for human: Similar to the rat PBPK model, 

the human PBPK model integrates non-chemical specific physiological parameters for 

humans from the literature. Compound-dependent parameters, such as partition coefficients 

and oral absorption parameters, were adapted from the rat PBPK model. Recently generated 

data by the CAPHRA were used for the remaining compound-dependent parameters, such as 

metabolic rate constants, protein binding and tissue permeability. With respect to metabolic 

constants, CYP and CES enzymes involved in metabolism of a given pyrethroid will be 

identified and the in vitro metabolic constants for those enzymes will be determined for 

integration into the PBPK model. Intrinsic clearance for each active enzyme will be scaled to 

in vivo using scaling factor data collected by the CAPHRA and the SIMCYP database. The 

ontogeny of enzyme expression (also from the CAPHRA data and the SIMCYP database) will 

be incorporated into the process of obtaining distributions of age-specific intrinsic clearance 

for each enzyme. Preliminary simulations have been conducted for deltamethrin to 

demonstrate the process used with the PBPK model. 

a. Within the context of understanding potential juvenile sensitivity, characterize the robustness 

of the PBPK model for extrapolating age-specific internal tissue exposures for humans. In 

your response, please comment on the structure and parameters used to build the model and 

include discussion of confidence, reliability, and uncertainties associated with the 

deltamethrin human model. Please include in your comments a discussion of the data from 

the McCarver/Hines Laboratory (submitted as part of the CAPHRA package) for providing 

ontogeny of CES enzymes.
22

The human PBPK model was developed with the intent of predicting human tissue exposure to 

deltamethrin. Parameters of the model included body weight, tissue volume, tissue body weight 

fraction, tissue permeability, partition coefficients, etc.

While appreciating CAPHRA’s effort to develop a human PBPK model, the Panel had several 

concerns, mostly regarding the model structure. Relative to the model parameters, the Panel 

highlighted the fact that there were discrepancies between the model parameters reported in the 

presentation and the overview documents making it appear as if the model was still under 

development.

For the model structure, the major concerns of the Panel were relative to the parameterization of 

the human brain in the PBPK model, especially in light of its influence on the Cmax predictions, 

and, the pathway of absorption that is structured to completely bypass the liver and portal system 

by switching to the lymphatic system. The Panel believed that the differences between rats and 

humans pose a major challenge especially with respect to the metabolism of deltamethrin. 

Further, the Panel was concerned about the capacity of the human PBPK model to reproduce the 

large variability observed among humans and was perplexed about the usage of data from 1-year 

olds and the use of the Monte Carlo approach since neither was justified or documented..

The Panel suggested revisions to the PBPK model focused on the definition of clearance, the 

definition and usage of age grouping when modeling the liver function, the usage of western blot

analyses,, and the determination of internal concentrations within an organ. Due to the various 

concerns relative to the model structure and status of the model parameter calibration, the Panel 

did not believe it had sufficient evidence to comment on the reliability, confidence, and 

uncertainty of the age-related brain Cmax predictions. 

b. Please comment on the proposed use of SIMCYP for providing enzyme ontogeny patterns and 

deriving population distributions for metabolic parameters in humans. Please include in 

your comments whether or not other tools with similar capacity to SIMCYP are available.

The Panel recognized that there is general acceptance of the SIMCYP Simulator by the modeling 

community as a tool to provide enzyme ontogeny patterns and derive population distributions for 

metabolic parameters in humans. On the other hand, the Panel pointed out potential challenges 

for the pediatric modeling of pyrethroids due to high-levels of protein binding, extensive 

metabolism by multiple enzyme systems, and potential involvement of active transport. In 

addition, there are knowledge gaps regarding the metabolism of pyrethroids in humans. These 

knowledge gaps include but are not limited to: (1) the involvement of both hydrolysis and 

oxidation in metabolism (whether and to which extent these two elimination pathways 

differentially contribute to the overall clearance of a pyrethroid is not clear); (2) the presence of 

both hydrolysis and oxidation enzymes in the liver and extrahepatic tissues (there is no 

information on organ-specific clearance of a pyrethroid, as well as age-dependency in this 

regard); and, (3) the potential for differential induction of cytochrome P450s over 

carboxylesterases in humans by pyrethroids, which are activators of pregnane X receptor (how 

the differential induction might lead to changes of the contribution of respective elimination 

pathways is not clear). The Panel also recommended that an alternative modeling platform 

should be used as a comparison with the SIMCYP Simulator. 
23

7. Physiologically-based pharmacokinetic models for humans with other pyrethroids: Thus 

far, the CAPHRA has focused its human PBPK efforts on deltamethrin and, to a lesser extent, 

permethrin. Soon laboratory efforts will turn to other pyrethroids. CAPHRA’s proposal is to 

conduct fewer studies and in lieu of such data, use read across and computational 

approaches.

a. Please comment on the appropriateness of the current human PBPK model to be used for 

other Type I, Type II, or mixed type pyrethroids. Please include in your response evaluation 

of the path forward provided by the CAPHRA regarding in vitro and in vivo studies for other 

Type I, Type II, or mixed type pyrethroids.

While the Panel provided comments on the appropriateness of the use the PBPK model to read 

across for different pyrethroids, it also pointed out that these comments and recommendations 

were provided on a concept that had not yet been thoroughly vetted. The model appeared to still 

be under development and the experimental work has not yet been completed. In theory, if the 

issues regarding rat and human models were addressed to decrease uncertainty in the models’ 

structures and increase accuracy of the models’ predictions, the Panel thought a read across with 

chemical-specific parameters would be appropriate. The Panel did not anticipate that the 

pharmacokinetic model structure would need to be different for the different pyrethroids; 

however, the parameter values would differ. As pointed out by one panel member, one challenge 

of modeling these compounds is the variability in metabolism, and that the metabolic clearance 

for individual pyrethroid compounds must be determined to allow models to be developed with 

accurate predictions. The Panel made several recommendations regarding the proposed path 

forward that was presented by CAPHRA with focus on partition coefficients, permeabilitysurface-area cross products, in vitro kinetics since isomeric forms will likely differ, and 

evaluation of PBPK models for all pyrethroids for which data is generated. The Panel also 

commented that the Adverse Outcome Pathway approach is a combination of pharmacokinetics 

and pharmacodynamics and both have to be adequately understood to read across (i.e. 

extrapolate) to other compounds. This charge question focused only on extrapolation of the 

pharmacokinetics across the family of pyrethroids.

8. Integration of lines of evidence:

a. The tissue dosimetry data from the rat suggest higher brain levels in juveniles compared to 

adults. In contrast, the preliminary PBPK modelling for human predicts slightly lower brain 

concentrations in young children compared to adults. Please comment on these differences, 

including comments on the key inputs that lead to this difference, human variability 

associated with the key parameters, and the confidence and uncertainties associated with the 

difference between the rat and human internal dosimetry.

The Panel raised several concerns regarding the human PBPK model in previous charge 

questions. Of particular concern was the model structure and parameters (e.g. brain permeability 

surface area cross product and blood flow to the liver) that highlighted the large uncertainties 

surrounding the findings obtained thus far. Therefore, the Panel was not confident in the 
24

conclusions that CAPHRA has reached with respect to the differences between rat and human 

internal dosimetry. 

The Panel strongly questioned the rat as a good model to learn about the response of the human 

brains to exposure to deltamethrin and suggested CAPHRA look into alternative experiments to 

better quantify internal dosimetry. In particular, the Panel suggested CAPHRA explore the 

possibility of using either guinea pigs or ES1 plasma carboxylesterase knockout mice as 

potentially more appropriate animal models for extrapolation to human. 

Further, to pursue a weight of evidence approach and combine findings from multiple disparate 

experiments, the Panel recommended considering the quantitative Weight Of Evidence (WOE) 

approaches that are being formalized by the Organization for Economic Co-operation and 

Development (OECD) for developing Adverse Outcome Pathways (AOPs) (OECD, 2014, 

Becker et al., 2015). 

b. The CAPHRA has proposed to continue in vivo behavioral testing for deltamethrin and 

permethrin in definitive dose-response evaluations. Pyrethroids have been studied for 

decades and thus there is a large body of evidence for these pesticides. Given your response 

to 8a along with 1) the extensive body of scientific literature on pyrethroid toxicity syndromes 

and high dose studies in juvenile rats (e.g., Sheets et al, 19943

); 2) neurolemma studies 

supported by the CAPHRA (Question 2) along with additional in vitro studies (e.g. Meacham 

et al., 2008); and 3) recent in vivo studies from the Vorhees & Bruckner labs, please comment 

on the additional scientific value that would be provided in conducting further in vivo rat 

experiments to assess potential for PD sensitivity of human infants and children.

From a general standpoint, the Panel thinks that CAPHRA’s plan to direct the research effort 

mostly on the pharmacokinetics aspect and the effort to increase the level of understanding of 

age-related metabolic differences between adults and novices seemed reasonable. However, the

Panel encouraged CAPHRA to reconsider the utility of the neurolemma studies, as the large 

uncertainties in the results seriously undermine the reliability of the results obtained, and the 

magnitude appear difficult to dissipate even with larger sample sizes. 

Moving forward, the Panel urged CAPHRA to: (i) Continue pursuing in vivo studies, focus on 

developing new assays, and determine a clinical endpoint that could assess the clinical relevance 

of deltamethrin (DLM) presence in the brain; (ii) Examine an additional type II pyrethroid, 

cyhalothrin, in addition to the core pyrethroids; (iii) Complete the permethrin studies, relating 

ASR and DCO response to brain levels, as was done for deltamethrin.

On a more general level, the Panel recommended that CAPHRA re-evaluate the approach 

undertaken thus far to evaluate the effect of exposure to pyrethroids on the nervous system. More 

specifically, the Panel suggested a more transparent approach that would: 1) review all the 

potential risks of pyrethroid exposure on juveniles, 2) define the degree or frequency of impact 

that is acceptable, 3) examine indications of potential endpoints and outcomes of disease 

states/potential impacts, and 4) provide the most definitive evidence for such endpoints. As an 

 3 Sheets, L.P., et al., Age-Dependent Differences in the Susceptibility of Rats to Deltamethrin. Toxicology and Applied 

Pharmacology, 1994. 126(1): p. 186-190.
25

illustration, the Panel suggested potential endpoints to consider for the nervous system including 

activation and impacts on neuronal development, DNA damage, oxidative stress, reduction of 

neurogenesis, and loss of dopaminergic (DA) neurons. Finally, the Panel believed that while the 

human pharmacokinetic model is a useful tool for estimating the potential for exposure, the 

information provided by this model needs to be paired with an understanding of the doseresponse relationship for an effect that implicates a specific outcome of a disease state.

c. If you believe there is additional scientific value to conducting additional in vivo experiments 

(8b), please comment on the CAPHRA's proposed experiments. In the context of your 

response to Question 3, please include in your comments a discussion of dose levels and/or 

additional study design elements to improve existing preliminary evaluations.

If in vivo neurotoxicity studies are continued, the Panel suggested that larger sample sizes be 

used and litter effects be considered, as proposed for further deltamethrin and permethrin studies. 

Relative to dose, the Panel recommended that 1 mg/kg dose be used in future deltamethrin 

studies, with unchanged time course and possible reduction in the number of trials per session to 

reduce habituation.

In regard to the use of corn oil as vehicle in previous experiments, the Panel suggested using the 

minimal volume necessary to enhance the rate of uptake and peak brain concentration in future 

experiments. Additional suggestions included: (i) performing additional experiments to monitor 

neuronal functions along with clinical observations of extracellular neurotransmitters release 

with brain microdialysis for better understanding age and dose dependent neurotoxicity of 

pyrethroids; (ii) considering ways to reduce tremor in rats as it might interfere with ASR; and 

(iii) examining how tissue concentration varies in the brain to provide better insight into what 

parts of the brain are more sensitive to pyrethroids. 
26

Detailed Panel Recommendations

1. High-throughput screening studies using human sodium channels expressed in 

mammalian cells with their regulatory Beta subunits (ChanTest Data): Human voltagegated sodium channels (NaVs 1.1, 1.2, 1.3, 1.6) were expressed in human embryonic kidney 

(HEK) cells and 9 pyrethroids were tested for their effects on channel conductance, as well as 

the effects of co-expressing the Beta 1 and 2 regulatory subunits. 

a. Please comment on the ChanTest studies conducted for nine pyrethroids. Please include in 

your comments a consideration of their robustness (i.e. reproducibility, controls, statistics, 

background information, NaV selection, etc). 

The Panel acknowledged that heterologous gene expression systems have been an invaluable tool 

for gaining a better understanding of how protein structure relates to physiological function. The 

value of ion channel expression has been especially informative in our quest to understand how 

specific regions of sequence contribute to specific aspects of ion channel function. This includes 

identifying domains essential for the millisecond to millisecond characteristics of ion channel 

currents, including activation, inactivation, deactivation, and the selectivity filter. In addition, 

heterologous expression models have helped identify key determinants of protein-protein 

interactions, some of which mediate post-translational modifications and longer-term adaptation 

(integration) to cellular activity. With respect to Voltage Gated Sodium Channel (VGSC), 

interactions with beta subunits appear to be important for optimal targeting of the NaV alpha 

subunit to the surface membranes and finer aspects of channel modulation. In this regard, NaVs

subunits can interact with any one of 3 beta subunits, but the degree to which they do largely 

depends on cellular context. 

Reproducibility

The Panel noted that comparative investigations based on expression of ion channel subunits in 

heterologous models must be tightly controlled in terms of the level of protein expression and 

targeting to the surface membrane. In the ChanTest case, the level of expression and targeting of 

each NaV isoform is not determined by western blotting or immunofluorescence. The highly 

variable (>10X based on blot shown) transient expression of the beta2 subunit β2 (Fig 1) and an 

unknown level of expression of the beta1 subunit β1 (antibodies did not work), suggest a lack of 

precision in the current measurements made by ChanTest. Lack of precision is evidenced by the 

>200% (-0.7-1.6nA) variation in Ipeak among NaV1.3 cells. The variability in NaV1.3/β1β2

expression may explain why Ipeak varies appreciably among cells (-1.4-2.7nA). Based on the 

information provided, and the degree of variability in the basic electrophysiological properties of 

each NaV clone (with or without electroporation of β1β2), comparisons among isoforms would 

permit only moderate to large differences to be discerned with confidence, whereas more subtle 

differences may go unresolved. In this regard, summary statistics are not given in any of the NaV

summary tables (Tables 1A&B; 2A&B).

The Panel raised several statistical concerns. For instance in regards to sample size, how many 

data points in each group are being compared? The Panel noted that the sample sizes that were 
27

presented were small, as low as n=3. When comparing the pyrethroids, there is uncertainty about 

the robustness of calculation for EC50s.

With regards to Controls

The Panel commented that inferences about the similarity or differences between human VGSC 

properties reported by ChanTest and those previously reported with the rat NaV1.3 expressed in 

oocytes are neither warranted nor valid unless the homologous rat NaV isoform lines (in the 

presence or absence of ß1ß2 electroporation) are recorded using the IonWorks Barracuda 

platform (at least NaV1.3 for validation). For valid cross species comparisons (human vs rat), it 

is necessary to make measurements under identical experimental conditions. The lack of such 

essential controls greatly weakens the usefulness of the ChanTest results with pyrethroids, 

especially when comparing results to other studies using completely different methodology.

For example, “CAPHRA concludes that results from 7 pyrethroids tested in this first phase are 

consistent with and extended the Tan and Soderlund (2009) data on tefluthrin showing that 

human VGSC are much less sensitive than those in the rat. Comparison of homologous rat and 

human, Nav1.3 VGSC isoforms showed that the rat isoform was 4-fold more sensitive than the 

equivalent human sodium channel to the pyrethroid tefluthrin. This suggests that, in contrast to 

the situation with rats, the pyrethroids tested are poor modulators of the human sodium channel, 

which in turn suggests that the rat is likely an overly sensitive model to predict the neurotoxicity 

of pyrethroids.”

However, the results from HEK cell expressed human clones vs. rat channels expressed in 

Xenopus oocytes show large divergence in inactivation times, with rat NaV1.3 channels

exhibiting a 3-fold larger fraction of channels exhibiting the slow component of inactivation and 

~3 fold slower kinetics at test pulse (Vt) of -15mV compared to human NaV1.3 (Fig 1C&D; Tan 

& Soderlund 2009). It is uncertain if this fundamental difference is a result of model differences 

or if they would be observed in native neuronal models (e.g., primary embryonic neurons from 

rat vs. human). If this difference is artificial it may drive the difference in pyrethroid sensitivity.

Key comparisons on inactivation parameters between human NaV1.3 expressed in HEK 

(ChanTest) and those reported by Tan and Soderlund (2009) are not possible because the data are 

not included in the ChanTest report. Furthermore recent work has demonstrated that tefluthrin 

and deltamethrin exert different effects on Nav1.6 channels expressed in HEK293 cells (He and 

Soderlund, 2011) and on Nav1.6 channels expressed in Xenopus oocytes (Tan and Soderlund, 

2010).

The Panel noted that the concentrations of pyrethroids used in the ChanTest experiments were a 

concern, especially with regard to the possible lack of specificity towards the intended Adverse 

Outcome Pathway (AOP) (i.e., selective alteration of VGSC kinetics). Tefluthrin concentrations

extend to 100μM (>3 log units above its solubility; Fig 3). Tefluthrin shows a NOEL towards 

NaV1.3 of 1μM, 20-fold above its aqueous solubility limit (ChemID). Allethrin, on the other 

hand is used within its solubility range (1-10μM). It is unclear if this fact explains the differential 

influence of the two pyrethroids on NaV1.3 ß1ß2 or other isoforms?
28

There are several published papers that report that NaV1.3 interacts with β1and/or β3, and at least 

one paper that indicates that β2 has no influence on NaV1.3 singly or in combination with β1 or 

β3 (Meadows et al 2002). It is unclear what influence this has on the results reported by 

ChanTest. 

There are some discrepancies in reporting the EC50 value for Itail between Table 1A/Fig 4 and 

summary Table 2A (Allethrin 5.4 vs 7.8; Tefluthrin 2.0 vs 3.2). Again this reduces confidence in 

the general reproducibility of the measurements and raises questions about the validity of the 

conclusions extending comparisons of human NaVs to other studies that were done using very 

different expression models. Again, summary statistics are not given in any of the NaV summary 

tables (Figures 1A&B; 2A&B).

The ChanTest does not take into consideration the occurrence of splice variants in individual 

sodium channel isoforms. For example, three splice variants of NaV1.3 have been found in the 

human brain (Thimmapaya et al., 2005). While it is unclear the role that splice variants play in 

the toxicity of pyrethroids in humans, splice variants of sodium channels in insects have been 

demonstrated to produce pharmacologically distinct channels and are considered to be the basis 

for resistance. 

Table 1 below summarizes quantitative measures of NaV1.3 and NaV1.6 channel function 

reported by ChanTest with addition of rat oral LD50 values and aqueous solubility for 6 

pyrethroids included in the ChanTest report (LD50 and solubilities obtained from ChemID PLUS 

(http://chem.sis.nlm.nih.gov/chemidplus/). Evident from Table 1 is the lack of definable 

association between NaV1.3 or NaV1.6 channel parameters and acute toxicity. Collectively these 

data raise several uncertainties of whether the ChanTest experiments adequately address the key 

issues related to AOP and juvenile susceptibility.

Table 1. Summary of quantitative measures of NaV1.3 and NaV1.6 channel function reported by 

ChanTest with addition of rat oral LD50 values and aqueous solubility for 6 pyrethroids 

included in the ChanTest report (LD50 and solubilities obtained from ChemID PLUS 

(http://chem.sis.nlm.nih.gov/chemidplus/).
29

b. CAPHRA thinks that additional data from this line of evidence is unlikely to contribute useful 

information to characterize the potential for juvenile sensitivity. Thus, the CAPHRA has 

proposed NOT to continue this line of research. Please comment on this proposal and degree 

to which the ChanTest data inform the issue of evaluating the potential for juvenile sensitivity.

The Panel agreed that the ChanTest approach is unlikely to contribute useful information to 

characterize the potential for juvenile sensitivity of pyrethroids. The approach is fundamentally 

limited by the heterologous expression system where the NaV isoforms are over-expressed out of 

context and exposed to acute challenges to most pyrethroids above their solubility range.

A more defensible approach for high-throughput screening would be to implement primary 

neuronal networks in cultures isolated from juvenile rodent or human iPSC derived neuronal 

cells grown in culture and measure electrical network activity using FluoVolt or patterns of 

synchronous Ca2+ oscillations, which are tightly coupled to membrane electrical activity as an

integral part of the AOP. 

c. The ChanTest experiments focus on human sodium channels and generally show weak response. In 

contrast, in vitro studies in rodents in the literature (e.g. Choi and Soderlund, 2006; Meacham et al., 

2008; Tan and Soderlund, 20094

) show stronger responses to pyrethroids. Please comment on the 

extent to which the expressed human sodium channels can be used in combination with these sources 

to infer relative pharmacodynamic sensitivity between rats and humans. 

The Panel agrees that the ChanTest experiments as described do not sufficiently discriminate 

among the electrophysiological properties of expressed NaV isoforms and do not discriminate 

differential influences of pyrethroids having large range of acute neurotoxicological potencies on 

NaV isoforms. The data reporting stronger response of rat NaV to pyrethroids from the above 

cited literature cannot be used in combination with the ChanTest data because those data were 

obtained from a non-mammalian expression system (i.e., Xenopus oocytes) that could potentially 

have differences in post-translational modification of proteins, in membrane composition, or in 

phosphorylation status that could influence interactions between pyrethroids and NaVs 

(Meacham et al., 2008). The weak effects of pyrethroids in the ChanTest protocol and the lack of 

neuronal and developmental contexts are unlikely to add knowledge about isoform sensitivity, 

unless the rat and humans clones are tested under identical experimental conditions with a more 

robust sample size.

2. Transplantation of adult & juvenile rat brain synaptic membrane into Xenopus oocytes:

Purified neurolemma membranes from adult and juvenile rats were separately micro-injected 

into Xenopus oocytes. Patch clamp testing was performed on the oocyte membranes versus 

various doses of pyrethroids to determine their EC50 values for NaV channel activation. 

 Choi JS and Soderlund DM. 2006. Structure-activity relationships for the action of 11 pyrethroid insecticides on rat Na v 1.8 

sodium channels expressed in Xenopus oocytes. Toxicol Appl Pharmacol. 2006 Mar 15;211(3):233-44. Epub 2005 Jul 26 

Meacham, C.A., et al., Developmentally-regulated sodium channel subunits are differentially sensitive to [alpha]-cyano 

containing pyrethroids. Toxicology and Applied Pharmacology, 2008. 231: p. 273-81. Tan, J. and D.M. Soderlund, Human and rat 

Nav1.3 voltage-gated sodium channels differ in inactivation properties and sensitivity to the pyrethroid insecticide tefluthrin.

NeuroToxicology, 2009. 30(1): p. 81-89
30

Inhibitors of competing channels were added to isolate the sodium channel conductance (i.e. 

chloride and calcium channels). 

a. Please comment on the synaptic membrane (“neurolemma”) studies conducted for deltamethrin and 

permethrin. Please include in your comments a consideration of the robustness of data from the 

synaptic membrane transplantation into oocytes (reproducibility, control compounds, channel 

modulator reagents, statistics, use of Na blocker tricaine as an anesthetic, etc) and the degree to 

which these data inform the issue of evaluating the potential for juvenile sensitivity. Please comment 

on the confidence and uncertainties and associated findings in the oocyte experiments.

The Panel recognized that the scientists performing the neurolemma experiments provided a 

detailed, thorough and comprehensive description of their procedures and results. A number of 

basic and preliminary experiments were conducted using appropriate channel modulator 

inhibitors reagents to assure reviewers that the model was a valid way of looking at rat sodium 

channels in a non-mammalian cellular system. The use of tricaine as an anesthetic for collection 

of Xenopus oocytes is well documented in the literature and deemed acceptable. Appropriate 

methodology was developed to look at sodium channels affected by pyrethroid insecticides. 

Concentration-related pyrethroid effects on brain neurolemma isolated from Postnatal Day 15 

(PND15) and PND90 rats were measured, but no differences in potency (EC50 values between 

ages) were detected nor inactivation and deactivation tau values. Differences were observed in 

efficacy values (βmax) but this was suggested to be due to differences in the channel densities 

between the two ages rather than any sensitivity differences between the channels from rats of 

the different age groups. 

The study does acknowledge the potential for incorrect orientation of the channel in the 

membrane and incorrect assemblage to the native state that are concerns with this methodology

(Ivorra et al., 2002; Eusebi et al., 2009). The scientists indicated that they injected TTX into the 

oocyte to block any incorrectly oriented NaV and the signal did not change. The scientists 

acknowledge that some yet to be characterized channels that are targets of some pyrethroids 

could be present in the preparation and that could influence the results obtained.

With respect to confidence in the findings of the oocyte experiments, the Panel raised the 

following concerns:

Sample size: Various statistical tests performed in the different stages of the experiments were 

based on a very small sample size. As an example, look at the effects of ion channel antagonists 

on outward ion currents measured during depolarization, one sample t-tests with n=3 were 

conducted (Figure 7, Appendix 2-2). Although the tests were significant, the very limited sample 

size raises concerns on the validity of the findings.

Fit of the response-concentration curve: Parametric curves were fit to the concentrationdependent response curves relative to NFA, KB-R7943 and TEA (see Figure 12, 14, 15 in 

Appendix 2-2 in the CAPHRA white paper). The fit in most cases were based on few sample 

points, with fits that in some cases looked appropriate (particularly, Figure 14) and fits that in 

other cases (especially TEA) looked more questionable. As IC50 are estimated from such curves, 

an increase in the sample size could remove some of the uncertainties related to these findings. 
31

Large uncertainties: There are very large error bars (e.g. SEM) in the plots showing the effect of 

permethrin and deltamethrin concentrations on TTX-sensitive, inward current, particularly in the 

plots relative to NFA (see Figure 28, Appendix 2-2 in the CAPHRA white paper). The large 

uncertainties appeared to increase as concentrations increased (see Figure 28, Appendix 2-2) and 

they seriously undermine the confidence in the findings. Increasing the sample size and using 

more data, as the scientists themselves conclude (page 54 Appendix 2-2), could help clarify 

whether there is a different sensitivity to permethrin and deltamethrin in adult vs juvenile rats. 

Robustness of the assay: The robustness of the assay is limited by the large variability of the 

primary data and by the requirement for multiple data transformations to obtain measurements 

that could be used for comparisons. The current assay also fails to take into account potential 

differences in the TTX sensitivity of the juvenile channel and how such differences can possibly 

impact data interpretation and differences between adults and juveniles with respect to pyrethroid 

sensitivity. The Panel recommended that additional validation studies are needed to clarify the 

relevance of this model. 

b. In the context of your response to 2a, the CAPHRA has proposed to collect dose-response data in 

adult and juvenile rat synaptic membrane transplanted into oocytes for five more pyrethroids 

(including Type I, Type II, and mixed). CAPHRA’s proposed path forward: If the additional five 

pyrethroids show similar patterns to deltamethrin and permethrin (i.e. no lifestage sensitivity 

observed), no additional pyrethroids will likely be tested in this system. Alternatively, if a different 

pattern is observed, additional pyrethroids are likely to be tested. Please comment on the CAPHRA’s 

proposed path forward.

The Panel acknowledged that the system is capable of examining age-related differences in the 

sensitivity of rat brain sodium channels. However, the results with deltamethrin and permethrin 

did not demonstrate any age-related pharmacodynamic differences. It appears that the NaV

components of juvenile and adult P2-neurolemma are structurally identical only differing in the 

levels of NaV1.3 and NaV 1.6 present. Perhaps there is some value in testing additional 

pyrethroids, but the expectation that any age-related pharmacodynamic differences will be 

detected appears to be small unless a pyrethroid specifically targets NaV1.3 or NaV1.6. Therefore, 

it was the Panel’s opinion that the overall usefulness of this system as the sole component for 

determining mechanisms associated with possible age-related differences in pyrethroid toxicities 

appears minimal.
32

3. Targeted in vivo studies in adult and juvenile rat: acoustic startle/detailed clinical observations: 

Preliminary experiments that measure acoustic startle and detailed clinical observations have been 

conducted by the University of Cincinnati. In these experiments, juvenile and adult male SpragueDawley rats were treated with deltamethrin and permethrin as model Type II and Type I pyrethroids, 

respectively. In addition, brain and plasma concentrations were measured in PND 15 and 90 rats 

exposed to deltamethrin. The preliminary results suggest that juvenile rats are more sensitive to 

deltamethrin based on changes in detailed clinical signs and, to less extent, acoustic startle. However, 

there was no greater sensitivity in juvenile rats exposed to permethrin. Preliminary data from whole 

brain tissue samples with deltamethrin indicated that PND 15 rats had higher deltamethrin 

concentrations as compared to adult rats given the same dose. The brain concentration and toxicity 

data from the CAPHRA studies for deltamethrin are consistent to those previously published by the 

Bruckner lab (Kim et al., 2010) which showed that at a similar dose (2 mg/kg deltamethrin) PND 10 

rats had increased severity of clinical signs and higher Cmax of deltamethrin in the brain as compared 

to PND 21, PND 40, and PND 90 rats.

a. The in vivo behavior studies reported thus far are preliminary evaluations. Please comment 

on the study design in the preliminary in vivo studies for deltamethrin and permethrin 

conducted at the University of Cincinnati (Vorhees lab). 

In general, the Panel believed that these studies were carefully planned and that the study design 

included many facets to address scientific rigor and reproducibility. Preliminary studies were 

conducted to examine potential confounders and to validate the test system. In addition, the 

Panel commented that the study design also permitted rigorous statistical analysis. Along with 

these positive observations, the Panel also noted the following shortcomings associated with the 

studies.

First, the Panel remarked that there is a need for more explanation on the selection of the 

Postnatal Day 15 (PND15) age in rats, especially as it relates to the development of the nervous 

system in a 1-3 year old human. This information is important since the Agency’s overall 

interpretation is that “there was no greater sensitivity in juvenile rats exposed to permethrin”. 

However, the rationale for the particular age chosen seems particularly important given 

that permethrin (PM) was shown to affect the ASR in PND17 and PND21 rats, but not 

PND15 rats. Thus, the presence of effects on PND17 and PND21 does not agree with the overall 

interpretation that “there was no greater sensitivity in juvenile rats exposed to permethrin” and 

leads to questions concerning the appropriateness of the selection of PND15 for testing. While it 

was indicated that the specific age chosen for juvenile testing was done in concurrence with 

EPA, the explanation behind this decision was not provided. However, it was revealed during 

the oral discussions at the public SAP meeting that the decision to use PND15 rats was based on 

the 2010 SAP where it was stated that the PND15 age was a compromise considering: 1) the 

neurobiological development required for the ASR tests, 2) brain development in a human child 

of the targeted age, and 3) age-related metabolic enzyme profiles in rats and humans. This 

rationale was not provided in the CAPHRA Overview Document and the rationale behind the use 

of only male rats in the CAPHRA in vivo studies was not discussed in the in vivo study reports. 

During the public meeting, it was stated that there were data demonstrating that there were no 

sex effects. The Panel noted that this data should have been discussed in the CAPHRA Overview 

Document.
33

Second, the Panel commented that the rationale for the dose selection in several studies was 

unclear. For example, there were higher permethrin doses in juveniles than adults. This led the 

Panel to question whether this range was expected to encompass doses that would cause no 

effect to doses that would cause a severe effect. However, the dose range finding studies do not 

appear to have demonstrated a dose that did not cause an effect.

Third, with regard to the pretesting of animals in order to improve performance, it is not clear 

how this was performed, and how this was used to match groups before the tests started. The 

potential for biases created by this design should be explained. For example, in Fig 1 of A2-4 the 

initial Vmax of control animals varied significantly. This observation raised the question of 

whether or not this was related to the pretest grouping. It also introduced the question of if the 

pretesting approach had the potential for causing habituation, thus decreasing the ability to detect 

differences and reducing the dynamic range. Although this does not appear to be the case with 

the PM experiments in adults, it is not clear what effect this may have on the overall 

experimental design. Habituation in the ASR studies with adults after 2 hours could prevent 

direct comparison with juvenile studies that do not seem to exhibit this phenomenon. The Panel 

suggested that this issue should be addressed.

Fourth, the Panel noted was there was no clear explanation regarding why Sprague Dawley rats 

(SD) were selected for the studies instead of Long Evans (LE) rats. Although the data presented 

in the CAPHRA document certainly show that the SD rats are suitable for the studies, it appeared 

in the pilot studies the LE rats were giving a more robust response to permethrin.

Finally, the Panel expressed concern that even though both control pups and treated pups 

originated from the same litter, the litter should have been included in the statistical analysis. The 

basis for this was since the pups and dams were shipped to the investigators, the dams were 

required to care for the pups during transit and the shipping could have induced some level of 

stress in the dams. The magnitude of the response to that stress could have varied between dams 

and resulted in differential levels of care which could have impacted the behavioral outcomes of 

both treated and control pups in that litter. Litter could have been included to account for this 

possibility. During the public meeting, CAPHRA acknowledged that this was not the optimal 

method to obtain pups but was selected because of the short time frame required to obtain the 

preliminary data. CAPHRA also stated that in future studies, the pups would be obtained from a 

breeding colony onsite and not be subjected to the potential stress during shipping. The Panel also 

noted that there was no chemical characterization information provided for permethrin.

b. In 2010, FIFRA SAP commented on some challenges that were anticipated with the use of auditory 

startle to compare the relative sensitivity of juveniles and adults. In the studies conducted by the 

CAPHRA, both auditory startle and detailed clinical observations were evaluated. Please comment on 

the degree to which the auditory startle and detailed clinical observations provide useful data for 

evaluating the potential for juvenile sensitivity. Please include in your comments discussion of the 

dynamic range; the direction of the response varying between Type I and IIs; and the type of data 

obtained (continuous, ranked).
34

One study indicated that the tactile startle response (TSR) test provided a somewhat larger 

dynamic range; however, more studies may need to be done across ages, compounds and doses, 

to support the robustness of this conclusion. Given that they both appeared to be CNS-mediated, 

the Panel was uncertain whether switching to TSR could be justified based on the possibility that 

this would address the problem of non-specific peripheral effects.

The use of ordinal Detailed Clinical Observations (DCO) data is valuable as a secondary 

observation. However, these data are subjective, and it is not constitute a rigorous unbiased 

method for decisions related to hazard assessment across many compounds. Relative to DCO, 

the automated ASR (or TSR) approach is a better primary method for assessing in vivo toxicity. 

However, one panel member noted that the DCO observations, and tremors in particular, might 

better fit the “adverse” description as opposed to a compound effect.

Adult rats exposed to PM and tested in the ASR did not exhibit any clinical signs of toxicity, 

including tremors. The Panel suggested that perhaps these tremors did not likely affect the ASR 

since a proportional change (25%) did not occur in ASR. This suggestion may be true, but it is 

speculative without more data to support such a proportional relationship. For the PM preweaning results, there seems to be no clear relationship between the magnitude of the tremor 

response and the magnitude of the ASR response. It is unclear which is the more relevant 

indicator of toxicity. With respect to the ASR data for DLM, CAPHRA concluded that DLM did 

not induce statistically significant effects on ASR in PND90 rats (Figure 2-5a) while DLM 

induced statistically significant effects on ASR in PND15 pups (Figure 2-5b). These data suggest 

the presence of age differences in rats treated with DLM. However, the CAPHRA document 

states that “This profile of age-dependent sensitivity to deltamethrin is consistent with published 

results, which show no difference in sensitivity of PND 21 and adult rats at low doses of 

deltamethrin, based on decreased ASR, and a marked increase in toxic response in juvenile rats 

as doses are escalated (Sheets et al., 1994).” In the CAPHRA document, the dosage of 1 mg/kg 

did not result in statistical significance in PND15 pups but it was not tested in the PND90 

animals. The lowest dosage used in both ages was 2 mg/kg that was statistically significant in 

PND15 rats but deemed to not be significant in PND90 rats at any time. However, the PND90 

ASR data suggest the presence of a biologically relevant effect. For example, ASR was reduced 

in PND90 rats at 2 hrs. (Figure 2-5a. of the CAPHRA Overview Document) by 40-48% with all 

three dosages of DLM. The statistical model used by the scientists did not indicate any statistical 

significance. However, when the 95% confidence intervals are calculated using the means and 

standard error of the data, there is no crossing of intervals between the treated groups and the 

control group suggesting that they may be different. In fact, the only time treated and control 

confidence intervals crossed was at 4 hrs. in the 2 mg/kg DLM group. This issue decreased the 

confidence of the Panel concerning the absence of significant effects on ASR in PND90 rats 

exposed to DLM. 
35

General Comment:

Historically the ASR study has been a measure of pyrethroid neurotoxicity and data generated by

this study should be comparable to values in the literature. However, the relevance of the ASR to 

either observed or potential effects in humans has not been established. The question is whether 

there are other measures of functional neurotoxicity that could be dissociated from non-specific 

effects, and that have more relevance to observed neurotoxicity in humans. It is recognized that 

there may not be enough or any data in humans to design such a model, so tremors and/or 

writhing in rats may be more sensitive and relevant to the human condition. Another primary 

question is whether ASR is linked to any known mechanism of action for pyrethroids, including 

their effect on sodium channels, or other ion channels.

As an overall conclusion, the Panel concurs that it does appear that use of the ASR for juveniles 

is problematic because of the overlap with non-specific effects. Going forward, the Panel 

recommends further testing with the TSR, either alone or in combination with the ASR, to see if 

the test is sensitive enough to separate out the specific effects from the non-specific effects. 

c. Please provide comments comparing the temporal pattern and magnitude of the brain and 

plasma concentration data from the Vorhees & Bruckner labs and utility of such data to aid in 

the interpretation of the auditory startle and detailed clinical observation data. 

For DLM, the time of the rise in plasma and brain levels did not match the peak in ASR effects;

however, the trends are consistent considering the slight delays. It is not expected that these 

should match perfectly since there are several unknown factors involved in the potential doseand time-related mechanisms underlying the effect of DLM on ASR.

A plan to continue investigating the comparison in levels of neurotoxicity in young and adult rats 

with similar levels of exposure (concentrations) within the brain may be appropriate. If the data 

support the available findings to date, the Panel would concur that there would be no further in 

vivo work needed to resolve the pharmacodynamics question.

While it may be important to examine the effects of pyrethroids in young and old rats at a 

matched internal dose (e.g. brain/plasma concentration), the difficulties in obtaining a high 

enough dose in adults and the temporal variability in these doses may be prohibitive. This 

difficulty must be weighed against the added value of these data to the overall question of 

whether young rats/humans are more affected by a given external dose, regardless of intrinsic 

differences. The Agency will need to consider what information will be critical for the final 

hazard assessment and regulatory activity.
36

d. Please comment on the use of a 5 ml/kg dosing volume in the CAPHRA studies and how any 

impact on pyrethroid kinetics affects correlations with behavioral effects. 

The Panel concluded that in the DLM experiments the dose volume did not significantly

influence the timing of ASR effects. However it was clear that a large volume delayed the effect 

on ASR in other studies, and overall, it was not clear how the results of the experiments varying 

dose guided a final decision of using 5 ml/kg in future testing. 

The Agency should consider the question: Is a slower release and delayed or extended period of 

the ASR desirable? For example, is it better to give a smaller volume and test at 2 hours, or 

larger volume and test at 4 or 6 hours? The answer may lie in the optimal study design for 

comparable results across compounds, as well as how best to design the model in order to 

simulate exposure in humans. The issue of habituation is relevant here as well since an extended 

period of exposure allows more time for habituation to occur. A particular concern is how 

differences in the apparent habituation responses of adult and juveniles contribute to 

understanding of physiological effects of pyrethroids.

Considering the Agency goal to modify toxicity testing not only to reduce animal use but also to 

test more human relevant doses, it might be worth a consideration going forward if it would 

really be necessary to push the doses of low potency compounds to such high levels. There does 

not yet appear to be a large database on this volume effect in vivo, but there could be a concern 

that for a potent compound an effect could be observed at a lower dose using the smaller volume, 

even with a later evaluation time for the larger dose volume. A positive control, such as DLM,

could be included in testing of low or unknown toxicity compounds at the limit of solubility or if 

a higher dose volume was used.

The Panel questioned the justification of using 5 mL/kg in the CAPHRA studies given that 

previous studies in the literature used 1 mL/kg. CAPHRA stated that the methods were 

developed to allow testing of additional pyrethroids in the future. Many of these pyrethroids have 

very low toxicity and very high dosages would be required for testing.

Several panel members were concerned that the 5 mL/kg dosage rate would be a large volume to 

administer especially to a PND15 pup. Other members were not as concerned given that a SD 

PND15 pup weighs on average 30 g and administering a 5 mL/kg solution would only be 0.15 

mL which is only about 5% of the total stomach volume (2.8 mL). However, the Panel agreed 

that there is a concern since this amount was administered to a pup with a full stomach of milk. It 

was stated that 15 day old rat pups were not removed from the dam prior to dosing which did not 

allow gastric emptying. This is different from the studies with adults who were removed from 

food to allow gastric emptying prior to administration of the pyrethroids. It is possible that the 

milk in the stomach altered the absorption and pharmacokinetics of the pyrethroid. Given how a 

larger volume (5 mL/kg) of corn oil used to administer 120 mg/kg permethrin to adults delayed 

the onset of behavioral outcomes as compared to the same dosage administered in a lower 

volume (1 mL/kg) (Appendix A2-4), the presence of milk could have delayed the onset of 

behavioral outcomes in juveniles following DLM exposure.
37

4. Pharmacokinetic studies: A number of pharmacokinetic studies using deltamethrin were 

performed by the CAPHRA in order to further refine and validate the developing rat PBPK 

model and construct a developing human PBPK model. For refinement of the rat PBPK 

model, tissue: plasma partition coefficients (in vivo measurements in PND 21 and adult rats), 

age dependent plasma protein binding (in vivo measurements in PND 10, 15, 21, and 90 rats), 

and cytochrome P450 (CYP) and carboxylesterase (CES) metabolism (in vitro measurements 

using rat liver and plasma preparations from PND 15, 21, and 90 rats) were evaluated. 

Further in vivo pharmacokinetic studies in rats were conducted with single IV (PND 90), 

single oral (PND 90), and multiple oral (PND 15, 21, and 90) doses to generate plasma and 

tissue data to validate the developing rat PBPK model. Additional experiments with 

deltamethrin were conducted for constructing the developing human PBPK model. 

Parameters evaluated in vitro included age dependent plasma protein binding derived in 

plasma from human donors aged from birth to adults, transport across the blood brain 

barrier using a human brain microvascular endothelial cell line, estimates of gastrointestinal 

absorption in caco-2 cells, and ontogeny data for CES, CYP1A2, and CYP2C8 determined in 

human liver tissue from donors (age 1-18 years old). Further in vitro experiments are 

currently underway by the CAPHRA to determine human adult and juvenile liver metabolism 

of deltamethrin by CYP and CES enzymes using human liver preparations or recombinant 

human enzymes. 

a. Please comment on the in vitro experiments to support the PBPK model development in the rat 

conducted thus far for deltamethrin. 

The in vitro work conducted so far by CAPHRA has generated important information for the 

development and validation of a PBPK model for deltamethrin in a developing human. Overall 

the Panel thought that the experiments that were either completed or underway were excellent in 

quality and appropriate for addressing the age-dependence of liver and plasma metabolism and 

protein binding. The age-dependence of these kinetic factors is particularly important to be 

captured in the model, and the experiments have quantitated important issues that are critical for 

the IVIVE approach such as looking at the age-related changes in the relative amounts of CES1 

and 2 in microsomal and cytosolic fractions.

The Panel commented that the plasma binding study was thorough and produced high quality 

data, because it differentiated binding by age as well as by albumin, total protein, and 

lipoproteins; therefore Bmax and Kd values were useful for the model development.

The experiments clearly showed the age-dependence of plasma binding in rats with progressively 

increasing levels of albumin and total proteins. The extent of binding of deltamethrin at 250 nM 

was similar among ages except for the 1wk and 4wk age-groups, suggesting that total binding, at 

exposures equal to or lower than 250 nM may not have significant impact. This was noted to be

true for total binding, protein binding, and albumin binding, but not lipoprotein binding. 

For the vitro metabolism studies, the CAPHRA program has made considerable progress with 

proposed enzyme kinetic work on the metabolism of deltamethrin with rat hepatocytes, liver 

microsomal and cytosolic fractions, and plasma from male Sprague Dawley rats. Hepatic 

metabolism of deltamethrin in microsomal and cytosolic fractions was shown to be comparable 

to that by primary hepatocytes, which was an important confirmation on the use of microsomes 
38

and cytosol as the surrogate for hepatocytes. The Panel thought this was critical information to 

support the IVIVE approach to be used for the development of the pyrethroid PBPK models. The 

Panel recommended that the CAPHRA program should complete the proposed metabolism work, 

in particular enzyme preparations from various age groups. The Panel also noted that the cell 

lines used for the blood-brain barrier penetration potential (hCMEC/D3) and for intestinal 

permeability and transport (Caco2) were cell lines that are widely used for the respective studies. 

A few areas where the Panel identified deficiencies in the work included the lack of data or 

experiments addressing age-related differences in permeability and active transport at the blood 

brain barrier. This will be crucial information for reducing uncertainty in model predictions, 

particularly since permeability-surface-area cross product was an influential parameter governing 

brain concentrations of deltamethrin in the PBPK model.

In addition the Panel was not able to find or identify in vitro studies that addressed possible agerelated changes in the absorption of deltamethrin. There was a lot of discussion during the 

meeting with regard to how absorption has been integrated into the model. The current PBPK 

model structure has absorption completely bypassing the liver and directly entering the 

lymphatic system. As these are critical model assumptions that affect model predictions of agerelated changes in the brain Cmax, the Panel recommended conducting studies with appropriate 

in vitro systems to support the modeling work and parameter values.

For the metabolism studies, the Panel recommended additional studies including: 1) tissues from 

female rats to address sex-related differences in metabolism; and 2) extrahepatic tissues 

metabolism including the gastrointestinal tract and the brain. Information on the metabolic 

clearance of pyrethroids, including deltamethrin by intestinal tissue, could be important and may 

present challenges for modeling of any pyrethroid compounds that undergo significant 

biotransformation through intestinal CYPs and carboxylesterases. Some panel members 

recommended monitoring both the loss of deltamethrin and the formation of products to gain 

additional confidence on the results.

b. Please comment on the in vivo experiments to support the PBPK model development in the rat 

conducted thus far. 

The CAPHRA program systematically carried out and collected high quality data for numerous 

toxicokinetic and tissue distribution studies, which had several strengths including: (1) use of 

various age-groups to determine age-related changes in toxicokinetics; (2) administration of 

deltamethrin by intravenous and oral dosing to investigate bioavailability (although some 

concern was expressed with regard to results); and (3) dosing over a range of deltamethrin 

concentrations doses to provide important information on the detoxification capacity (saturation).

However, the Panel expressed some concerns about the study design and the data analysis. While 

single intravenous (IV) or oral dosing for pharmacokinetics studies are necessary for establishing 

kinetic parameters, it does not reflect true environmental exposure to deltamethrin or other 

pyrethroids. The charge question mentioned multi-dose studies as part of the suite of in vivo

studies conducted, but Panel members were unable to locate the information in either the review 

documents or the presentations. Repeated administration with doses relevant to true 

environmental exposure should be considered. In addition the experiments also showed that 
39

PND15 and PND90 but not PND21 groups had dose-dependent increases in Cmax values 

(Figure 3-2), but there was no explanation for this outcome, which should be addressed. The 

review documents indicated that male rats were used for the PND90 group, mixed sex was used 

for the PND21 group and unspecified sex was used for the PND15 group, but did not provide 

justification for the differences in sex selection. 

One issue of concern to the Panel was the effect of delivery vehicle on absorption, in particular 

the absorption route to the systemic circulation (i.e. lymphatic system versus the portal system). 

There was also concern about the results and analysis of the kinetics from the IV versus oral 

exposure. As noted by one panel member, comparison of the Dose/AUC ratios would suggest 

less than 10% of the administered dose was bioavailable, but CAPHRA members stated that 30 

to 40% of the administered dose is eliminated fecally. If first pass metabolism is not occurring 

due to absorption into the lymphatics, then CAPHRA needs to resolve the difference in measured 

fecal levels versus estimated bioavailability from the kinetic studies. This is important because 

the assumption of bypassing the liver in model may partly contribute to the modeling results, 

which predicted a higher brain Cmax in the adults versus children.

CAPHRA provided an explanation in their Overview Document about the limitations of in vitro

or algorithm-derived partition coefficients for deltamethrin. Therefore, the Panel deemed the in 

vivo method to be appropriate for measuring the tissue plasma partition coefficients for various 

types of tissues. One area that was noted as needing explanation was the lack of age-related 

differences for permethrin as compared with deltamethrin. The lack of difference had been noted 

in the Overview Document, but no further information was provided. These differences in agerelated effects among pyrethroids will be important to understand in order to reduce uncertainty 

in predictions for extrapolating the PBPK model across the family of pyrethroids. The Panel 

pointed out that although there are limitations and issues for the predictive ability of algorithms, 

these comparisons of the measured values with those derived empirically will be important as the 

modeling efforts advance to other pyrethroids that have not been proposed to have partition 

coefficients measured experimentally.

c. Please comment on the in vitro experiments in the human tissue conducted thus far. Please 

include in your comments a discussion of the ongoing in vitro experiments with recombinant 

enzymes for use in PBPK models and associated confidence and uncertainty with the use of 

such data.

The Panel noted the approach that CAPHRA was using for the IVIVE with recombinant 

enzymes seemed to be thorough and a logical method strategy to predict metabolism in the liver 

across age groups. The Panel noted several areas of confidence for the approach including: (1) 

Ontogeny data (expression) on human liver carboxylesterases (performed by Ronald Hines and 

colleagues) were consistent with data reported in the literature; (2) Ontogeny data (expression) 

on various CYP2C8 and CYP1A2 enzymes (performed by M Yoon and colleagues) in the liver 

were consistent with data reported in the literature as well; and (3) Enzyme kinetics will be 

determined with recombinant CYPs and CESs.

There were some aspects of the experiments that were of concern to the Panel and would 

generate uncertainty in the use of the data including: (a) lack of studies determining hepatic 
40

clearance with primary human hepatocytes from various age populations (cryopreserved 

hepatocytes); and (b) determination of ontogeny expression of various enzymes by different 

laboratories. The Panel questioned why the same samples were not used for all types of enzymes 

to minimize variations raised from different laboratories. Although either purified recombinant 

enzymes or lysates from overexpression likely produce robust signals and the results from these 

preparations are informative, the recombinant enzymes may differ from native enzymes, for 

example with respect to posttranslational modifications and the cellular environment in which 

the enzyme resides. Therefore, some data from recombinant enzyme studies should be validated 

with native enzymes. Human liver enzyme preparations with different expression of an enzyme 

of interest are usually used for this purpose. In addition it would be interesting to have seen a 

proof of concept with using the scaling from recombinant to either a human hepatocyte or a 

combined microsomal/cytosolic incubation. 

5. Physiologically-based pharmacokinetic model in rat: Using data described in Question 4, 

the CAPHRA has developed a PBPK model for deltamethrin using age-specific metabolism 

parameters in rats to simulate plasma and brain internal exposures in young and adult rats. 

This PBPK model relies on in vitro to in vivo extrapolation (IVIVE) to use age-specific 

metabolic data collected in vitro to estimate hepatic metabolic clearance in vivo. The 

deltamethrin brain or plasma concentrations estimated by the model in rats are then 

compared to measured concentrations from in vivo rat studies to verify the model. Nonchemical specific physiological parameters for rats were obtained from the published 

literature, including body weight, cardiac output, hematocrit levels, tissue volumes, and tissue 

blood flows. As discussed in Question 4, recently generated data by the CAPHRA and 

published data were used for compound-dependent parameters, including partition 

coefficients, metabolic rate constants, absorption rates, protein binding, compartments and 

tissue permeability.

a. Please comment on the robustness of the rat PBPK model for simulating internal exposures in the 

developing rat. In your response, please include evaluation of the structure and parameters used to 

build the model, as well as its ability to accommodate different oral absorption scenarios (i.e. different 

vehicles used for in vivo studies) and discussion of confidence, accuracy and uncertainties associated 

with the deltamethrin developing rat model. Please also comment on the sensitivity analyses of 

parameters CAPHRA has completed thus far.

Comments concerning the model were based on CAPHRA’s presentations during the public 

meeting and the information given in the overview. Panel members did have some concern about 

commenting on the confidence, accuracy and uncertainty of a model that did not appear to be in 

the final form. However, the Panel recognized the fluidity of the modeling process and 

commended the researchers and modelers on the iterative nature of the work with the 

experiments informing the model and vice versa. 

The model-predicted concentrations of deltamethrin in the brain and plasma of PND10, PND15 

and PND90 rats have been simulated with a PBPK model structure, which built upon the model 

by Tornero-Velez et al. (2010) and expanded it to account for: (1) vehicle-specific absorption of 

deltamethrin; (2) plasma metabolism; (3) age-specific plasma protein binding; and (4) the 

metabolic contribution of CES to pyrethroid hydrolysis. As shown in Tables 3-1 and 3-2 of the 
41

overview document, parameters for the rat PBPK model were derived from a variety of sources, 

including both values reported in the literature and CAPHRA-generated values. The Panel felt 

the incorporation of this additional information increased the confidence and accuracy of the 

model.

With regard to CAPRHA’s sensitivity analyses conducted thus far, the only local sensitivity 

analysis (LSA) that was available in either the presentation or the overview was for the human, 

and not the rat. The Panels’ comments relating to the results of that analysis were included in 

Charge question 6a as it related to confidence and uncertainty in certain model parameters with 

regard to the human. As a general statement, the Panel considered conducting sensitivity 

analyses as an important component during model development and parameter calibration, but 

one that has to be interpreted in light of the existing model structure and parameter values, since 

it is a local sensitivity analysis. For example the sensitivity coefficients (SC) may have changed 

based on the dose or within the range of plausible values for the parameters. Therefore a global 

sensitivity analysis was recommended for the modeling work. In addition with regard to the 

LSA, the SC will change over time, therefore inclusion of other times may be important to gain 

confidence over the entire time profile of pyrethroid concentrations in the brain. 

Model Structure:

There were two places where the comparison between the model simulations and the data raised 

concern among the Panel about either the adequacy of the model structure or the parameters used 

for the model. Slides 40 to 44 presented the model comparisons with literature and newly 

generated data for different age rats. There was deviation of the model fit from the data both with 

regard to prediction of Cmax and Tmax and with comparisons during the terminal regions of the 

concentration time profiles, particularly for the brain concentrations in the older rats. The altered 

model structure of a two-compartment absorption system was an appropriate method to 

accommodate delayed absorption when administered orally and sought to address the difference 

in vehicle-related kinetics CAPHRA noted in the rats. However the parameters appear to need to 

be calibrated to provide better correlation of predicted versus observed plasma concentrations.

There was some concern among panel members about the model structure switch from uptake to 

the portal system to completely bypassing the liver via the lymphatic system. Not enough 

evidence was presented to support this model structure assumption in humans, nor were 

modeling comparisons given to allow the panel to judge the effect on model predictions in 

humans. Therefore, there was low confidence in model predictions because of the uncertainty in 

the validity of this structure. As demonstrated during the CAPHRA slide presentations, 

lymphatic absorption would bypass the liver and the pyrethroid would not be subjected to first 

pass metabolic degradation by hepatic CES and CYP, which have age-related differences. 

Therefore, a model with only lymphatic absorption eliminates the potential for age-related 

differences in the systemic concentrations of the pyrethroids due to first pass metabolism, and 

the Panel was concerned that could lead to errors in comparing age-dependent levels of 

pyrethroid reaching the brain. However, when one panel member briefly played with the model 

and the parameters, there appeared at least for the PND 90 rat to not be a large difference in 

assuming gut uptake vs lymphatic uptake on brain concentrations. The Panel was unable to 

successfully load the human model, which may be different from the rat.
42

Model Parameters:

In addition, there was some uncertainty with regard to how the absorption parameters were 

determined for the various vehicles. The documentation seemed to only show the values for corn 

oil. In the model there were three parameters that could be changed to affect the rate of 

absorption either to the gut or through the lymphatics. The Panel was uncertain from the 

documentation whether better fits could be obtained for the early time point data for the different 

vehicles. There should be a quantitative versus visual assessment conducted to determine 

whether the parameter values are vehicle-specific. The lack of a quantitative assessment of the 

adequacy of fit for the absorption parameters decreased the confidence in the model predictions.

With regard to the discrepancy in model predictions at the terminal region of the brain 

concentration versus time curve, one parameter that could have influenced the predicted brain 

concentrations was the permeability-surface-area cross product. The model assumed there was 

no age-dependent change in the blood-brain barrier permeability, however it was curious to note 

that the model provided better predictions in the younger rats (PND10 and 15) than in PND 90. 

Upon working briefly with the rat model code, one panel member found that decreasing the 

PABRC (the permeability-surface-area cross product for the brain) resulted in prolonged 

retention of deltamethrin in the brain. Therefore there was a lack of confidence in the assumption 

of no age-dependent changes for the permeability-surface-area cross products, which affects the 

assumptions regarding humans. This appeared to be a parameter that was highly uncertain and 

the Panel recommended the penetration of the blood-brain barrier as a function of age should be 

investigated and incorporated in the model.

In briefly looking through the rat model code, the majority of the code looked appropriately 

described. One minor note from the Panel: In the model code, the percentage of brain attributed 

to blood volume in the tissue was slightly higher than that reported in Brown et al. 1997. Also 

the volume in Brown et al. (1997) is for total blood, therefore the volume in the model needs to 

be adjusted to represent the plasma volume, since the models used tissue: plasma partition 

coefficients. The cardiac output and tissue flows were adjusted to represent plasma flow to the 

tissues.

In addition, as one panel member noted, the robustness of the rat PBPK model could have been 

evaluated by comparing the predicted concentration curve of deltamethrin in plasma and brain in 

PND10, PND15 and PND90 rats with both data reported in published studies and data collected 

by CAPHRA-supported studies (the new CAPHRA data).) As noted in the document, the 

modality of administration used was different in the two sets of studies, the first used glycerol as 

the oral gavage vehicle at a volume of 1ml/kg body weight, while the new CAPHRA studies 

used corn oil at 5 ml/kg body weight. Also the dosage used in the two sets of studies was 

different: 2 and 10 mg/kg in the published studies for PND90 and 0.4, 2 and 10 mg/kg in for 

PND10. 
43

b.In the context of your response to 5a, please comment on the extent to which additional data are/are not 

needed to refine the developing rat model.

The Panel recommended several studies that were needed to better validate the predictions of the 

current model and provide further refinement,particularly with respect to predicted brain 

concentration of deltamethrin in older versus younger rats. In charge question 4, there was 

mention of multi-dose studies, but the Panel could not locate the study results in the main 

document, presentations, or appendices. The model has been compared only with single oral 

doses; however, data from repeated dosing of pyrethroids are important to look at the potential 

accumulation in the brain and as a source to validate model predictions. The Overview 

Document stated there was no expected accumulation of deltamethrin, but it did not present 

supporting data. Since the neurological effect of pyrethroids are correlated to Cmax, this 

information is important to document, as well as model accuracy. In addition repeated exposure 

may result in induction of enzymes, which will affect the toxicokinetic profile. The induction 

also would need to be performed with hepatocytes from different ages to assess possible agerelated changes.

The model structure with the absorption directly into the lymphatic system was regarded by the 

Panel as plausible but not well supported by the current experimental data or by model 

predictions of the data. The Panel thought that information on the impact of the lymphatic 

absorption in the model would be useful if it is to be incorporated into the final structure, and in 

vivo measurement of the lymphatic transport as supporting evidence would be crucial. Another 

aspect of the bioavailability that was not incorporated into the model was metabolism in the GI 

tissues. Extrahepatic metabolism, and in particular potential age-related differences, will be 

important to address, since it will affect plasma and brain concentrations. The model currently 

assumes 100 percent bioavailability as a worst case scenario, but is also predicting higher brain 

concentrations in the adult versus infant. Portal system absorption with greater metabolism in the 

adult versus infant, may result in model predictions of greater concentrations of pyrethroids in 

the brains of infants versus adults.

Data are also needed to evaluate the possibility of age-dependent differences in the blood-brain 

barrier. Based on the discussions of the Panel and the demonstrated influence of the model’s 

brain permeability-surface-area cross product on predicted brain concentrations, this will be 

important data to provide confidence in the use of the PBPK model for risk assessment for 

children.

6. Physiologically -based pharmacokinetic model for human: Similar to the rat PBPK model, 

the human PBPK model integrates non-chemical specific physiological parameters for 

humans from the literature. Compound-dependent parameters, such as partition coefficients 

and oral absorption parameters, were adapted from the rat PBPK model. Recently generated 

data by the CAPHRA were used for the remaining compound-dependent parameters, such as 

metabolic rate constants, protein binding and tissue permeability. With respect to metabolic 

constants, CYP and CES enzymes involved in metabolism of a given pyrethroid will be 

identified and the in vitro metabolic constants for those enzymes will be determined for 

integration into the PBPK model. Intrinsic clearance for each active enzyme will be scaled to 

in vivo using scaling factor data collected by the CAPHRA and the SIMCYP database. The 
44

ontogeny of enzyme expression (also from the CAPHRA data and the SIMCYP database) will 

be incorporated into the process of obtaining distributions of age-specific intrinsic clearance 

for each enzyme. Preliminary simulations have been conducted for deltamethrin to 

demonstrate the process used with the PBPK model. 

a. Within the context of understanding potential juvenile sensitivity, characterize the robustness 

of the PBPK model for extrapolating age-specific internal tissue exposures for humans. In 

your response, please comment on the structure and parameters used to build the model and 

include discussion of confidence, reliability, and uncertainties associated with the 

deltamethrin human model. Please include in your comments a discussion of the data from 

the McCarver/Hines Laboratory (submitted as part of the CAPHRA package) for providing 

ontogeny of CES enzymes.

The Panel appreciated the effort of CAPHRA to develop a human PBPK model to extrapolate 

internal tissue exposure to pyrethroids (specifically deltamethrin) in humans, with the ultimate 

goal to assess whether juveniles experience an increased sensitivity to pyrethroid neurotoxicity. 

Following on the same construct of the rat PBPK model, the human PBPK model for 

deltamethrin estimated/predicted tissue exposure to deltamethrin by modeling age-related 

physiological and metabolic processes that affect the chemical kinetics. Several age-specific 

parameters were used to represent the various physiological processes: body weights, tissue 

volume, tissue body weight fraction, partition coefficients, tissue permeability, etc. Values for 

these parameters were derived from various sources including among others the published 

literature, CAPHRA-supported studies, and rat models. In some cases, as for example, for the 

metabolic rate constants, the values refer to adults and not juveniles. The Panel expressed the 

following concerns regarding some of these model parameters:

1- The Panel noted some discrepancies between some of the model parameters/model structure 

reported in CAPHRA’s presentation during the public meeting and the parameter values/model 

structure reported in the Overview Documents (specifically, with respect to the GI uptake and 

the permeability-surface-area cross products). The Panel recognizes that a possible justification 

for the discrepancy could be the continuous efforts of CAPHRA to update and improve the 

model. The Panel also encountered difficulty in accessing the model files from the provided 

Dropbox folder because some of the files, in particular the executable for the human model in the 

software acs1X, did not load properly. 

2- The Panel noted a difference in the values for the permeability-surface-area cross products 

listed in the overview document and the parameters presented by CAPHRA during the public 

meeting. The Panel could not understand the reason for a discrepancy in the values. The values 

in the presentation did not match the values listed in the m files for the model, which the Panel 

noted as fitted. As noted in the CAPHRA’s overview document, brain permeability was an 

influential parameter in the model, since accurately predicting brain kinetics will be necessary to 

correctly predict toxicity. 

The Panel had the following concerns regarding the model structure specifically:
45

1- The Panel raised concerns regarding the fact that the model switched from uptake to the portal 

system to bypassing the liver completely via the lymphatic system. 

In addition, the Panel was concerned about whether there were differences between rats and 

humans in the role of transporters in gut absorption. 

2- The Panel expressed concerns regarding how the structure of the brain was parameterized in 

the model and how that would impact Cmax predictions. CAPHRA appears to have conducted 

experiments to address this aspect of deltamethrin pharmacokinetics and the summary data 

provided appears to not support significant involvement of transporters. However the low brain: 

plasma concentration ratio from the steady state experiments seemed to suggest that there is 

more than just a permeability limitation for the distribution.

3- The Panel recognized as a major challenge the difference among rats and humans in the 

metabolism of deltamethrin. While hydrolysis seems to be more important than oxidation in 

humans, the opposite is true in rats. In light of this observation, the Panel raised concerns about 

whether the hydrolytic clearance was over-estimated in humans, and they advised CAPHRA to 

investigate whether the estimated clearance supported by human hepatocytes varies with age.

4- The Panel highlighted the fact that the available human data showcased large variability 

among individuals, presumably and at least in part due to induction.

5- The Panel found it unclear why 1-year olds were used since the significant differences were 

found for 1 month-old. Additionally, the Panel was unclear on how the Monte Carlo approach 

was implemented (distributions used, number of simulations, etc).

The Panel recognized that the model does appear to incorporate reliable sources for the average 

age-specific physiological parameter values. One parameter that the Panel encourages CAPHRA 

to look at more closely is the percentage body fat: a fairly narrow range is given for children 

compared with adults. The Panel believed that there should be more variability in body fat 

percentage especially if sex is taken into account: the common approach in the literature on 

percentage body fat algorithms is to account for both age- and sex-specific variation. 

The Panel suggested that CAPHRA consider the following suggestions in the next modeling 

endeavors:

1. Change the definition of clearance: The use of average clearance for an age group with large 

individual variations tends to exaggerate the capacity of clearance and under-estimate the risk 

for those whose clearance capacity is at the bottom end. Thus, the Panel suggested replacing 

average clearance with the 10th percentile of clearance in the model.

2. Change the age grouping when modeling the liver function: During the first year of life, the 

liver undergoes a functional switch: from a blood-production organ to a metabolic organ. Based 

on the expression of carboxylesterases, it is not appropriate to group children altogether. Rather, 

the Panel believed it is reasonable to have several age subgroups: one month, 2-3 month, 3-6 

month and 7-12 months.
46

3. A void using western blotting: Western blotting can detect a relatively narrow range of 

variations (struggling with even 10-fold). In comparison, the activity can detect a large dynamic 

range of variation. In particular, for a given group of samples, the range of variation detected by 

Western blotting tends to be much smaller than the range of variation detected by activity. 

Therefore, a western-blotting based approach tends to exaggerate the capacity of clearance. 

Given the issues mentioned above, and the fact that the Panel was not provided with sufficiently

updated information, neither in the background material nor in the presentations, the Panel found 

it very hard to comment on the confidence, reliability and uncertainty of the predictions of agerelated increase in Cmax in the brain.

One aspect of the modeling effort that the Panel expressed confidence in was the expertise of the 

individuals involved in the work: the investigators involved in the modeling have extensive 

expertise and knowledge in the field and are experienced in extrapolating among species and 

with IVIVE.

6b. Please comment on the proposed use of SIMCYP for providing enzyme ontogeny patterns and 

deriving population distributions for metabolic parameters in humans. Please include in your 

comments whether or not other tools with similar capacity to SIMCYP are available.

The Panel recognized that there is a general acceptance of the SIMCYP Simulator by the modeling 

community as a tool to provide enzyme ontogeny patterns and derive population distributions for 

metabolic parameters in humans. This Simulator has a pediatric module that integrates 

developmental physiology and age-related drug elimination pathways. As for the elimination 

pathways, the SIMCYP Simulator is equipped with well-characterized enzyme systems such as 

cytochrome P450s and UDP-glucuronosyltransferases as well as some transporter systems such as 

P-glycoprotein. Generally, using SIMCYP Simulator, an adult model is built and then scaled down 

to children. This approach has been used to build models for drugs and some of them are quite 

predicative. 

The Panel believed that pediatric modeling with the SIMCYP Simulator was robust particularly 

for drugs that are 1) primarily excreted through the kidney, 2) undergo minimal metabolism or are 

metabolized by a single enzyme system, 3) show some but not extensive protein-binding and 4) 

have limited involvement of active transport. The Panel, at the same time, pointed out potential 

challenges for the pediatric modeling of pyrethroids. These challenges are raised from two 

perspectives. First, pyrethroids, as a class of chemicals, exhibit the unique modeling-challenging 

characteristics including high-levels of protein binding, extensive metabolism by multiple enzyme 

systems and potential involvement of active transport as suggested by CAPHRA for uptake. 

Second, there are several knowledge gaps regarding the metabolism of pyrethroids in humans. 

1. Both hydrolysis and oxidation are involved in the metabolism of pyrethroids such as 

deltamethrin. It is not clear whether and to which extent these two elimination pathways 

differentially contribute to the overall clearance of a pyrethroid. 

2. Both hydrolysis and oxidation are present in the liver and extrahepatic tissues, probably varying 

depending on an organ. There is no information on organ-specific clearance of a pyrethroid as 

well as age-dependency in this regard. 
47

3. Pyrethroids are activators of pregnane X receptor that supports differential induction of 

cytochrome P450s over carboxylesterases in humans. It is not clear how the differential 

induction leads to changes of the contribution of respective elimination pathways. The 

complication is that induction of drug-metabolizing enzymes is inversely related to age, 

meaning the pediatric induction has a greater impact on the changes of drug clearance.

Throughout the public SAP meeting, particularly during the pharmacokinetic discussion, the Panel 

made some suggestions that will likely enhance the confidence and performance of the pediatric 

model for this class of insecticides.

1. Incorporation of organ-specific clearance (CLint, app) into the modeling (liver, brain and 

intestinal mucosa): The clearance rates should be estimated based on the metabolism of a 

pyrethroid with subcellular fractions or intact cells if possible such as pediatric and adult 

human hepatocytes. 

2. Specification of differentially regulated expression of hydrolysis and oxidation pathways by 

pyrethroids: If possible, induction studies such as in hepatocytes should be performed to 

ascertain how the hydrolytic and oxidative elimination pathways are differentially impacted 

by pyrethroids. 

3. Subgrouping of children at 1 year of age or younger: The first year of life undergoes 

tremendous changes in metabolic capacity. This age group should be sub-grouped further to 

reflect these changes to minimize the impact of large variations.

4. Alternative modeling platform: In addition to the SIMCYP Simulator, an alternative modeling 

platform should be used for comparison. There are other softwares for modeling and simulation 

as reported by Jamei M et al, 2009 and by Khalil and Läer, 2014.

7. Physiologically -based pharmacokinetic models for humans with other pyrethroids: Thus 

far, the CAPHRA has focused its human PBPK efforts on deltamethrin and, to a lesser extent, 

permethrin. Soon laboratory efforts will turn to other pyrethroids. CAPHRA’s proposal is to 

conduct fewer studies and in lieu of such data, use read across and computational 

approaches.

a. Please comment on the appropriateness of the current human PBPK model to be used for 

other Type I, Type II, or mixed type pyrethroids. Please include in your response evaluation 

of the path forward provided by the CAPHRA regarding in vitro and in vivo studies for other 

Type I, Type II, or mixed type pyrethroids.

The Adverse Outcome Pathway is a combination of pharmacokinetics and pharmacodynamics. 

Both have to be adequately understood to read across (i.e. extrapolate) to other compounds. The 

Panels comments on this charge question were focused only on extrapolating the 

pharmacokinetics across the family of pyrethroids. The Panel pointed out that it was difficult to 

make statements on a model that appeared to still be in development. The model structure and 

results differed between material provided in the overview document and the presentation of the 

work during the meeting. The Panel felt that they were being asked to make comments and 

recommendations on a concept that had not yet been thoroughly vetted.
48

The Panel noted that in theory, if the issues raised with regard to the rat and human model were 

addressed to decrease uncertainty in the model and increase accuracy of model predictions 

compared with data, then a read across with chemical-specific parameters would be appropriate. 

The Panel did not anticipate that the pharmacokinetic model structure would need to be different 

for the different pyrethroids, but the parameter values would differ. As pointed out by one panel 

member, one challenge of modeling these compounds is variability in metabolism. For example, 

bioallethrin (type I) and deltamethrin (type II) are both preferably hydrolyzed by CES1. 

Bioallethrin, compared with deltamethrin, is a better substrate of CYP3A4. Adding to the 

complication is the fact that hydrolysis varies depending on the isoform. For example, cispermethrin is hydrolyzed comparably by CES1 and CES2. However, trans-permethrin is 

predominately hydrolyzed by CES2. Metabolic clearance for individual pyrethroid compounds 

must be determined to allow models to be developed with better predictive ability:

Based on the presentation of the path forward that was presented by CAPHRA, the Panel made 

the following recommendations:

1. The proposed path for quantitation of the partition coefficients would be appropriate if 

QSAR or predictive algorithms are demonstrated to be useful for predicting measured values 

from the early stage pyrethroids. In addition it is not just partition coefficients that need to be 

assessed among the pyrethroids but also the permeabilities that are influential in determining 

the tissue concentrations. These permeabilities, particularly in the brain, need to be 

determined.

2. The in vitro kinetics were recommended to be determined for the juvenile microsomal and 

cytosolic fractions for at least permethrin because of its isomeric forms. This may affect the 

proposed IVIVE approach with recombinant enzymes. Deltamethrin does not have isomeric 

forms, therefore, permethrin may serve as a prototype to guide modeling of other pyrethroids.

3. The Panel recommended developing the PBPK models in adult rats for all pyrethroids if the 

data is generated. This would provide further support for the approach for comparison of the 

adult with the juvenile rats. The data would be available and the modeling would not require 

extensive effort once the approach is refined with deltamethrin and permethrin.

8. Integration of lines of evidence:

a. The tissue dosimetry data from the rat suggest higher brain levels in juveniles compared to adults. In 

contrast, the preliminary PBPK modelling for human predicts slightly lower brain concentrations in 

young children compared to adults. Please comment on these differences, including comments on the 

key inputs that lead to this difference, human variability associated with the key parameters, and the 

confidence and uncertainties associated with the difference between the rat and human internal 

dosimetry.

The Panel recognized that, through both the overview material and the background information 

provided for each project, CAPHRA has generated a significant volume of information on 

pyrethroid toxicity in addition to having compiled, discussed, and presented historical data and

current research by others. The data generated constitutes different lines of evidence that may 

point in different directions with respect to the ultimate goal of evaluating juvenile sensitivity to 
49

pyrethroids. Tissue dosimetry from the rat model suggest that there is an expectation of higher 

brain levels of pyrethroid concentrations in juvenile compared to adults, while the PBPK model 

for humans predicts lower concentrations in young children vs. adults’ brain.

 

Combining these two lines of evidence to form a conclusion using a weight of evidence approach 

requires a better understanding of the uncertainty, confidence, and reliability of each line of 

evidence since weight of evidence is an approach that, by means of qualitative or quantitative 

methods, integrates individual lines of evidence to form a conclusion (Linkov et al., 2009). A 

fundamental issue in the weight of evidence approach is a critical understanding of the 

reliability, robustness, and relevance of all the evidence.

The Panel has already addressed issues regarding the uncertainty and confidence on the 

individual lines of evidence in charge questions 2, 3 and 6. 

In particular, with respect to the human PBPK model, while the Panel recognized that the model 

has the advantage of predicting internal tissue concentration for humans, the Panel also 

expressed skepticism with regard to the PBPK modeling results that showed increasing Cmax 

with age in humans, thus implying that children potentially would be less susceptible to adverse 

neurological responses from exposure to deltamethrin than adults. The Panel hypothesized that 

the PBPK model-predicted Cmax in the brain was dependent on the model structure and 

parameter values. In particular, at the exposure conditions simulated, the brain concentration is 

mainly influenced by 1) the brain permeability surface area cross product, the parameter 

controlling diffusion across the blood brain barrier; and 2) the blood flow to the liver, as 

systemic clearance is predicted to be blood flow limited, and not metabolically limited. Since 

children have a higher blood flow per gram of liver, they would clear deltamethrin from the 

plasma more rapidly than adults if the protein binding is assumed to be the same for the ages 

simulated. Therefore, the brain permeability surface area cross product and the blood flow to the 

liver are key inputs to the model. The Panel, as stated in responses to other charge questions, has 

concerns about the values of these parameters and the model structure, and therefore, is not 

confident in the conclusions that CAPHRA has made thus far with respect to the differences in 

the rat and human internal dosimetry. 

The Panel recognized that tissue dosimetry was a measurable endpoint, but it can only be 

measured in rats and the concordance between rats and humans may be questionable. In 

particular, as humans have fewer genes than rats, members of the Panel believe that a rat model 

cannot be a good model for humans. For future studies, members of the Panel have 

recommended the following two alternatives for internal dosimetry experiments: 1) to use guinea 

pigs as they might provide a better model for humans than rats or 2) use ES1 plasma 

carboxylesterase knockout mice, now available. The latter have the advantage of being 

physiologically more relevant to predict neurotoxic effect of pyrethroid to humans. Although this 

type of mouse lacks the ES1 gene, they have normal levels of carboxyleterase in liver, intestine, 

lung, and other organs.

Finally, as discussed in other charge questions (charge question 3 and 6), another major obstacle 

to integrating the two lines of evidence is the significant uncertainty associated with both the 

PBPK model and the experimental data. In light of the above points, the Panel does not believe 
50

that there is enough understanding or confidence in the results obtained from the two lines of 

evidence – e.g. the experimental data and the human PBPK - to utilize a weight of evidence 

approach and draw a conclusion regarding internal tissue dosimetry.

The Panel recommended considering quantitative WOE approaches being formalized by the 

Organization for Economic Co-operation and Development (OECD) for developing Adverse 

Outcome Pathways (AOPs) (OECD, 2014, Becker et al., 2015). The Bradford-Hill (BH) 

considerations (Hill, 1965), originally developed for the evaluation of causality of associations 

observed in epidemiological studies, provide a useful approach for evaluating the extent of 

support for hypothesized AOPs. A formalized framework would have to be applied in order to 

compare the confidence and uncertainty associated with the differences in the rat and human 

dosimetry. Following Becker et al. (2015), it is critical to evaluate the reliability, robustness, and 

relevance of all the evidence using a formal weight of evidence (WoE) approach. The purpose of 

a WoE evaluation is to document certainty in inferring responses beyond interpolation within the 

range of empirical observations in a transparent manner. Confidence in inference is underpinned 

by the degree of certainty that the lines of evidence support the hypothesized inference.
51

b. CAPHRA has proposed to continue in vivo behavioral testing for deltamethrin and permethrin 

in definitive dose-response evaluations. Pyrethroids have been studied for decades and thus 

there is a large body of evidence for these pesticides. Given your response to 8a along with 1) 

the extensive body of scientific literature on pyrethroid toxicity syndromes and high dose 

studies in juvenile rats (e.g., Sheets et al, 19945

); 2) neurolemma studies supported by the 

CAPHRA (Question 2) along with additional in vitro studies (e.g. Meacham et al., 2008); and 

3) recent in vivo studies from the Vorhees & Bruckner labs, please comment on the additional 

scientific value that would be provided in conducting further in vivo rat experiments to assess 

potential for PD sensitivity of human infants and children.

The CAPHRA summary document and presentations clearly indicated that CAPHRA’s proposed 

focus moving forward would be on pharmacokinetics and on the in vitro neurolemma system, 

with minimal if any further behavioral testing (limited to completing definitive studies on 

permethrin and deltamethrin). The conclusions presented in the documents were that there was 

no evidence for any differences in pharmacodynamics in juvenile versus adult rats, but there was 

clear evidence from the deltamethrin studies that brain levels in juvenile rats were higher than 

those in adults at a given dose. The current human model predicts the opposite, with equivalent 

or higher brain levels predicted for the adult. In discussions about the model, CAPHRA pointed 

out that the model prediction of lower brain levels in humans was only for deltamethrin, and it 

will be interesting to see modeled concentrations for other planned test pyrethroids. 

Panel discussions highlighted the great uncertainty surrounding the neurolemma and ion channel 

studies (charge question 2) as well as the need for further work on the PBPK model (charge 

question 6). In particular, the great levels of uncertainty surrounding the neurolemma and ion 

channel studies raised concerns among panel members on the usefulness of (more) neurolemma 

and ion channel studies. A large majority of the Panel believes that the uncertainty in the results 

of the neurolemma studies would not be dissipated even with a very large sample size.

Although the Panel, in general, agreed with CAPHRA’s plan to focus on the pharmacokinetics 

and expand the information on age-related metabolism differences in juveniles and adults, the 

Panel also recognized that in vivo studies should not be abandoned. In particular, the Panel 

suggested that CAPHRA explores new assays that would facilitate an understanding of the 

clinical relevance of the presence of DLM (deltamethrin) in the brain. The Panel emphasized that 

it is fundamental that a clinical endpoint that measures the level of DLM in the brain and its 

clinical relevance be investigated by CAPHRA. Clearly, data that would increase confidence in 

and expand to other pyrethroids the prediction of the equivalent or lower levels of pyrethroids in 

the brains of young children at a given exposure level is the most critical. This would also 

impact the judgment of how relevant the rat studies are to assessing the relative risks of 

pyrethroid exposure in children and adults.

The Panel suggested that in addition to core pyrethroids, CAPHRA should also examine another 

type II pyrethroid, cyhalothrin, whose toxicity is not associated with the same symptoms 

associated with deltamethrin. For example, deltamethrin produces choreoasthetosis, while 

 5 Sheets, L.P., et al., Age-Dependent Differences in the Susceptibility of Rats to Deltamethrin. Toxicology and Applied 

Pharmacology, 1994. 126(1): p. 186-190.
52

cyhalothrin does not. Several references on the differential actions of cyhalothrin on 

neurotransmission are available (Hossain et al., 2004, 2006, 2006, 2013).

.

As that database is expanded, the need for further behavioral testing of additional pyrethroids can 

be reconsidered. It was stated that the blood and tissue level work conducted in parallel with the 

permethrin rat study is nearly completed, and the data from that study should be evaluated in a 

manner similar to that used for the deltamethrin data, relating ASR and DCO responses to brain 

levels, before deciding if and how to proceed with the definitive studies with permethrin and 

deltamethrin. 

The Panel recommended framing the question about the value of additional studies that can be 

conducted to reduce uncertainties in the knowledge related to pyrethroid risks, using a formal 

Value of Information (VoI) framework (Keisler et al., 2014). The Panel found that the approach 

taken in the CAPHRA report suffered from a limited focus and from an evidence-driven line of 

inquiry and did not discuss contribution of individual lines of evidence to the overall goal. 

Specifically, although the Adverse Outcome Pathway to detect an effect on nerves is critical for 

understanding the mechanism and is useful in predicting a response to exposure along that 

specific pathway, it is only one piece of information that should be considered in the decision 

process and collecting additional information should be brought into the perspective of the 

decisions at hand.

A more transparent process for pyrethroid risk assessment would be to enumerate all the 

potential consequences of exposure and the level of effect that is tolerable given the benefits of 

exposure to these compounds. This decision-focused approach ensures that tolerable or 

intolerable endpoints are agreed to before the evidence of the exposure and consequences are 

reviewed. For example, the increased startle response in juvenile rats may indicate risks in 

children of neurosensitivity, attention disorders, autism, or other long-term impacts. The 

relationship between startle repose and these other endpoints should be researched. Once these 

connections are understood or hypothesized, the potential risk can be calculated for each 

endpoint. Where the evidence is unavailable for specific endpoints or exposure levels, those 

studies can be designed to inform the decision- making process based on an understanding of the 

limitations of specific models or approaches. When these data are available to inform the 

process, their role in the decision process can be clearly identified and the studies reviewed for 

relevance and quality.
53

Figure 1: A schematic diagram for a decision-focused approach for evaluating the existing 

research on the effect of exposure to pyrethroids on children with the ultimate goal of evaluation 

and regulation of pyrethroids.

The first step in the process would be to review all the potential risks of pyrethroid exposure to 

juveniles and agree to the degree or frequency of impact that is acceptable. Based on the 

outcomes of such a review, there should be a discussion on what is the most definitive evidence 

for that impact/endpoint (i.e. discussion on what is/is not a good model for that endpoint, and 

what supplementary information is deemed important). In this context, the relevance of the high 

throughput screening or Xenopus oocyte models should be considered (Questions 1 & 2). Does 

the binding and activation of the sodium receptor provide adequate evidence of the effects that 

are indicative of increased risk? In addition to the issue of juvenile susceptibility to 

neurotoxicity, there are several other endpoints of concern according to EPA’s TEACH technical 

summary for Permethrin and Resmethrin (2007). Endpoints to consider in the nervous system 

include activation and impacts on neuronal development, DNA damage and oxidative stress, 

depression of neurogenesis and loss of dopaminergic (DA) neurons. Other endpoints that should 

be considered include skin irritation, respiratory irritation, and immune system activation. The 

mechanism(s) to consider for these endpoints may be outside the scope of the Adverse Outcome 

Pathway for sodium channel activation.

With each of these endpoints, the tolerable level of impact should be designated. It may be 

important to consider the understanding of the long-term impact of small differences in juveniles, 

such as the relationship between DA neuron and Parkinson’s disease. When these decisions are 

set in advance, then the application of research or the identification of the necessary research to 

make a clear decision becomes transparent. With the designation of the tolerable levels of risk 

associated with each endpoint, there should be a discussion of the research/data necessary to 

evaluate that endpoint. Specifically, what exposure levels are common, by what means, and in 

what models.
54

The human pharmacokinetic model is a useful tool for estimating the potential for exposure. 

However, the information provided by the PBPK model needs to be paired with an 

understanding of the dose-response relationship for an effect that implicates a specific outcome 

of a disease state. The AOP for juvenile nervous system activation is a piece of evidence that 

informs the endpoint(s) of interest associated with neuronal activation. If the AOP does not 

predict the level of activation designated as not indicative of an outcome/disease state requiring a 

change in regulation, then the activation endpoint is considered “safe” given the current 

understanding of exposure levels and consequences. The rest of the available literature should be 

reviewed for relevance to the evaluation of the endpoint and the risk of research in the designated 

level of effect. Gaps in the literature can be clearly identified, as well as the necessary 

assumptions for making a decision given the currently available information. One benefit of this 

approach is that it should facilitate subsequent review of future research and be able to identify 

those aspects of understanding that would change the decision to alter the regulation of these 

chemicals.

c. If you believe there is additional scientific value to conducting additional in vivo experiments (8b), 

please comment on the CAPHRA's proposed experiments. In the context of your response to Question 

3, please include in your comments a discussion of dose levels and/or additional study design elements 

to improve existing preliminary evaluations.

If in vivo neurotoxicity studies are continued, the Panel suggested that the proposed study 

designs for the permethrin and deltamethrin definitive studies discussed by CAPHRA are 

pursued. CAPHRA has discussed in the public presentation some strategies to enhance the study 

design, including larger sample sizes and consideration of litter effects. (The basis of the 

determination of the optimal sample size should be presented with the study report.) The doses 

selected appeared reasonable. It would not appear critical to expand the time course. In fact, 

given the considerable discussions concerning the effect of the corn oil vehicle, it may be worth,

in future experiments, to consider using the minimal volume necessary to enhance the rate of 

uptake and peak brain concentration. Reducing the number of trials per session to reduce 

habituation, as presented, may improve the ability to measure the response.

If further in vivo studies with deltamethrin are continued, the inclusion of a 1 mg/kg dose group 

(and maybe lower) in the PND15 rats should be considered. According to the overview 

document, one study was done with PND15 rats with 0, 1, 2, 4 mg/kg dose levels. Although not 

significant at 1 mg/kg, the authors pointed out that the 1 mg/kg group showed a trend of 

decreasing ASR at 8 hrs. (Figure 10, Appendix A2-4) when data from two studies with PND 15 

rats were pooled. A larger number of rats should be used to detect any effects. Note that some 

mortality was observed at 3 mg/kg with mortality increasing at 5, 6, and 8 mg/kg in PND 15 rats. 

At 2 mg/kg in PND15 rats, mobility was mildly affected and rats had mild tremor. The 

observation of DCO at the dose of 1 mg/kg and possibly lower (not previously done) could also 

provide information on adverse neurological effects. 

As neurotoxicity of pyrethroid insecticides is reflected in the alteration of neuronal functions, the 

Panel suggested that CAPHRA conduct additional experiments to monitor neuronal functions 

along with clinical observations of extracellular neurotransmitters release with brain 

microdialysis for better understanding age and dose dependent neurotoxicity of pyrethroids.
55

CAPHRA stated that tremor might reduce ASR since it overlaps with ASR. Further, a suggestion 

for future in vivo studies would be to counter dose it with zonisamide (no specific sodium and 

calcium channeler blocker) to prevent the tremor by blocking of Nav.. 

Finally, as a general comment, the Panel emphasized that Questions b) and c) asked about the 

value of additional studies that can be conducted to reduce uncertainties in our knowledge related 

to pyrethroid risks. Generally, additional evidence is only material if it will reduce the 

uncertainty in a way that changes the decision at hand. A formalized “value of information” 

(VoI) approach can be used to determine the value of an additional study, specifically, what 

would be needed to ensure that information is definitive (statistical power, timing, verification of 

exposure over what time course and with what sensitivity, etc.)? Also the amount of evidence 

needs to be considered. Specifically, what kind of replication of any new study is critical 

(different labs, rat strains, behavioral assays)? To reduce uncertainties regarding the effects of 

pyrethroid exposure, a more transparent process in the context of pyrethroid risk assessment 

should be undertaken. 
56

Literature Cited

Eusebi, F., Palma, E., Amici, M., and Miledi, R.. (2009) Microtransplantation of ligand-gated 

receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: A potent tool for 

expanding functional information. Progress in neurobiology 88 (1), 32-40.

He, B. and Soderlund, D.M. (2011). Differential state-dependent modification of rat Nav1.6 

sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid 

insecticides tefluthrin and deltamethrin. Toxicology. Applied. Pharmacology. 257(3):377-387. 

PMID: 21983428.

Hossain, M.M., Suzuki, T., Unno, T., Komori, S., Kobayashi, H. (2008). Differential presynaptic 

actions of pyrethriod insecticides on glutamatergic and GABAergic neurons in the hippocampus. 

Toxicology. 243:155-163.

Hossain, M.M., Suzuki, T., Sato, N., Sato, I., Takewaki, T., Suzuki, K., Tachikawa, E., Kobayashi, 

H. (2006). Differential effects of pyrethroid insecticides on extracellular dopamine in the striatum 

of freely moving rats. Toxicology. Applied. Pharmacology.217: 25-34.

Hossain, M.M., Suzuki, T., Sato, I., Takewaki, T., Suzuki, K., Kobayashi, H. (2004). The 

modulatory effect of pyrethroids on acetylcholine release in the hippocampus of freely moving 

rats. NeuroToxicology 25(5):825-833.

Hossain, M.M., Suzuki, T., Kobayashi, H., Richardson, J.R. 2013. Acute effects of pyrethroids on 

serotonin neurotransmission in the striatum of awake rats: in vivo microdialysis study. J. Biochem. 

Mol. Toxic. 27(2):150-156.

Ivorra, I., Henriquez, M., Lax, P., Riquelme, G., and Morales, A. (2002). "Functional 

transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane 

to Xenopus oocytes." Pflügers Archiv 444 (6): 685-691.

Jamei, M, Marciniak, S, Feng, K. et al (2009) The Simcyp population-based ADME simulator. 

Expert Opin Drug Metabolism Toxicology, 5, 211-223.

Keisler, J. M., Collier, Z. A., Chu, E., Sinatra, N., & Linkov, I. (2014). Value of information 

analysis: the state of application. Environment Systems and Decisions, 34(1), 3-23.

Khalil & Läer (2014) Physiologically Based Pharmacokinetic Models in the Prediction of Oral 

Drug Exposure Over the Entire Pediatric Age Range—Sotalol as a Model Drug

American Association of Pharmaceutical Sciences Journal Mar; 16(2), 226–239.

Linkov, I., Loney, D., Cormier, S., Satterstrom, F. K., & Bridges, T. (2009). Weight-of-evidence 

evaluation in environmental assessment: review of qualitative and quantitative approaches. 

Science of the Total Environment, 407(19), 5199-5205.
57

Meadows L.S., Chen Y.H., Powell A.J., Clare J.J., & Ragsdale D.S. (2002) Functional 

modulation of human brain Nav1.3 sodium channels, expressed in mammalian cells, by auxiliary 

beta 1, beta 2 and beta 3 subunits. Neuroscience. 114(3)745-753.

Meacham, C.A., Brodfuehrer, P.D., Watkins, J.A., Shafer, T.J. (2008). Developmentallyregulated sodium channel subunits are differentially sensitive to α-cyano containing pyrethroids. 

Toxicology. Applied. Pharmacology 231(3)273-281. PMID: 18538810.

Organization for Economic Co-operation and Development (OECD), (2014) Adverse Outcome 

Pathways, Molecular Screening and Toxicogenomics. Available online at:

http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screeningand-toxicogenomics.htm.

Tan, J. and Soderlund, D.M. (2010). Divergent actions of the pyrethroid insecticides Sbioallethrin, tefluthrin, and deltamethrin on rat NaV1.6 sodium channels. Toxicology Applied

Pharmacology 247(3):229-237. PMID: 20624410.

Thimmapaya, R., Neelands, T., Niforatos, W., Davis-Taber, R.A., Choi, W., Putman, C.B., 

Kroeger, P.E., Packer, J., Gopalakrishnan, M., Faltynek, C.R., Surowy, C.S., Scott, V.E. 2005. 

Distribution and functional characterization of human NaV1.3 splice variants. Eur. J Neurosci. 

22(1):1-9. PMID: 16029190.