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 Mi l bourn

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

, M PH

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 , 201 5 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 creat e or confer legal rights or impose any legally bi nding 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

..................................................................................................... 563 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 environ ment. 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 faci ng 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, t he 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 necessari ly 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 S ecretary. 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

- 201 5

- 01 30 , 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 Biochem ical 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, MD6 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, M I

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 Sci ence Focus Area Lead US Army Engineer Research and Development Center Concord, MA7 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, RI8 L IST OF C OMMONLY U SED A CRONYMS AND A BBREVIATIONS

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

D CO: 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: Pharmacodynamic9 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 A gency the authority to establish tolerances for residues on food and/or f eed resulting from use of a pesticide. The studies required to allow the A gency 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 21 st 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

d irection 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 f or 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 neurot oxicity (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) review ed 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 21 st Century using a combination of in vitro studies , targeted in vivo studies , and phy siologically

based pharmacokinetic (PBPK) models.

Based on feedback from the SAP and the A gency, 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 w ith 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 t ra nsplantation 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, has10 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 construc tive. 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 A gency sought the FI FRA 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 publi c 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 studie s using human sodium channels expressed in mammalian cells with their regulatory b eta subunits (ChanTest Data)

- The Panel concurred that the proposed high

- throughput screening approaches would not be useful in depicting the potential for juvenile sensitivity f rom pyrethroids. They noted that there

were several factors that diminish ed the strength of the data ( e.g. the large variability in protein expression and many of the electrophysiological parameters).

• Topic 2) N eu rolemma studies: Transplantation of adult & juvenile rat brain synaptic membrane into (Xenopus) \ oocytes

- The Panel expressed a lack of confidence i n 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

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

dose selection was lacking . The Panel found that the d etailed 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 need ed

. However, the Panel detailed a few deficiencies

. . One deficiency

noted was the lack of data in addressing age

- related differences.

• Topic 5 ) Physiological ly : based pharmacokinetic model in rat

- The Panel had concerns regarding the confidence, accuracy, and uncertainty associated with this model. They also

noted th at the model appear ed to be in a preliminary form

. They also recommended

further research to better valid ate the model.

• Topic 6 ) Physiological ly : 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) Physiological ly : based pharmacokinetic models for humans with other pyrethroids

- The Panel concurred that using the PBPK model to evaluate other12 pyreth r oids was no t 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. The y recommended exploration of alternative experiments to learn about

internal dosim et ry. Also the Panel recommended the consideration of the Weight of Evidence (WOE) a pproaches 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 voltage

gated sodium channels (Na Vs 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, Na V selection, etc). Please comment on the confidence and

uncertainties in t he ChanTest experiments and related findings.

The FIFRA SA P 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 fundamental ly limited by the heterologous expression system where the Na V 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 (I peak ), normalized tail current,

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

extends to all Na V 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 ar e necessary before conclusion can be drawn

about differential influences on Na V isoforms and juvenile toxicity. Importantly, many of the most acutely toxic pyrethroids in vivo failed to show detectable influences on Na V channel parameters, bringing into qu estion 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

Ca 2+ oscillations, which are tightly coupled to membrane electrical activity as an integral part of

the AOP.14 b. CAPHRA thinks that add itional 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 agree d with CAPHRA that the ChanTest approach using expressed Na V 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; Meacha m et al., 2008; Tan and Soderlund, 2009 1 ) 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

betwe en rats and humans.

The Panel unanimously concluded that the ChanTest experiments do not sufficiently discriminate among the electrophysiological properties of expressed Na V isoforms. Moreover, the ChanTest approach does not have the sensitivity to iden tify modest to moderate differential influences of pyrethroids of sodium channel gating kinetic parameters . T his 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 Na V isoforms measured by ChanTest. The data reporting stronger response of rat Na V 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 Na V s thereby making comparisons difficult and possibly misleading. The weak effects of pyrethroids in the ChanTest protocol and th e 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 EC 50 values for Na V 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 channel s 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 conduc ted 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 b locker 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 size s used were too s mall, 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 comp ared.

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 obser ved, 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, th e 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 conc luded that the usefulness of using this system for determining age

related 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 pyrethroid s, 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 2 ) which showed that at a similar dose (2 mg/kg deltamethrin) PND 10 rats had increased severity of clinical signs and higher C
max 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 Pa nel 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 sensitivit y to the Acoustic Startle Response (ASR) observed in PN D 15 rats was not observed in PN D 17 and PN D 21 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 shou ld 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 b een 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 f or permethrin.

2 Kim KB

, Anand SS

, Kim HJ

, White CA

, Fisher JW

, Tornero

- Velez R

, Bru ckner 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 det ailed 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 discussio n 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 o ne study indicates that the Tactile Startle Response (TSR) test provides a somewhat larger d ynamic 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

su bjective 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 tha t 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 magn itude 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 PND1 5 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 neur otoxicity 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, ha s not been established. The Panel commented that tremors and/or writhing in rats m ight 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

comb ination 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 D L M, 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 unknow n factors involved in the potential dose

and time

- related mechanisms underlying the effect of D L M 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 data18 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 exami ne 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 A gency 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 tha t the dos e volume does not seem to appreciably influence the D L M 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 h ou r s , 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 exposur e 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 d ependent 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 evaluat ed. 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 experime nts 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

- bra in19 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 appea rs 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 e xcellent 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 quantitate d 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. T he 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 ch anges 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 developmen t 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, t he 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 s tudy, lack of adequate discussion for resu lts 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 dist ribution. 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 experime nts in the human tissue conducted thus far. Please include in your comments a discussion of the ongoing in vitro experiments with recombinant20 enzymes for use in PBPK models and associated confidence and uncertainty with the use of

such data.

The Panel not ed 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: o ntogeny data on human liver carboxylesterases; o ntogeny 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 wou ld generate uncertainty in the use of the data . These included l ack of estimation in hepatic clearance in primary human hepatocytes from various age populations (cryopreserved hepatocytes)

, and

, use of different laboratories to determine ontogeny expressi on, 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 si mulate 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

deltamethri n 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. Non

chemical 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 coeffi cients, 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 associ ated with the deltamethrin developing rat model. Please also comment on the sensitivity analyses of parameters CAPHRA has completed thus far.

While t he 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 v ehicle

- specific absorption of

deltamethrin

, p lasma metabolism a ge

- specific plasma protein binding

, and t he 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 certain21 model structures ( e.g. lymphatic versus portal uptake of deltamethrin). The lack of a quantitative

assessme nt 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 prov ide 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 mo deling 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 t he 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 d etermine 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 fo r 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 paramet ers, 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 CAPH RA 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 PB PK 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 param eters 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.

Whil e 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 r eported 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, espec ially in light of its influence on the C
max predictions,

and

, the pathway of absorption that is structured to completely bypass the liver and p ortal system by switching to the lymphatic system. The Panel believed that the differences between rats and

human s 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 da ta from 1

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

.

The Panel suggest ed revision s to the PBPK model focused on the definition of clearance, the definition and usage of age grouping when modeling the live r 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 s ufficient evidence to comment on the reliability, confidence

, and uncertainty of the age

- related brain C
max 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 t o 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, extens ive 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) t he involvement of b oth hydrolysis and

oxidation in metabolism (w hether and to which extent these two elimination pathways differentially contribute to the overall clearance of a pyrethroid is not clear ) ; (2) t he presence of both hydrolysis and oxidation enzymes in the liver and extrahepatic tissues (t here is no information on organ

- specific clearance of a pyrethroid, as well as age

- dependency in this regard ) ; and

, (3) t he potential for differential induction of cytochrome P450s over carboxylesterases in humans by pyrethroids, which are activators of pregnane X receptor (h ow 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 co mparison 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 provide d comments on the appro priateness 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 app e ared to still

be under development and the experimental work has not yet been completed. In theory, if the issues regard ing 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 ou t 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, permeability

surface

- 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, hu man 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 questi ons

. Of particular concern was t he 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 the24 conclusions that CAPHRA has reached with respect to the differences between rat and human internal dosimetry.

The Panel strongly question ed the rat as a good model to learn about the response of the human brains to exposure to de ltamethrin 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 potential ly more appropriate animal model s 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) approache s 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 liter ature on pyrethroid toxicity syndromes and high dose studies in juvenile rats (e.g., Sheets et al, 1994 3 ); 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 standpoin t, 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 encourage d 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 siz es.

Moving forward, the Panel urge d CAPHRA to: (i) C ontinue pursuing in vivo studies, focus on developing new assays

, and determin e a clinical endpoint that could assess the clinical relevance

of deltamethrin ( DLM ) presence in the brain; (ii) E xamine 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 t hat CAPHRA re

- evaluate the approach

undertaken thus far to evaluate the effect of exposure to pyrethroids on the nervous system. More specifically, the Panel suggest ed a more transparent approach that would : 1) r eview all the potential risks of pyrethroid exposure on juveniles , 2) d efine the degree or frequency of impact that is acceptable, 3) e xamine indications of potential endpoints and outcomes of disease

states / potential impacts , and 4) p rovide the most definitive evidence for such endpoint s . 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 believe d 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 dose

response relationship for an effect that impli cates 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 evalu ations.

If in vivo neurotoxicity studies are continued, the Panel suggest ed that larger sample sizes be used and litter effects be considered, as proposed for further deltamethrin and permethrin studies. Relative to dose, the Panel recommend ed 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 suggest e d using the minimal volume necessary to enhance the rate of uptake and peak brain concentration in future experiments. Additional suggestions include d : (i) p erform ing 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) c onsider ing ways to reduce tremor in rats as it might interfere with ASR; and (iii) e xamin ing how tissue con centration 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 voltage

gated sodium channels (Na Vs 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, sta tistics, background information, Na V selection, etc).

The Panel acknowledge d 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 V olta ge Gated Sodium Channe l ( VGSC )

, interac tions with beta subunits appear to be important for optimal targeting of the Na V alpha subunit to the surface membranes and finer aspects of channel modulation. In this regard, Na Vs 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 c omparative investigations based on expression of ion channel subunits in heterologous models must be tightly controlled in terms of the l evel of protein expression and targeting to the surface membrane. I n the ChanTest case, the level of expression and targeting of

each Na V 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 I peak among Na V 1.3 cells. The variability in NaV1.3 /β1β2 expression may explain why I peak varies appreciably among cells (

- 1.4

- 2.7nA). Based on the information provided, and the degree of variability in the basic ele ctrophysiological properties of

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

The Panel raised s everal statistical concerns

. For instance in regards to s ample size, how many data points in each group are being compared ? The Panel noted that the sample size s that were27 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 i nferences about the similarity or differences between human VGSC properties reported by ChanTest and those previously reported with the rat Na V 1.3 expressed in
oocytes are neither warranted nor valid unless the homologous rat Na V isoform lines (in the

pres ence or absence of ß1ß2 electroporation) are recorded using the IonWorks Barracuda platform (at least NaV 1 .3 for validation ) . For valid cross species comparisons (human vs rat)

, it is necessary to make measurements under identical experimental conditions. Th e 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 pyr ethroids 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 V GS C 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 sodiu m 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 Na V 1.3 channels exhibiting a 3

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

~3 fold slower kinetics at test pulse (V t ) of

- 15mV compared to human Na V 1.3 ( Fig 1 C& D; Tan & Soderlund 2009)

. I t 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 Na V 1.3 expressed in HEK (ChanTest) and those reported by Tan and Soderlund (2009) are not possible because the data are

not included in the ChanTest r eport. Furthermore r ecent 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 kineti cs)

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

Na V 1.3 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 explain s the differential

influence of the two pyrethroids on Na V 1.3 ß1ß2 or o ther isoforms?28 There are several published papers that report that Na V 1.3 interacts with β1 and/or β3

, and at least one paper that indicates that β 2 has no influence on Na V 1.3 singly or in combination with β1 or β3 ( Meadows et al 2002 ). I t is unclear what influence this has on the results reported by

ChanTest

.

There are some discrepancies in reporting the EC 50 value for I tail 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 Na V s to other studies that were done using very different expression models. Again, summary statistics are not given in any of the Na V summary tables ( Figures 1A&B; 2A&B).

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

sodium channel isoform s. For example, three splice variants of Na V 1.3 have been found in the human brain ( Thimmapaya et al., 2005 ). While it is unclear the role t hat 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.

T able 1 below summar izes quantitative measures of Na V 1.3 and Na V 1.6 channel function reported by ChanTest with addition of rat oral LD 50 values and aqueous solubility for 6 pyrethroids included in the ChanTest report (LD 50 and solubilities obtained from ChemID PLUS ( http://chem.sis.nlm.nih.gov/chemidplus/ ). Evident from T able 1 is the lack of definable

association between Na V 1.3 or Na V 1.6 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 Na V 1.3 and Na V 1.6 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 agree d that the ChanTest approach is unlikely to contribute useful information to characterize the potential for j uvenile sens itivity of pyrethroids

. T he approach is fundamentally limited by the heterologous expression system where the Na V isoforms are over

- expressed out of context and exposed to acute challenges to most pyrethroids above their solubility range.

A m ore defensibl e approach for high

- throughput screening would be to implement primary

neuronal networks in c ulture s isolated from juvenile rodent or human iPSC derived neuronal cells grown in culture and measure electrical network activity using FluoVolt or patterns of synchronous Ca 2+ 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 i n the literature (e.g. Choi and Soderlund, 2006; Meacham et al.,
2008; Tan and Soderlund, 2009 4 ) show stronger responses to pyrethroids. Please comment on the extent to which the expressed human sodium channels can be used in combination with these source s 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 Na V isoforms and do not discriminate differential influences of pyrethroids having large range of acute neurotoxicological potencies on

Na V isoforms. The data reporting stronger response of rat Na V 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 ph osphorylation status that could influence interactions between pyrethroids and Na V s (Meach a m 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 iso form 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 EC 50 values for Na V 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 pyrethroi d insecticide tefluthrin. NeuroToxicology, 2009. 30 (1): p. 81

- 8930 Inhibitors of competi ng 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 considera tion 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 l ooking 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 cha nnels affected by pyrethroid insecticides.

Concentration

- related pyreth r oid effects on brain neurolemma isolated from Postnatal Day 15 ( PND15 ) and PND90 rats were measured

, but no differences in potency (EC 50 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 Na V and the signal did not change. The scientists acknowledge that some yet to be characterized channel s 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 oocy te experiments, the Panel raised the following concerns:

S ample size : V arious 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 : P arametric curves were fit to the concentration

dependent 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 : T here are very large error bars ( e.g. SEM) in the plots showing t he effect of permethrin and del tamethrin 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 increase d (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 clarif y 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 tr ansformations 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 interpretatio n and differences between adults and juveniles with respect to pyrethroid sensitivity. The Panel recommended that a dditional validation studies are needed to clarify the

relevance of this model.

b. In the context of your response to 2a, the CAPHRA has pro posed 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 pa tterns 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 comm ent on the CAPHRA’s proposed path forward.

The Panel acknowledge d 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 Na V components of juvenile and adult P2

- neurolemma are structurally identical only differing in the

levels of Na V 1.3 and Na V 1.6 present. Perhaps there is some value in testing additional

pyr ethroids, but the expectation that any age

- related pharmacodynamic differences will be detected appears to be small unless a pyrethroid specifically targets Na V 1.3 or Na V 1.6. 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 Sprague

Dawley rats were treated with deltamethrin and permethrin a s 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 c hanges 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 s howed that at a similar dose (2 mg/kg deltamethrin) PND 10 rats had increased severity of clinical signs and higher C

max 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 prelim inary 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 a nd 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

Pa nel commented that t he study design also permitted rigorous statistical analysis. Along with these positive observations, t he 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 info rmation is important since the A gency’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 PN D 17 and PN D 21 rats, but not

PN D 15 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 app ropriateness 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, i t was revealed during

the oral discussions at the public SAP meeting that the decision to use PN D 15 rats was based on

the 2010 SAP where it was stated that the PN D 15 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 t he rationale behind the use of only male rats in the CAPHRA in vivo studies was not di scussed in the in vivo study reports. During the public meeting , it was stated that there were data demonstrating that there were no

sex effects. T he Panel noted that this data should have be en 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, w ith regard to the pretesting of animals in order to improve performan ce, 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

. F or example

, in Fig 1 of A2

- 4 the

initial V
max of control animals var ied signif icantly. This observation raise d the question of

whether or not this was related to the pretest grouping

. It also introduce d the question of if the pretesting approach ha d 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 e ffect this may have on the overall experimental design. Habituation in the ASR studies with adults after 2 h ou r s 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) w ere 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, t he Panel expressed c oncern that even though both control pups and treated pups originated from the same litter, the litter should have been included in the statistical analysi s. 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 stre ss 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 pub lic 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

bre eding colony onsite and not be subjected to the potential stress during shipping. T he 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 audit ory 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 th e type of data obtained (continuous, ranked).34 One study indicate d that the tactile startle response ( TSR ) test provide d a somewhat larger dynamic range; however, more studies may need to be done across ages, compounds and doses,

to support the robustnes s of this conclusion . Given that they both appear ed 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

, th ese 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 note d that the DCO observ ations, and tremor s in particular, might better fit the “adverse” description as opposed to a compound effect.

A dult rats exposed to PM and tested in the ASR did not exhibit any clinical signs of toxicity, including tremors. The Panel suggested that perha ps 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 pre

weaning results, there s eems 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 ind uce 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 CAPHR A 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 incr ease 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. o f 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 erro r 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 is sue 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 pyreth r oid 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 sen sitive 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 o verall c onclusion , the Panel concurs t hat i t 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 sensi tive 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 a id in the interpretation of the auditory startle and detailed clinical observation data.

For D L M, the time of the rise in plasma and brain levels d id 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 dose

and time

- related mechanisms underlying the effect of D L M 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 nee ded to resolve the pharmacodynamics question.

While i t 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 an d 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

dif ferences. The A gency 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 D L M experiments t he dose volume d id not significantly

influence the timing of ASR effects. However it was clear that a large volume delay ed 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 testi ng

.

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 h ou r s , or larger volume and test at 4 or 6 hours? The answer may lie i n 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 fo r 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 on ly to reduce animal use but also to test more human relevant doses, it m ight 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 data base 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 p anel members were concerne d 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 ab out 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 whi ch 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 pharma cokinetics 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 val idate 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 meas urements 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 i n 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 cons tructing 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 CAP HRA 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 d eveloping 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 the se 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 mic rosomal 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 ex cept 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. T his 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 microsomes38 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 propose d metabolism wor k, in particular enzyme prepara tions 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 stu dies that addressed possible age

related 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 completel y bypassing the liver and directly entering the lymphatic system. As these are critical model assumptions that affect model predictions of age

related 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, t he 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 pyret hroid compounds that undergo significant biotransformation through intestinal CYPs and carboxylesterases. Some panel members recommended monitoring both the loss of deltamethrin and the formation of pr oducts to gain

additional confidence on the results.

b. P lease 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. Whil e single intravenous (I V ) or oral dosing for pharmacokinetics studies are necessary for establishing kinetic parameters, it does not reflect true environmental exposure to deltamethrin or other

pyre throids. 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 that39 PND15 and PND90 but not PND21 group s had dose

- dependent increases in Cmax values (Figure 3

- 2), but there was no explanation for th is 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 P anel 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 resul ts 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 adminis tered 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 impor tant 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 explanati on in their O verview D ocument 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. On e area that was noted as needing explanation was the lack of age

- related differences for permethrin as compared with deltamet h rin. The lack of difference had been noted

in the O verview D ocument

, but no further information was provided

. T hese differences in age

related 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,

th ese 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 experi mentally.

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 c onsistent 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 t he Panel and would generate uncertainty in the use of the data including: (a) lack of studies determining hepatic40 clearance with primary human hepatocytes from various age populations (cryopreserved hepatocytes); and (b) determination of o ntogeny expression of various enzymes by different

laboratories

. The Panel questioned why the same samples were not used for all ty pes 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 enz ymes 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 l iver 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

com bined 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. Non

chemical specific physiological parameters for rats were obtained from the publish ed 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 par tition 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, plea se 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 uncertain ties 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 i nformation 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 an d

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 simu lated 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

metabo lic contribution of CES to pyrethroid hydrolysis. As shown in Tables 3

- 1 and 3

- 2 of the41 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 value s. 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, t he 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 co nfidence 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 parameter s. 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 pyret hroid 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 r egions 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 amon g 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 comparis ons 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,

l ymphatic 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 me mber 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, wh ich 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 di screpancy 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

- surfac e

- 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 del ta methrin 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 human s . 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 inc orporated in the model

.

In briefly looking through the rat model code, the majority of the code looked appropriately

described. One minor note from t he Panel: In the model code, the percentage of brain attributed

to blood volume in the tissue was sligh tly 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 deltamethri n 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 p ublished 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 P anel recommended several studies that were needed to better validate the predictions of the

current model and provide further refinement,particular ly with respect to predicted brain concentration of deltamethrin in older versus younger rats. In charge que stion 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 pyrethroid s are important to look at the potential accumulation in the brain and as a source to validate model predictions. The O verview

D ocument stated there was no expected accumulation of deltamethrin, but it did not present supporting data. Since the neurologica l 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 age

related 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 exper imental data or by model predictions of the data. The P anel 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 trans port 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 t o 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

- brai n 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 f or risk assessment for

children.

6. Physiological ly

- 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 t he 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 involv ed 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 fac tor data collected by the CAPHRA and the SIMCYP database. The44 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 e nzyme. 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 appreciate d the effort of CAPHRA to develop a human PBPK model to extr apolate internal tissue exposure to pyrethroids (specifically deltamethrin) in humans, with the ultimate goal to assess whether juvenile s experience an increased sensitivity to pyrethroid neurotoxicity

. Following on the same construct of the rat PBPK model , the human PBPK model for

deltamethrin estimate d /predict ed 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

- suppo rted 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 expresse d the following concerns regarding some of these model parameters:

1

- The Panel noted some discr epancies 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 O verview D ocuments (specifically, with respect to the GI uptake and

the perme ability

- 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 d id not match the values listed in t he 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 P anel had the following concerns regarding the model structure s pecifically:45 1

- The Panel raise d concerns regarding the fact that the model switche d from uptake to the portal
system to bypassing the liver completely via the lymphatic system.

In a ddition , the Panel was concerned about whether there were differences between rats and humans in the role of transporters in gut absorption.

2

- The Panel expresse d 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:

p lasma concentration ratio from the steady state experiments seem ed to suggest that there is more than just a permeability limitation for the distribution.

3

- The Panel recognize d 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 raise d 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 highlight ed the fact that the available human data showcase d large variability among individuals, presumably and at least in part du e 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 simula tions, etc).

The Panel recognize d 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 c hildren compared with adults. The Panel believe d that there should be more variability in body fat percentage especially if sex is taken into account: the common approach in the literat ure on percentage body fat algorithms is to account for both age

- and s ex

- specific variation.

The Panel suggested that CAPHRA c onsider 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 exagger ate 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 10 th 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 gro up children altogether. Rather

, the Panel believe d 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 (str uggling 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 det ected 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 sufficient ly 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 age

related increase in Cmax in the brain.

One aspect of the mo deling effort that the Panel expresse d 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 specie s 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 cap acity 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 t he 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 ga ps 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 t o 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 support s 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 durin g 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 (CL int, app ) into the modeling (live r, 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 regulate d 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. Subg rouping 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. Physiological ly

- 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

co nduct 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 difficul t 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 a sked to make comments and recommendations on a concept that had not yet been thoroughly vetted.48 The Panel noted that i n theory, if the issues raised with regard to the rat and human model were

addressed to decrease uncertainty in the model and increase ac curacy 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 t he 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, cis

permethrin is hydrolyzed comparably by CES1 and CES2. However, trans

- permethrin is predo minately 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 P anel m ade 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 coeff icients 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

determine d

.

2. The in vitro kinetics were recommende d 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, permet hrin may serve as a prototype to guide modeling of other pyrethroids.

3. The P anel 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

ad ult 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

k ey 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 recognize d 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 to49 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 concentration s 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, in tegrates 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 al l the evidence.

The Panel has already a ddressed 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 recognize d that the model has the advantage of predicting in ternal 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 r esponses 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 concentrati on 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 me tabolically limited. Since children have a higher blood flow per gram of liver, they would clear del ta methrin from the plasma more rapidly than adults if the protein binding is assumed to be the same for the ages simulated. Therefore, the brain permeabilit y 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

confiden t in the conclusions that CAPHRA has made thus far with respect to the differences in the rat and human internal dosimetry.

The Panel recognize d that tissue dosimetry was a measurable endpoint, but it can only be

measured in rats and the concordance betw een 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 P anel have

recommended the following two alter native s 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 rele vant to predict neurotoxic effect of pyrethroid to humans. Although this type of m ouse lack s the ES1 gene , they have normal levels of carboxyleterase in liver, intestine, lung, and other organs

.

Finally, as discussed in other charge questions (charge ques tion 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 believe50 that there is enough under standing 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 recommend ed consi dering 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

comp are the confidence and uncertainty associated with the differences in the rat and human dosimetry. Following Becker et al. (2015), it is cr itical to evaluate the reliability, robustness

, and

relevance of al l 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 b ody of scientific literature on pyrethroid toxicity syndromes and high dose studies in juvenile rats (e.g., Sheets et al, 1994 5 ); 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.

Th e 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 s tudies 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 predi ction 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

s tudies (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 o f (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

, agree d with CAPHR A’s plan to focus on the pharmacokinetics and expand the information on age

- related metabolism differences in juveniles and adults, the Panel also recognize d that in vivo studies should not be abandoned. In particular, the Panel
suggest ed that CAPHRA explores new assays that would facilitate an understand ing of the clinical relevance of the presence of DLM (deltamet h rin) in the brain. The Panel emphasize d 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 suggest ed that in addition to core pyrethroids, CAPHRA should a lso 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 differenti al actions of cyhalothrin on

neurotra nsmi ssion 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 fr om 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 re commend ed 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 tha t the approach taken in the CAPHRA report suffer ed from a limited focus and from an evidence

- driven line of inquiry and d id 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 add itional 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 e ndpoints 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 impac ts. 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 endpoi nts 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 proc ess 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 pyrethro ids 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 releva nce 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 n euronal 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, b y 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 speci fic 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 indica tive 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 benefi t 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 scien tific 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 el ements to improve existing preliminary evaluations.

If in vivo neurotoxicity studies are continued, the Panel suggest ed that the proposed study designs for the permethrin and deltamethrin definitive studies discussed by CAPHRA are pursued. CAPHRA has disc ussed in the public presentation some strategie s 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 dos es selected appear ed 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 volum e 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 contin ued, the inclusion of a 1 mg/kg dose group (and maybe lower) in the PND15 rats should be considered. According to the overview document, o ne 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 hr s. (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 PND 15 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 suggest ed 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 p yrethroids.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 bloc king of Na
v. .

Finally, as a general comment, the Panel emphasized that Questions b) and c) ask ed 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 c ourse 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

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