cc: 

EPA 

Stephen Owens (OCSPP) 

James Jones (OCSPP) 

Steven Bradbury, Ph.D. 

William Diamond 

Vicki Dellarco, Ph.D. 

William Jordan 

Margie Fehrenbach 

Joan Harrigan-Farrelly 

Jack Housenger 

Mike McDavit 

Donald Brady 

Tina Levine, Ph.D. 

Steve Knizner 

Jack Fowle, Ph.D. 

Oscar Morales 

Richard Keigwin, Jr. 

Lois Rossi 

Anna Lowit, Ph.D. 

Edward Scollon, Ph.D. 

Rogelio Tornero-Velez, Ph.D. (ORD) 

Jimena Davis, Ph.D. (ORD) 

R.Woodrow Setzer, Ph.D. (ORD) 

OPP Docket 

Doug Parsons (OCSPP) 

Enesta Jones (OPA) 

Vanessa Vu, Ph.D. (SAB) 

FIFRA SAP Members 

Janice Chambers, Ph.D., DABT, ATS 

Steven Heeringa, Ph.D. (FIFRA SAP Chair) 

Gerald LeBlanc, Ph.D. 

Carey Pope, Ph.D. 

Kenneth Portier, Ph.D. 

Daniel Schlenk, Ph.D. 

FQPA Science Review Board Members 

Mary Jane Alcorn, Ph.D. 

John Bowyer, Ph.D. 

Abby Collier, Ph.D. 

Marion Ehrich, Ph.D. 

Penelope Fenner-Crisp, Ph.D., DABT 

Mari Golub, Ph.D. 

Gaylia Jean Harry, Ph.D. 

Dale Hattis, Ph.D. 

Wendy Heiger-Bernays, Ph.D. 

Sastry Isukapalli, Ph.D. 

Dallas Johnson, Ph.D. 

Teresa Leavens, Ph.D. 

Nu-may Ruby Reed, Ph.D., DABT 

Sonya Sobrian, Ph.D. 

 

2 




5

Panel Members for the Meeting of the Federal Insecticide, Fungicide and 

Rodenticide Act Scientific Advisory Panel (FIFRA SAP)

to consider and review

Comparative Adult and Juvenile Sensitivity Toxicity Protocols for 

Pyrethroids

July 23, 2010

PARTICIPANTS

FIFRA SAP Chair

Steven G. Heeringa, Ph.D.

Research Scientist & Director for Statistical Design

University of Michigan Institute for Social Research

Ann Arbor, MI 

Designated Federal Official

Sharlene R. Matten, Ph.D.

US Environmental Protection Agency

Office of Science Coordination & Policy

FIFRA Scientific Advisory Panel

EPA East Building, MC 7201M

1200 Pennsylvania Avenue, NW

Washington, DC 20460

Tel: 202-564-8450, Fax: 202-564-8382, matten.sharlene@epa.gov

FIFRA Scientific Advisory Panel Members

Janice E. Chambers, Ph.D., DABT, ATS 

William L. Giles Distinguished Professor 

Director, Center for Environmental Health Sciences 

College of Veterinary Medicine 

Mississippi State University 

Mississippi State, MS

Gerald A. LeBlanc, Ph.D.

Professor and Department Head

Department of Environmental & Molecular Toxicology

North Carolina State University 

Raleigh, NC

Carey N. Pope, Ph.D.

Professor, Head & Sitlington Chair of Toxicology
6

Department of Physiological Sciences

Oklahoma State University College of Veterinary Medicine

Stillwater, OK

Kenneth M. Portier, Ph.D.

Program Director, Statistics

American Cancer Society

National Home Office

Atlanta, GA 

 

Daniel Schlenk, Ph.D.

Professor of Aquatic Ecotoxicology & Environmental Toxicology 

Department of Environmental Sciences 

University of California, Riverside 

Riverside, CA

 

FQPA Science Review Board Members

Mary Jane Alcorn, D.V.M., Ph.D. 

Associate Professor of Pharmacy 

Pharmacy Graduate Chair 

University of Saskatchewan

Saskatoon, Saskatchewan

CANADA 

John F. Bowyer, Ph.D.

Research Pharmacologist

Division of Neurotoxicology

National Center for Toxicological Research

Department of Health & Human Services 

Food & Drug Administration 

Jefferson, AR

Abby C. Collier, Ph.D.

Assistant Professor of Pharmacology

John A. Burns School of Medicine

University of Hawai’i at Manoa, Kaka’ako campus

Honolulu, HI

Marion F. Ehrich, Ph.D., DABT, ATS

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
7

Penelope A. Fenner-Crisp, Ph.D., DABT

Private Consultant

North Garden, VA

Mari S. Golub, Ph.D., DABT 

Director, Murine Behavior Assessment Laboratory

California Regional Primate Research Center

University of California, Davis

Davis, CA

Dale B. Hattis, Ph.D.

Research Professor

The George Perkins Marsh Institute

Clark University

Worcester, MA

Wendy J. Heiger-Bernays, Ph.D. 

Associate Professor

Department of Environmental Health 

Boston University School of Public Health 

Boston, MA

Gaylia Jean Harry, Ph.D.

Head of Neurotoxicology Group 

Division of Intramural Research

National Institute of Environmental Health Sciences

Research Triangle Park, NC

Sastry S. Isukapalli, Ph.D.

Assistant Professor

Department of Environmental and Occupational Medicine

University of Medicine and Dentistry

New Jersey Robert Wood Johnson Medical School

Piscataway, NJ

Dallas E. Johnson, Ph.D. 

Professor Emeritus

Department of Statistics

Kansas State University

Manhattan, KS

Teresa L. Leavens, Ph.D.

Research Assistant Professor 
8

College of Veterinary Medicine

Center for Chemical Toxicology Research and Pharmacokinetics

North Carolina State University

Raleigh, NC

Nu-may Ruby Reed, Ph.D., DABT

Staff Toxicologist

Department of Pesticide Regulation

California EPA

Sacramento, CA 

Sonya K. Sobrian, Ph.D.

Associate Professor

Department of Pharmacology

Howard University College of Medicine

Washington, DC 
9

INTRODUCTION

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

(SAP) has completed its review of the Agency’s analysis of Comparative Adult and Juvenile 

Sensitivity Toxicity Protocols for Pyrethroids. Advance notice of the SAP meeting was 

published in the Federal Register on April 30, 2010. The review was conducted in an open 

Panel meeting on July 23, 2010 at One Potomac Yard, Arlington, Virginia. Materials for this 

meeting are available in the Office of Pesticide Programs (OPP) public docket or via 

Regulations.gov, Docket No. EPA-HQ-OPP-2010-0378. Steven Heeringa, Ph.D., chaired the 

meeting. Sharlene Matten, Ph.D., served as the Designated Federal Official. Stephen Bradbury, 

Ph.D., Director, Office of Pesticide Programs (OPP), and Tina Levine, Ph.D., Director, Health 

Effects Division, OPP provided opening remarks at the meeting. Presentations of technical 

background materials were provided by Edward Scollon, Ph.D., OPP, and the Pyrethroid and 

Pyrethrin Technical Working Group (PPTWG).

Synthetic pyrethroids and naturally occurring pyrethrins1 have seen increased usage over the past 

decade as replacements for organophosphate and N–methyl carbamate insecticides. At the 

present time, OPP is actively evaluating the human health risks associated with pyrethroids 

through its registration review program (http://www.epa.gov/oppsrrd1/reevaluation/pyrethroidspyrethrins.html). In addition, OPP is assessing pending new uses of pyrethroids requested by 

pesticide registrants through the Pesticide Registration Improvement Act (PRIA).

The Agency has proposed that pyrethroids share the same mode of action (MOA), namely the 

ability to interact with voltage-gated sodium channels (VGSCs) ultimately leading to 

neurotoxicity (USEPA 2009). In June 2009, the SAP met to evaluate the Agency’s preliminary 

conclusions which were provided in the document, “Draft Science Policy Paper: Common 

Mechanism Grouping (CMG) for Pyrethrins and Synthetic Pyrethroid Pesticides.” The panel 

members agreed with the Agency that pyrethroid insecticides share the VGSC as a common 

molecular target site. Pyrethroids modify the sodium channel kinetics, resulting in a delayed 

channel closing and altered nerve cell transmission ultimately leading to fine tremors or 

choreoathetosis and salivation. Furthermore, the Agency proposed subdividing the pyrethroids 

into two subgroups, Type I and Type II, largely based on structure (the absence or presence of 

aα-cyano moiety) and distinct toxicity syndromes. The Panel agreed with the Agency that there 

was sufficient scientific evidence to subgroup the pyrethroids into Type I and Type II based on 

structure, the nature and extent of sodium channel modification, ability to interact with calcium 

and chloride voltage gated channels, and behavioral manifestations at high doses (SAP 2009, 

USEPA 2010). However, there were a few pyrethroids that did not fit neatly into either 

subgroup due to intermediate effects and were considered “mixed” pyrethroids. 

As part of the pesticide registration process, numerous guidelines studies2

 1 This document applies to the naturally occurring as well as synthetic pyrethroids. For ease of discussion, herein, 

the naturally occurring pyrethrins and the synthetic pyrethroids will be called ‘pyrethroids’ collectively.

evaluating the 

exposure and toxicology of pyrethroids have been conducted and submitted to EPA for review. 

2 40 CFR Part 158 Toxicology Data Requirements for Conventional Pesticides. 

http://www.epa.gov/lawsregs/search/40cfr.html
10

Among these registration studies, experimental laboratory studies using a variety of durations 

(acute to chronic), routes (oral, dermal, inhalation), species (rat, rabbit, mouse, dog), and 

lifestages (juvenile, adult) are available for virtually all of these pesticides registered in the U.S., 

and are of particular interest for considering the potential for age-dependent sensitivity. In 

addition, six developmental neurotoxicity (DNT) studies in rats also are available for evaluation.

EPA recently has reviewed these DNT studies (i.e., deltamethrin, bifenthrin, esfenvalerate, betaand lambda-cyfluthrin, fenpropathrin) and concluded that they did not provide consistent 

evidence of neurotoxicity or show any increased juvenile sensitivity. Furthermore, they did not 

contribute significantly to the selection of points of departures (PoD) as part of risk 

characterization of these chemicals (USEPA 2010). Based on this review, the Agency believed

that the results of these six studies could be applied to other members of the class and that no 

additional DNT studies need to be conducted for the other pyrethroids.3

 This conclusion, 

however, does not alleviate concern for potentially increased sensitivity to juveniles, particularly 

from high dose post-natal exposure, as reported in the scientific literature (Shafer et al. 2005,

USEPA 2010).

Through decades of research, much is known about the exposure, MOA, and toxicological 

profiles of pyrethroid insecticides (e.g., Soderlund et al. 2002, Shafer et al. 2005, USEPA 2009). 

However, even with the robust scientific literature on pyrethroids, gaps in knowledge exist in 

understanding the potential for postnatal age-dependent sensitivity. In order to assess this 

potential, the Agency solicited proposals4 from the pesticide registrants to evaluate differential 

sensitivity between juveniles and adults. The Agency stated that it would consider protocols that 

included data from in vivo, in vitro, and/or in silico studies (or combinations thereof).

The Agency received only one proposal, a joint proposal from the PPTWG on May 21, 2010. 

The PPTWG is a consortium of 24 registrants who hold almost all of the U.S. registrations for 

pyrethrins and the synthetic pyrethroid insecticides. The Working Group proposal consisted of 

three areas or “study blocks” to address the data gaps: Block 1 – Further studies of possible agerelated sensitivity in the rat, Block 2 – Experimental determination of parameters for PBPK 

models, and Block 3 – Age-dependent in vitro metabolism of pyrethroids in human liver tissue. 

The one day SAP meeting focused on specific aspects of the PPTWG proposal that were most 

critical to the PPTWG commencing their experiments. These were the in vitro experimental 

studies to inform the PBPK models, development of the PBPK models, and a review of the use 

of the acoustic startle reflex as a measure of neurotoxicity potential. The charge questions for 

this meeting were organized into two broad areas: 1) behavior and 2) PBPK model development. 

The Agency expects to follow up this SAP meeting with another SAP meeting in 2011/2012 that 

will focus on issues not addressed at this meeting, such as, the ontogeny of sodium channels. At 

that time, the PPTWG will provide the Panel and the public with an update on its research 

efforts. 

 3 http://www.epa.gov/oppsrrd1/reevaluation/pryethroids-pyrethrins.html 4 Letter from T. Levine, February 16, 2010; http://www.epa.gov/oppsrrd1/reevaluation/pyrethroids-pyrethroids.html
11

PUBLIC COMMENTERS

Oral statements were presented by:

On behalf of the PPTWG:

Thomas Osimitz, Ph.D.; Chair, PPTWG

Larry Sheets, Ph.D., Bayer Crop Science

David Kim, Ph.D., Syngenta

Ronald Hines, Ph.D., Medical College of Wisconsin

Written statements were provided by:

1. J. TerBush, private citizen

2. Thomas Osimitz, Ph.D.; Chair, PPTWG
12

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS

1.0 Auditory Startle Response or Acoustic Startle Reflex (ASR) 

1.1 Auditory Startle Response to Assess Age-Dependent Sensitivity to Pyrethroids

The PPTWG is proposing that the ASR provides a robust and sensitive measure of 

neurotoxicity and is well suited to assess age-dependent sensitivity to pyrethroids. 

Please comment on the appropriateness of the ASR technique as a measure of

pyrethroid induced toxicity, including suggestions to assure quality of the study design 

(i.e., appropriate time-to-peak response, variability of peak response, etc.) and resulting 

data. 

The Panel concluded that the acoustic startle response is not the best measure for juvenile 

sensitivity to pyrethroids because the response itself varies with age. Therefore, it would be 

difficult to discern if any observed change was related to toxicity, variability, or maturity.

The Panel stated that the use of an acute dosing paradigm and the ASR as an endpoint to 

assess peak-time of exposure effects and age-dependent sensitivity to pyrethroids was not 

sufficient to address the concerns of age-dependent vulnerability. The Panel raised concerns 

that a peak time for chemical concentration within the brain can differ across ages and thus, 

makes comparable assessments difficult. The Panel identified problems regarding the use of a 

neurobehavioral endpoint as an acute response to exposure to determine relative potency of

compounds across ages. Making such comparisons across systems at very different levels of 

maturation does not take into consideration the changing dynamics of the integrated 

sensory/motor systems in childhood development. These limitations would be present in any 

neurobehavioral endpoint. Additionally, the Panel was concerned with relying on a response 

to an acute exposure as a surrogate for an “effect” on the nervous system. 

1.2 Age-Dependent Toxicity – Choice of Rat Life Stage to Conduct ASR Studies

The PPTWG has proposed to use conduct ASR studies on 21-day old rats. Please 

comment on the appropriateness of this age group in regards to i) assessing agedependent toxicity and ii) assessing whether the 21-day old rat will adequately inform 

the Agency regarding toxicity as it relates to children three years of age and younger. 

The Panel concluded that the inability to determine a consistent response among different age 

groups, and most importantly, in a potentially more sensitive age group, makes the utility of 

the ASR, or any integrated neurobehavioral endpoint, in this context problematic. There was 

general agreement among the Panel that trying to identify a comparable age for each stage of 

brain development in the human and the rodent (rat) would be difficult. Measurements of 

such reflex oriented responses as the ASR require not only an integration of the cellular 

circuitry of the sensory system, but also the development and maturation of the motor 

system. While the rodent brain undergoes similar developmental events as those occurring in 

the human, the process is significantly accelerated and can be difficult to definitively map 
13

(Rice and Barone 2000, Clancey et al. 2007). Based on the available published data and the 

expertise of the Panel members, the Panel agreed that at weaning, the 21-day old rat would 

not serve as an equivalent model for determining the susceptibility of the human at an age of 

concern for the Agency, i.e., a toddler between the ages of 1-3 years. Given the differential 

pattern of development across species, the majority of Panel members suggested that it was 

imperative to know the developmental ontogeny of any specific functional process prior to 

using data to assess age-dependent toxicity. For the pyrethroids, this would include 

information on the ontogeny of sodium channels. 

2.0 Physiologically-Based Pharmacokinetic (PBPK) Modeling 

2.1 Proposed Modifications to the Tornero-Velez et al. PBPK Model

Please comment on the proposed modifications to the Tornero-Velez et al. (2010) model 

as described in sections 4.3 of the PPTWG proposal. Please include in your comments 

consideration for balancing potentially improved performance resulting from the 

increased complexity with model parsimony. 

The Panel generally supported the proposed efforts to further develop and refine the TorneroVelez et al. family of pyrethroid PBPK models with the aid of new measurements of partition 

coefficients and protein binding. In general, the Panel indicated that obtaining values for 

age-specific, chemical-specific parameters in a PBPK model as a function of lipophilicity, 

steric bulk, electrostatic interactions, polarity, hydrogen bonding, and ionization state (e.g., 

addressed in Zhang 2005) and tissue descriptors (tissue weight fractions for lipid, protein, 

and water) (Balaz et al. 1999) will aid in reducing uncertainty in these parameters. The Panel 

recommended that experiments focus on those parameters to which the relevant model 

responses (e.g., brain concentrations) are most sensitive, and also explore the relative rates of 

metabolism in tissues. The Panel was split about the prospective usefulness of the proposed 

Ussing Chamber techniques and human Caco-2 cells. The Panel stated that the six

pyrethroids proposed by PPTWG for study were too limited to be representative of all the 

pyrethroids in this class. 

2.2 Microsomal Incubation Studies

Please comment on the strengths and weaknesses of the PPTWG proposal to use 

hepatocytes in the PBPK effort, including the potential for hepatocytes to decrease 

uncertainty of model predictions in light of limited data. 

The Panel expressed enthusiasm for both the use of hepatocytes and hepatic microsomes in 

metabolic studies to inform the PBPK model. In vitro to in vivo extrapolations of data using 

hepatocytes are considered superior to extrapolations using microsomes. The Panel stated 

that there was some limitation in the availability of hepatocyte samples particularly within 

pediatric age groups, but not adult age groups, which might constrain the ability of the 

measurement program to appropriately represent the full diversity of the human population. 

However, microsome availability is not as limited as hepatocyte availability.
14

Considerations that favor the use of hepatocytes included: 1) measurements that help assure 

that the enzyme activities observed will reasonably reflect in vivo conditions and 2) cytosolic, 

Phase II enzymes, as well as directly oxidative Phase I microsomal enzymes. The Panel 

commented that the data from in vitro hepatocyte experiments should be related to the

corresponding whole liver function by considering heterogeneities in the liver (e.g., Andersen 

et al. 1998, Allen et al. 2005). Some panelists were concerned with PPTWG’s proposal to 

pool hepatocytes before measuring enzyme activities. These panelists stated that such 

pooling would obscure any information which may be obtained on human inter-individual 

variability among the pooled samples. 

Panel members noted that there is considerable experience with the use of microsomes for 

measurements of CYP enzyme activities, but the broad applicability of scaling this approach 

to clearance for other enzyme families (including the carboxylesterases) is far less well 

established. Enantiomers can also be evaluated easily with both microsomal and hepatocyte

systems. With regard to extrapolation from adult to juvenile or other early life stages, another 

approach would be to model the infant response based on adult data. Simcyp Limited and 

Simulation Plus, Inc., for example, provide simulation and modeling software that can model 

the metabolism of "virtual" children based on the input of "adult" parameters. Panel 

members offered a number of recommendations with regard to future microsomal studies.

3.0 Alternative Study Design(s) For Evaluating Age Differences in Pharmacokinetics 

Are there alternative approaches using empirical or data generation techniques 

potentially requiring less time than the PBPK effort proposed by the PPTWG for 

evaluating the potential for post-natal sensitivity, particularly with respect to 

differences in pharmacokinetic profiles, that could be used by the Agency?

The Panel did not recommend any specific approach to evaluate the potential for post-natal 

sensitivity, particularly with respect to differences in pharmacokinetic profiles. The Panel 

agreed that development of the PBPK model to facilitate the animal-to-human extrapolation 

was very important and should be continued. While these efforts are ongoing, the Panel

identified several changes/alternatives in the current approach to develop an in silico/ in 

vitro/ in vivo (SVV) PBPK model that could ultimately be used to predict the 

pharmacokinetics of pyrethroids in mammals, particularly in humans. These included: 1) 

data-mining efforts to target testing, 2) in vitro approaches, e.g., cassette dosing, 3) 

pharmacokinetic and pharmacodynamic considerations for in vitro studies, and 4) non-human 

primate (monkey) in vivo studies. Although these suggestions and alternatives may initially 

require some additional experiments, they may, in the long term, reduce the time necessary to 

develop the SVV model of the pharmacokinetics of pyrethroids in humans and enable a more 

feasible and accurate means of determining the viability of the PBPK model. 
15

DETAILED RESPONSES TO CHARGE QUESTIONS

(Note: Information in italics is verbatim of what is written in the Agency transmittal 

memorandum dated, June 21, 2010.)

1.0 Auditory Startle Response or Acoustic Startle Reflex (ASR) 

1.1 Auditory Startle Response to Assess Age-Dependent Sensitivity to Pyrethroids

Measurement of the auditory startle reflex response is a commonly used technique to assess 

neurobehavioral effects in rats. Auditory startle reflex is a motor reflex characterized by a 

sequence of reflexive muscle movements elicited by sudden and intense acoustic stimuli 

measured by a change in motor output. The proposed reflex path is short, consisting of the 

auditory nerve, posteroventral cochlear nucleus, the nucleus reticularis pontis caudalis, and 

motor neurons in the spinal cord (Davis et al. 1982). This mechanism is susceptible to a 

variety of drugs and toxicants making the reflex a useful model of sensorimotor reactivity 

across animal taxa, including rat and human (Lee et al. 1996). With regard to pyrethroids, 

auditory startle data in adult rats have demonstrated differing response patterns related to 

pyrethroid structure (Crofton and Reiter 1984; Tilson et al. 1985; Crofton and Reiter 1988; 

Hijzen et al. 1988; Hijzen and Slangen 1988); Type I pyrethroids produced an increase in 

startle amplitude and Type II pyrethroids produced a decrease in startle amplitude. In 

addition, ASR has been used to demonstrate age-dependent toxicity in rats following high 

oral doses of pyrethroids (Sheets et al. 1994; Sheets 2000). Therefore, ASR is a potentially 

sensitive measure to evaluate differences in neurobehavioral effects between adults and pups. 

Since ASR is a behavioral measurement, it is important to consider development, doseresponse and variability during interpretation of the results. In rats, the onset of ASR 

response corresponds to the development of the external auditory meatus. In the rat, this 

usually occurs between 13 and 16 days of age. Sheets et al. (1988) have shown the ability of 

rats to respond to ASR as early as PND 13; however, the amplitude of response continued to 

increase through PND 21. Pyrethroids modify the voltage gated sodium channels in the 

central nervous system and therefore the brain is considered the major target organ for 

toxicity. Kim et al. (2010) determined the distribution of deltamethrin, a Type II pyrethroid, 

in brain, fat, liver, plasma, and muscle in PND 10, 21, 40, and 90 rats for up to 510 hours 

following dosing. Brain concentrations in PND 10 pups were elevated for a longer time 

relative to the adults. This suggests that pyrethroid kinetics in the brain of pups may not 

mirror those of adult rats. The Kim et al. (2010) study emphasizes the importance of 

determining the appropriate time course of effects (i.e., time-to-peak-effect and/or time-totissue-recovery) in both adult and non-adult lifestages prior to measuring ASR responses. 

Additionally, the standard deviation for peak amplitude, the ASR measure proposed by the 

PPTWG, can vary greatly in guideline DNT studies (20-125%) and literature reviews 

(Raffaele et al. 2004). However, this variability can be reduced down to 20-30% if the studies 

are conducted in proven laboratories (Sette et al. 2004). 

The PPTWG is proposing that the ASR provides a robust and sensitive measure of 

neurotoxicity and is well suited to assess age-dependent sensitivity to pyrethroids. 
16

Please comment on the appropriateness of the ASR technique as a measure of 

pyrethroid induced toxicity, including suggestions to assure quality of the study design 

(i.e., appropriate time-to-peak response, variability of peak response, etc.) and resulting 

data. 

Panel Response

The Panel concluded that the acoustic startle response is not the best measure for juvenile 

sensitivity to pyrethroids because the response itself varies with age. Therefore, it would be 

difficult to discern if any observed change was related to toxicity, variability, or maturity.

The overall conclusion of the Panel was that the use of an acute dosing paradigm and the ASR 

as an endpoint to assess peak-time of exposure effects and age-dependent sensitivity to 

pyrethroids was not sufficient to address the concerns of age-dependent vulnerability. The 

Panel identified problems regarding the use of a neurobehavioral endpoint as an acute 

response to exposure to determine relative potency of compounds across ages. Making such 

comparisons across systems at very different levels of maturation does not take into 

consideration the changing dynamics of the integrated sensory/motor systems in childhood 

development. These limitations would be present in any neurobehavioral endpoint. 

Additionally, the Panel was concerned with relying on a response to an acute exposure as a 

surrogate for an “effect” on the nervous system. 

According to the EPA/OECD DNT testing guidelines, exposure is to occur during a prolonged 

developmental window under a general assumption that the broader age window would 

suffice to cover various developmental windows of vulnerability. The Agency noted in its 

background document that numerous toxicity testing studies, including DNT studies and twogeneration reproductive toxicity studies, have not detected a differential sensitivity following

in utero or pre-weaning exposure to the pesticide class under question. Furthermore, the

Agency’s recent review (Scollon 2010) suggested that data developed under GLPs following 

the current DNT guideline and submitted by the registrants demonstrate a high level of 

variability. This raises the question of the experimental quality of the datasets and their utility 

for adequately evaluating developmental neurotoxicity. In the review by Shafer et al. (2005), 

independent published studies and unpublished data examining the effect of pyrethroid 

exposure during various developmental windows suggested an elevated concern for 

neurological effects. Moreover, the recent work by Kim et al. (2010) reported severe clinical 

signs of toxicity, including death, in 10-day old rats orally dosed with 2 mg deltamethrin/kg, 

with a decrease in severity observed with increasing age, i.e., PND 21, 40, or 90, at the time of 

dosing. Further work demonstrated that a higher 4 mg/kg dose of deltamethrin produced 

higher brain levels at PND 21 as compared to PND 72; yet, a 50% deficit in ASR was at a 

maximum for both age groups. In the human population, a higher level of pyrethroid 

exposure is observed in toddlers due primarily to increased hand-to-mouth activity. The Panel 

commented that the Agency was justified in its concern for early childhood exposure which 

led to its publically soliciting proposals that would evaluate the potential for differential 

sensitivity between juveniles and adults.

The Panel discussed the level of documentation supporting the Agency’s request for proposals 

concerning study designs and protocols to evaluate potential differential sensitivity of 
17

juveniles and adults to pyrethroid insecticides and the proposal submitted by the PPTWG in 

response to the Agency’s solicitation. The Panel was in agreement that the written materials 

provided by the Agency and the PPTWG were sparse in details regarding the PPTWG 

proposal and the overall goals of the Agency solicitation. This lack of detail created difficulty 

for the Panel to review the PPTWG proposal and to provide a focused response to the 

Agency’s charge questions. While the Panel concluded that the ASR has many excellent traits 

for evaluating neurotoxicity in the adult, the Panel was concerned about the high level of 

variability in the data submitted by the pesticide registrants to fulfill the developmental 

neurotoxicity testing requirement. In a review provided by the Agency, it was mentioned that 

this variability decreased with a greater level of experience and expertise in the testing lab 

(Sette et al. 2004). The Panel agreed that testing for ASR requires expertise and optimization 

of experimental procedures that may be lacking in many of the pesticide registrant studies. A 

number of points raised by the Panel were related to the details of the testing conditions as a 

general measure to decrease variability in the ASR data. The Panel provided a number of 

suggestions to improve the quality of ASR data and to decrease variability, in general. These 

are summarized in Appendix 1. These suggestions relate to the ASR testing within a specific 

age group and are not presented as an endorsement of the proposed study plans for 

comparison between young and adult animals. 

The Panel found the scientific/biological logic behind the PPTWG proposal to be less than 

optimal. While a peak time-to-effect approach has been successful for pesticides displaying 

cholinesterase inhibition as a mode of action, equating ASR as an acute biomarker of effect 

lacks a scientific basis. The Panel also was concerned with the testing of only a limited 

number of compounds and with the logic of dose selection. The PPTWG proposes to develop 

data for only six of the nearly two dozen or so pyrethroids currently registered in the U.S. 

The rationale for their selection of the four Type I, one Type II and one mixed Type 

pyrethroids included the observation that these were the ones to which young children were 

more likely to be exposed, based upon existing indoor use patterns. The Panel indicated that 

the proposed number and distribution across types of substances to be tested will not be 

adequate to provide credible answers for the class of pyrethroids as a whole. The Panel 

recommended that in addition to more Type I and Type II pyrethroids, all compounds of an 

identified mixed type (i.e., esfenvalerate, in addition to fenpropathrin) should be evaluated in

any comparison. The proposal submitted by the PPTWG would evaluate a peak-time to overt 

toxicity in the young and adult rats. At peak-time, changes in the ASR would be evaluated at 

lower dose levels. A number of members of the Panel expressed concern that examination of 

a behavior occurring at a specific time following acute exposure, whether a response observed 

at higher dose levels or at peak concentration of chemical in the brain remains a measurement 

of “acute response.” In addition, the Panel was concerned that an adverse effect on the 

nervous system, especially during development, would not be reflected in a change in ASR, or 

any neurobehavioral endpoint, following a relatively short period of acute chemical exposure. 

The Panel emphasized that this approach would miss the potential for subclinical toxicity and 

effects on the nervous system that would become detectable only with complete maturation. 

The Panel noted that a similar problem would exist for multiple functional endpoints given the 

developmental differences of the brain and the ontogeny of the various neurobehavioral 

endpoints used within a DNT study.
18

A robust body of data is required to derive equivalent doses and produce a credible risk 

assessment under a cumulative risk assessment. The Panel viewed the PPTWG proposal as an 

ambitious testing program. Alternative approaches were presented by various panel members 

including examination of additional neurobehavioral endpoints; yet, any such neurobehavioral 

test requiring the integration of the nervous system would face the same limitations and 

concerns as mentioned for ASR, especially if used as a response to acute exposure. A number 

of the Panel members suggested various additional endpoints; however, they were based upon 

either more extended exposure or time following exposure to demonstrate a delay in the 

normal developmental pattern or a long-term perturbation. Alternative targets considered 

included motor activity and perinatal endocrine programming events such as delayed time to 

vaginal opening, as reported for esfenvalerate (Pine et al. 2008). However, these endpoints 

were measured in the DNT and in the two-generation reproductive toxicity studies submitted 

to the Agency. According to the Agency, these studies have provided no indication of an 

increased susceptibility from in utero and pre-weaning pyrethroid exposure.

The Panel suggested alternative approaches for consideration by the Agency in determining

the potential for age-related susceptibility of the nervous system to pyrethroids. Any 

evaluation must be conducted when peak concentrations of the relevant chemical occur in the 

brain. Given the age-related differences in absorption and disposition processes during 

development, the peak concentration in the brain may occur at different times following a 

dose in different age groups and species. One suggestion was to consider endpoints other than 

neurobehavioral, given the limitations of examining an integrated system during development 

and comparing the response to the mature system. In this case, targeted studies could be 

performed on dose levels and/or at specific developmental windows to provide appropriate 

data for the Agency. Such endpoints could include morphological, molecular, or 

neurochemical developmentally-regulated events occurring in the brain. Also to be 

considered is the postnatal developmental curve for some of the xenobiotic metabolizing 

enzymes (Atterberry et al. 1997) and the impact this could have on endpoints of neurotoxicity. 

For example, data for deltamethrin indicated a 50% decrease in ASR amplitude in both PND 

21 and PND 72 rats when exposed to the same dose, despite a higher brain concentration of 

the compound in the weanling rats. 

1.2 Age-Dependent Toxicity – Choice of Rat Life Stage to Conduct ASR Studies

Age-dependent toxicity has been observed in rat studies following high doses (i.e., LD50 

studies resulting in 50% mortality of test subjects) of Type II pyrethroids (Cantalamessa 

1993; Sheets et al. 1994; Sheets 2000). However, in sublethal studies using the ASR as a 

measure of toxicity, ED50 (dose at which 50% of the test subjects are affected) values were 

similar between postnatal day (PND) 21 and adult rats. 

These findings suggest that age-dependent toxicity may only be observed at high doses. 

Based on in vivo (Cantalamessa 1993) and in vitro (Anand et al. 2006) studies, the apparent 

discrepancy between high- and low-dose age-dependent toxicity is likely attributable to 

incomplete maturation of the enzymes that detoxify pyrethroids in immature animals, 

particularly the carboxylesterases and cytochrome P450s. These clearance mechanisms are 

overwhelmed in younger animals given LD50 doses, leading to increased accumulation of 
19

the pyrethroids in nervous tissue and ultimately increased toxicity. 

Carboxylesterases and P450 enzymes are the two major enzyme families responsible for 

metabolism of pyrethroids. In the rat, it has been shown that carboxylesterase activities are 

below adult levels at weaning (Moser et al. 1998; Karanth and Pope 2000; Anand et al. 

2006; de Zwart et al. 2008; Yang et al. 2009). Information on the ontogeny of 

carboxylesterase development in the human is more limited. However, increased plasma 

esterase activity during postnatal maturation has been reported (Ecobichon and Stephens 

1973). In contrast, Pope et al. (2005) found carboxylesterase activity in hepatic tissues was 

similar for humans ranging in age from 2 months to 36 years; however, the sample sizes 

were small and variability among the age groups was high. Maturation of the P450 enzymes 

shows a similar trend. 2C19, a CYP450 enzyme which has shown high pyrethroid metabolic 

activity (Godin et al. 2006), increases rapidly in the human during the first 2 years of life, 

whereas numerous P450s examined in the rat have minimal expression levels through 

gestation and do not approach adult levels of expression until PND10 days or later (de 

Zwart et al. 2008).

Comparisons between lifestages in the rat and human are difficult because of the ontogeny of 

the brain development and metabolizing enzymes are not an exact match. However, PND 11 

rats are considered to be close in development to newborn humans and PND 17 rats are 

believed to be closer developmentally to human toddlers (Davision and Dobbing 1966; 

Dobbing and Smart 1974; Benjamins and McKhann 1981). From the aspect of exposure, 

previous experience with developing cumulative risk assessments for other insecticide 

groups, ongoing work on HED's Standard Operating Procedures for Residential Pesticide 

Exposure Assessment, and the Agency's Guidance on Selecting Age Groups for Monitoring 

and Assessing Childhood Exposures for Environmental Contaminants 

http://www.epa.gov/raf/publications/guidance-on-selecting-age-groups.htm, the Agency 

believes that children three years old and younger, particularly those who are mobile 

(crawling, walking) and who exhibit hand-to-mouth behavior, have the potential for the 

greatest exposure to pyrethroids. Based on 1) the current understanding that the two major 

enzyme families responsible for the metabolism of pyrethroids are below adult activity levels 

at weaning (i.e., PND 21); 2) PND 17 rats are approximately comparable to human toddlers 

in terms of development; and 3) children younger than 3 years of age are expected to have 

the greatest exposures to pyrethroids, the Agency is concerned with the PPTWG’s proposal 

to conduct ASR studies in PND 21 rats. Instead, PND 15 to 17 rats, which have been shown 

to respond to ASR stimuli (Sheets et al. 1988), may better represent the most susceptible 

human lifestage. 

The PPTWG has proposed to use conduct ASR studies on 21-day old rats. Please 

comment on the appropriateness of this age group in regards to i) assessing agedependent toxicity and ii) assessing whether the 21-day old rat will adequately inform 

the Agency regarding toxicity as it relates to children three years of age and younger. 

Panel Response
20

There was general agreement among the Panel members that trying to identify a comparable 

age for each stage of brain development in the human and the rodent (rat) would be difficult.

What is critical is that the cellular and anatomical development of the brain and its circuitry 

are interdependent. Measurements of such reflex oriented responses as the ASR require not 

only an integration of the cellular circuitry of the sensory system, but also the development 

and maturation of the motor system. While the rodent brain undergoes similar developmental 

events as those occurring in the human, the process is significantly accelerated and can be 

difficult to definitively map (Rice and Barone 2000; Clancy et al. 2007). Based on the 

available published data and the expertise of the Panel members, the Panel agreed that at 

weaning, the 21-day old rat would not serve as an equivalent model for determining the 

susceptibility of the human at an age of concern for the Agency, a toddler between the ages 

of 1-3 years. Given the differential pattern of development across species, a majority of 

Panel members stated that it was imperative to know the developmental ontogeny of any 

specific functional process prior to using data to assess age-dependent toxicity. For the 

pyrethroids, this would include information on the ontogeny of sodium channels. 

Extrapolation from rodent to human on any developmentally-related biological endpoint

requires a specific understanding of the developmental features of the endpoint under study.

With specific reference to the ASR, while a response to an auditory stimulus can be detected 

as early as PND 13 in the rat, the developmental onset of this response varies and matures 

over the subsequent 25-30 days. This occurs along with the maturation of the internal 

circuitry as well as maturation of the motor system. In the DNT guidelines (OPPTS 

870.6300), the developmental landmarks include body weight, age of vaginal opening and 

preputial separation. No development landmarks are included in the published guidelines for 

neurological development such as onset of a startle response. The Panel recommended a reevaluation of the available literature on pyrethroids to determine whether data exists showing 

a developmental delay in the startle response, or in other comparable endpoints. 

A differential ASR to Type I and Type II pyrethroids has been demonstrated in the adult rat,

with an increased response to Type I and a decreased response to Type II pyrethroids. In the 

developing rat, this differential response has not been reported. Given that the ASR is a reflex 

response dependent upon a motor response to a sensory stimulus, the ability to detect lower 

ASR in immature rats may be difficult due to the immaturity of the motor system. Thus, Type 

II specific effects may not be detected in the immature rat due to the normally low ASR 

amplitude and would be more difficult to detect with younger and younger ages. Moreover, 

given the neural circuitry involved in the ASR, changes detected in an immature animal may 

potentially have a different basis than responses in the adult. The Panel concluded that the 

inability to determine a consistent response among different age groups, and most 

importantly, in a more sensitive age group, makes the utility of the ASR, or any integrated 

neurobehavioral endpoint, in this context problematic. 

2.0 Physiologically-Based Pharmacokinetic (PBPK) Modeling 

The PPTWG is proposing to use a model developed collaboratively by EPA’s Office of 

Research and Development (ORD) and the University of Georgia as a starting point in their 

modeling effort. EPA’s ORD has published a series of papers that describe the development 
21

and enhancement of pyrethroid PBPK models starting with a deltamethrin model in rats by 

Mirfazaelian et al. (2006) , improved by Godin et al.(2010), modified for permethrin by 

Tornero-Velez et al. (in prep.), and finally expanded to include age- and chemical-dependent 

parameters by Tornero-Velez et al. (2010). In 2007, ORD and OPP jointly presented an issue 

paper to the SAP (USEPA 2007) which described an approach for using a generic model 

structure with chemical specific parameters for pyrethroids. The “family modeling” 

approach was endorsed by the SAP and has been successfully applied in the above PBPK 

efforts. The Agency believes that it is both reasonable and scientifically sound to use the 

Tornero-Velez et al. (2010) PBPK model as the starting point for the PPTWG effort to build 

PBPK models for pyrethroids to assess young children. Furthermore, the PPTWG is 

proposing to develop PBPK models using in vitro and in vivo rat data, and then using human 

in vitro data to inform the model to predict human internal dosimetry, similar to the 

approach which was previously supported by the 2007 SAP. 

2.1 Proposed Modifications to the Tornero-Velez et al. PBPK Model

The PPTWG proposes to increase the complexity of the Tornero-Velez et al. (2010) PBPK 

model by modifying some aspects. For example, the PPTWG is proposing to: 

a) Predict intestinal permeability through the use of the Ussing Chamber technique with 

rat cells and human Caco-2 cells, with the potential to increase the number of 

compartments within the intestinal tract 

b) Determine partition coefficients using in vitro techniques

c) Obtain estimates of protein binding 

Please comment on the proposed modifications to the Tornero-Velez et al. (2010) model 

as described in sections 4.3 of the PPTWG proposal. Please include in your comments 

consideration for balancing potentially improved performance resulting from the 

increased complexity with model parsimony. 

Panel Response

The Panel generally supported the proposed efforts to further develop and refine the TorneroVelez et al. family of pyrethroid PBPK models with the aid of new measurements of partition 

coefficients and protein binding. In general, the Panel indicated that obtaining values for 

age-specific, chemical-specific parameters in a PBPK model as a function of lipophilicity, 

steric bulk, electrostatic interactions, polarity, hydrogen bonding, and ionization state (e.g., 

Zhang 2005) and tissue descriptors (tissue weight fractions for lipid, protein, and water) 

(Balaz et al. 1999) will aid in reducing uncertainty in these parameters. The Panel 

recommended that experiments focus on those parameters to which the relevant model 

responses (e.g., brain concentrations) are most sensitive, and also explore the relative rates of 

metabolism in tissues.

The Panel had a more mixed view of the prospective usefulness of the proposed Ussing 

Chamber techniques and human Caco-2 cells. Many panel members supported the use of the 

Caco-2 cell line because it has been widely used for many years by the pharmaceutical 

industry as a reliable screening tool to assess, in particular, paracellular and transporter-
22

mediated intestinal permeability. In addition, data obtained from this system generally 

correlates with absorption characteristics in vivo. Apparent permeability estimates from the

Caco-2 system can provide information concerning the absorption potential of compounds 

across the gastrointestinal tract. Estimation of the apparent permeability of the different 

pyrethroids in this cell culture system will provide an opportunity to rank order these 

compounds according to their permeability, and hence, absorption potential in vivo.

Calculation of an “efflux ratio” (the ratio of apparent permeability in the basal-to-apical 

direction to apparent permeability in the apical-to-basal direction) may provide information 

regarding the potential contribution of active uptake transporters or efflux transporters 

(Efflux ratio >2). There was also considerable support for using this system to explore the 

consequences of stereochemistry for possible differences in behavior of different pyrethroid 

isomers. With recent improvements, i.e., the 3-day Caco-2 culture, very rapid screening of 

pyrethroids can occur with this system.

On the other hand, some of the Panel expressed skepticism with these in vitro absorption 

measurements using Caco-2 cells due to concerns about limited solubility of the pyrethroids 

in water, the need to explore multiple in vitro systems (Balimane et al. 2006, Cheng et al.

2008), and effects of extensive cell culturing on comparative in vivo functions. As with all 

cell lines maintained in culture for an extensive time, the comparative in vivo functions could 

be compromised and should be considered. However, the Panel noted that there are no data 

to indicate that this has happened in the Caco-2 cells. These panelists were also concerned 

that the Caco-2 cells may not reveal possible differences related to age because they are of 

adult origin. Other panel members did not agree with these concerns.

As noted above, many on the Panel were confident in the use of the Caco-2 system and its 

ability to accurately assess the permeability potential of a series of compounds when 

permeability is due to passive diffusion mechanisms. The Caco-2 system can identify active 

processes, but the transporter expression profile is not that similar to the in vivo situation. 

Although age-dependent differences in cell membrane composition may result in differences 

in the permeability coefficient, these are likely minor. The Caco-2 system can provide some 

valuable information on permeability (and hence, absorption) without having to resort to in 

vivo absorption determinations (this is very costly in terms of animal resources). 

As an alternative to Caco-2 cells, some panel members suggested a preference for more 

extensive in vivo measurements of the detailed time course of pyrethroid transfer first within 

compartments of the gastrointestinal tract and then into the systemic circulation. Panel 

members also noted that extensive in vivo measurements would certainly be costly from an 

animal welfare perspective.

The Panel noted that the primary goal of the PBPK model is to assess the concentrations of 

the parent pyrethroid compounds in the brain because the parent compounds are the toxic 

moieties. The metabolites, as far as is known, do not contribute appreciably if at all to the 

neurotoxicity. In particular, the PBPK model discussed here is needed primarily for relating 

pyrethroid concentrations to differences between juvenile to adult sensitivities to their

neurotoxic effects. The prediction of expected and observed biomonitoring results and 

reverse dosimetry are secondary applications for the model. Some Panel members suggested 
23

that the priorities for measurements of individual compounds should be: 1) metabolism rates, 

2) partition coefficients and 3) membrane transport across the intestinal barrier or the bloodbrain-barrier. The Panel was in general agreement that partition coefficients should be 

measured in vitro. One panelist suggested that the model not be limited to those results as 

unchanging values, even for specific age and gender groups. This panelist indicated that 

partition coefficients should be varied within the model with time to recognize, for example, 

diurnal and postprandial changes in blood lipid content. In contrast, another panelist stated 

that by not fixing the values for partition coefficients and blood lipid content, and making 

these values change according to age and time of day would add a significant amount of 

complexity to the PBPK model and significantly reduce model parsimony. In addition, this

panelist expressed concern about the proposed methods for measuring partition coefficients 

in vitro given the very limited solubility of the pyrethroids in water. Another Panel member 

noted that partition coefficient measurements should not be a problem even if pyrethroids 

were insoluble in water. Membrane transport or transport across the intestinal or blood-brain 

barriers using in vitro methods could be problematic because it could be difficult to get them 

in the cultures. Partition coefficients are often done with water insoluble substances as this is 

just a ‘test tube’ test. 

Panelists also stated that the proposed number of pyrethroids to be included in the proposed 

experiments was too limited. An examination of six pyrethroids in detail, as proposed, might 

not be fully representative of the whole group of pyrethroids registered in the U.S. This

limited series of proposed experiments might not even be extensive enough to adequately 

explore differences between Type I and Type II pyrethroids, as well as the consequences of 

possible “mixed types” not adequately encompassed within these two categories.

2.2 Microsomal Incubation Studies

Microsomal incubation studies have been used to inform the pyrethroid PBPK models 

developed by ORD (Mirfazaelian et al. 2006; Scollon et al. 2009; Godin et al. 2010; 

Tornero-Velez et al. 2010; Tornero-Velez in prep.). The PPTWG has proposed to use intact 

hepatocytes instead because they may provide a better prediction of metabolism compared to 

microcellular fractions (Hewitt et al. 2007). Additionally, the PPTWG is proposing to 

compare the clearance activity of human hepatocytes and microsomal fractions for several 

age groups. However, the Agency notes that there are a limited number of human hepatocyte 

samples available to inform the PBPK model. Pooled human microsomes are available 

representing larger segments of the population relative to hepatocyte availability. The 

Working Group suggests characterization of the hepatocytes (i.e., CYP450 and esterase 

composition) and establishing a relationship between hepatocytes and microsomal fraction 

activities to reduce model uncertainty in light of limited data. 

Please comment on the strengths and weaknesses of the PPTWG proposal to use

hepatocytes in the PBPK effort, including the potential for hepatocytes to decrease 

uncertainty of model predictions in light of limited data. 

Panel Response
24

1) Hepatocytes

Panel members expressed considerable enthusiasm for the use of human hepatocytes. 

Considerations in favor of the use of hepatocytes included: 1) measurements to help assure 

that the enzyme activities observed will reasonably reflect in vivo conditions and 2) cytosolic, 

Phase II enzymes, as well as directly oxidative Phase I microsomal enzymes. Furthermore,

studies have demonstrated that the collection (i.e., time post-mortem), processing, and 

storage protocols for hepatocytes do not substantially alter metabolic enzyme activities 

compared to fresh in vivo hepatocytes (Guilbouzo et al. 1993). In addition, data gained from 

using hepatocytes will implicitly include transport properties (such as the ATP-binding 

cassette transporters), which may be very important for the disposition of pyrethroids, but 

have not yet been directly considered in the written application or verbal presentation of the 

proposed experiments.

While supporting the use of hepatocytes for enzyme activity measurements, one panelist 

observed that a weakness in using in vitro experimental data on hepatocytes is that the liver is 

not just a collection of hepatocytes. Additionally, there is spatial heterogeneity in several 

factors such as oxygen tension, receptor distribution, binding activity, glycolysis, etc. 

(Lamers et al. 1989). Therefore, this panelist observed that it would be desirable to focus 

efforts on relating data from in vitro hepatocyte experiments to corresponding whole liver 

function by considering heterogeneities in the liver (e.g., Andersen et al. 1998, Allen et al.

2005). Some panelists were concerned with the PPTWG’s proposal to pool hepatocytes 

before measuring enzyme activities. These panelists stated that such pooling would obscure 

any information, which may be obtained on human inter-individual variability among the 

pooled samples. The Panel discussed whether availability of hepatocyte samples within 

specific age groups would constrain the ability of the measurement program to appropriately 

represent the full diversity of the human population. Several companies were cited as 

examples of commercial suppliers of these preparations, e.g., XenoTech, LLC. Puracyp, Inc., 

KaLy-Cell, Cellz Direct, BD Biosciences5

. Panelists believed that commercial availability of 

adult hepatocytes is fairly constant since additional samples are continuously being collected 

and produced. However, the numbers across the various companies are limited to perhaps a 

couple of dozen individuals at any one time. The commercial availability of pediatric 

hepatocytes is far less than for adult hepatocytes, with current availability limited to less than 

10 individuals. The Panel commented that it is expected that new samples will only be 

available from 6-10 individuals per year.

2) Microsomes

The Panel expressed significant support for the proposed microsomal studies, although in 

vitro to in vivo extrapolations are considered more superior with hepatocytes than with 

microsomes. However, microsome availability is not as limited as hepatocyte availability. 

Panel members noted that there is considerable experience with the use of microsomes for 

measurements of CYP enzyme activities, but the broad applicability of scaling this approach 

up to clearance for other enzyme families (including the carboxylesterases) is far less well 

 5 Disclaimer: The Panel makes no endorsement of any company nor any product. Mention of any company or 

product is for illustration purposes only. 
25

established. Enantiomers can also be easily evaluated with microsomal systems and

hepatocyte systems. “Juvenile” and “toddler” liver microsomes with the full array of specific 

isoforms can be made commercially as a custom product by companies like BD Biosciences 

(BD GentestTM branded products), among others. Thus, Vmax and Km for specific metabolites 

(particularly the hydrolytic cleavage products that can have interaction with parent 

compounds) for each enantiomer can be obtained which will provide a better understanding 

of potential inhibitory interactions. With regard to extrapolation from adult to juvenile or 

early life stages, another approach would be to model the infant response based on adult data. 

Simcyp Limited and Simulation Plus, Inc., for example, provide simulation and modeling 

software that can model the pharmacokinetics of "virtual" children based on the input of 

"adult" parameters. 

On the other hand, analysis of metabolite production using microsomes can be complex. For 

example, there are two diastereomers of permethrin (cis and trans), each has a R- and a Senantiomer for a total of four enantiomers with their own set of metabolites that are also 

likely inhibiting biotransformation of the other enantiomers. Gaughan and Casida (1978)

reported more than 30 metabolites of permethrin using TLC analytical methods and only the 

two diastereomers of permethrin. EPA’s Tornero-Velez et al. (2010) PBPK model did not 

separate out the enantiomers which would likely lead to even more metabolites to measure. 

Many of these metabolites are conjugates. One panel member suggested that the metabolic 

stability could be assessed using the substrate depletion approach. Such an approach will 

generate metabolic rate information without a need for identification and quantitative 

determination of metabolites in microsomal systems. Alternatively, another panel member 

stated that if the most toxic enantiomer is not undergoing depletion because its metabolism is 

being inhibited by a primary metabolite or another enantiomer then the concentration of the 

enantiomer primarily responsible for the toxicity will be underestimated. This would be the 

case although the overall concentration of the four enantiomers is measured. The Panel 

advised that both the advantages and disadvantages of the substrate depletion approach and 

the metabolite formation approach be considered.

If most Phase II reactions are eliminated from the evaluation through the use of microsomal 

systems, then the Phase I metabolites may undergo additional secondary reactions with Phase 

I enzymes. For example, permethrin is hydrolyzed to 3-phenoxybenzoic acid (3-PBA), a

reaction mediated by both CYP enzymes and carboxylesterases. With hepatocytes, 3-PBA is 

likely conjugated via a Phase II reaction (to a sulfate or glucuronide) and would not undergo 

a second Phase I reaction. Thus, even though concentrations may be below Km values for 

the enzymes, the formation of multiple metabolites that are also substrates have the potential 

for inhibitory and possible stimulatory (CYP3A) reactions. Therefore, the Panel concluded 

that it is likely that a CYP approach would misclassify the precise paths of metabolism and 

the relative abundance of metabolites.

The Panel considered whether there was an adequate supply of microsomes to represent the 

full diversity of the human population. However, the Panel recommended that measurements 

in microsome samples be retained as individual samples rather than pooled, as an integral 

part of the measurement program. The Panel noted that microsomes from the collection of 
26

Dr. Ronald Hines

6

, Medical College of Wisconsin, are available and that the repository is 

diverse enough to allow for measurements of sufficient power to represent the human 

population.

Panel members offered a number of recommendations with regard to future microsomal 

studies.

1) The Panel noted that while the liver makes the greatest contribution to microsomal 

oxidation, ignoring other sources introduces uncertainty in metabolic measurements in 

microsomes. To reduce this uncertainty, some panelists stated that quantification of 

extrahepatic metabolism would be an important use of microsomal measurements. 

Therefore, panel members strongly recommended that microsomal oxidation through 

CYP isoforms in the intestine should be incorporated into the model. Very little is known 

regarding ontological studies of enzyme expression in the small intestine. The Panel 

recommended that Vmax and Km comparisons between CYP and carboxylesterases be 

evaluated in enterocytes of the small intestine. For example, one panelist pointed out that 

gut CYP3A4 levels are relatively high in human enterocytes and that CYP1A1 activity is 

also apparent (Thelen and Dressman 2009).

2) Some panelists expressed concern about the exclusive use of Michaelis-Menten (M-M) 

enzyme kinetics and scaling in the current pyrethroid modeling family. For example, 

Kenworthy et al. (1999) and others (Houston and Kenworthy 2000; Uchiapichat et al.

2004) have observed that M-M kinetics may not always hold either for single compounds 

or for mixtures of chemicals. The Panel suggested that the Agency carefully consider the 

metabolic scaling algorithms and enzyme kinetics required and adjust the model 

accordingly, particularly for analysis of pyrethrin mixtures. Being a composite of 

enantiomeric compounds, pyrethroids undergo complex Phase I and Phase II 

biotransformation, and therefore, it is expected that multiple CYP isoforms are involved 

as well as uridinediphosphate-glucuronosyltransferases (UGTs) and sulfotransferases 

(SULTs). However, at least some Panel members cautioned the Agency that if all 

possible factors that can influence the activity of an enzyme and how it is modeled, the 

complexity of the process would preclude the development of a practical and useful 

model. Added levels of complexity to the model must be weighed carefully as these 

decrease model parsimony.

3) Some panelists recommended that specific stereoselective interactions be examined

further. Incubations would be conducted with individual enantiomers, diastereomers 

(e.g., cis and trans permethrin), and the racemate for non-hepatic microsomes in the 

mixture that is in the commercial pyrethroid product. 

4) One panelist suggested consideration of experiments in non-human primates (i.e., 

monkeys) to elucidate pharmacokinetic parameter values. This would likely better reflect 

humans than observations made in rodents, although post speciation differences in 

enzyme action may be limiting.

 6 Ronald Hines, Ph.D., provided public comments during the meeting on behalf of the PPTWG.
27

5) Some panelists thought that the unusually low ratio of concentrations observed for the 

brain versus the blood for some pyrethroids should be further evaluated. To unravel this 

apparent anomaly in partitioning, the Panel recommended further investigation of the 

possibility of active efflux of pyrethroids from the brain back to blood, as well as specific 

in vitro measurements of equilibrium partitioning in the brain versus analogs to the 

plasma.

3.0 Alternative Study Design(s) For Evaluating Age Differences in Pharmacokinetics 

The Agency gives special consideration to the potential pre- and postnatal lifestages 

regarding potential exposure to pesticides. Pre-natal exposure to pyrethroids has been 

evaluated extensively in over 80 developmental toxicity, reproductive toxicity, and DNT test 

guideline studies and no sensitivity from in utero exposure has been observed. As previously 

described, there are gaps in knowledge surrounding the potential for post-natal sensitivity 

and, as described in Question 1.2, the Agency considers children less than 3 years of age to 

be the most susceptible population. The PPTWG has proposed a robust PBPK model 

development effort to describe pyrethroid dosimetry across several lifestages; however, these 

models will not be ready for use by the Agency until approximately 2013. 

Are there alternative approaches using empirical or data generation techniques 

potentially requiring less time than the PBPK effort proposed by the PPTWG for 

evaluating the potential for post-natal sensitivity, particularly with respect to 

differences in pharmacokinetic profiles, that could be used by the Agency?

Panel Response

The Panel did not recommend a specific approach to evaluating the potential for post-natal 

sensitivity, particularly with respect to differences in pharmacokinetic profiles. The Panel 

agreed that development of the PBPK model to facilitate the animal-to-human extrapolation 

was very important and should be continued. While these efforts are ongoing, the Panel

identified several changes/alternatives in the current approach to develop an in silico/ in 

vitro/ in vivo (SVV) PBPK model that could ultimately be used to predict the 

pharmacokinetics of pyrethroids in mammals, particularly in humans. These included: 1) 

data-mining efforts to focus future testing programs, 2) in vitro approaches, e.g., cassette 

dosing, 3) pharmacokinetic and pharmacodynamic considerations for in vitro studies, and 4) 

non-human primate (monkey) in vivo studies. Although these suggestions and alternatives

may initially require some additional experiments, they may, in the long term, reduce the 

time necessary to develop the SVV model of the pharmacokinetics of pyrethroids in humans 

and enable a more feasible and accurate means of determining the viability of the PBPK 

model. 

Data-mining and modeling

The Panel indicated that a more targeted testing approach could be developed to guide the 

choice of endpoints incorporated into the design of future toxicity studies used to 

quantitatively characterize age-related sensitivities. One way to focus in vitro studies would 
28

be a critical and thorough review of the existing pyrethroid database pertinent to the agespecific acute toxicity question at hand (e.g., pharmacokinetics, pharmacodynamics, acute 

toxicity, pre- and post-natal developmental toxicity). This new data-mining effort would 

update the pre-existing comprehensive review paper by Shafer et al. (2005) to include new 

studies conducted post-publication and include studies that were submitted to the Agency for 

pesticide registration. This effort should include comments on the strengths and limitations 

of each study. From this analysis, one would compile a database on the dose-response 

relationships of all relevant endpoints for each pyrethroid and perhaps identify age-specific 

sensitivity data as well as any data gaps. These data would not be limited to acute 

neurotoxicity as proposed by the PPTWG. For example, another possible endpoint might be 

long-term effects on reproductive functioning caused by developmental alterations in 

endocrine signaling reported for pyrethroids (see Shafer et al. 2005). 

Modeling the dose-response relationships (PBPK/PD) and time course pattern for all relevant 

toxicity endpoints for which data are available would also be a valuable exercise. These data 

may include the time course from time-to-peak-effect evaluation which is associated with 

specific neurobehavioral endpoints, e.g., motor activity. Useful data for modeling 

application can also come from studies showing differential severity of toxicological 

response solely due to different dosing volume. The expectation is that these exercises can 

provide insights into model integrity as well as inform the choice of dose-metrics pertinent to 

each specific endpoint that can potentially be used to establish the point of departure for risk 

assessment. A parallel effort to ensure reliable modeling of target age groups with 

significant exposure, in this case, young children, is also essential for a successful application 

of PBPK model for risk assessment. 

Possible in vitro approaches

The Panel had the following caveat regarding additional in vitro studies: All future testing 

should be designed to determine whether there are any quantitative differences in sensitivity 

between adults and juveniles. The Agency reminded the Panel that, currently, the mandatory 

default 10X safety factor is being retained in the assessment process for all pyrethroids, 

because the question of whether there are age-related differences in sensitivity to members of 

this class remains unanswered. The Panel indicated that what is important would be to 

identify those “critical studies” for which the Agency has characterized a NOAEL and/or 

LOAEL in the adult then use these studies to focus on determining whether the juvenile 

would be expected to be more, less, or similarly susceptible at the same dose levels. 

The Panel considered in vitro approaches that could generate data for input parameters to the 

rat and human models, especially those with significant impact to model outcome as 

indicated by sensitivity analysis, and pertinent to the potential candidates of dose metrics for 

interspecies extrapolation. The Panel did not prioritize their suggestions.

1) One suggestion was to measure the blood and brain levels of pyrethroids over the first 

four hours after either intravenous (i.v.) or subcutaneous (s.c.) (use s.c. if it is more 

feasible/reliable at PND 10) administration of a prototypical Type II pyrethroid (e.g.,

deltamethrin) in PND 10, PND 21 and PND 90 rats. High pyrethroid blood levels in 

neonate rats within the first couple of hours after oral dosing have previously been 
29

proposed to be due primarily to the combined result of rapid absorption, low first pass 

metabolism, rapid distribution into the brain, and limited subsequent metabolism. At

least one effect and one no-effect dose should be tested in all age groups, although a 

wider range and number of doses (e.g., two or three effect level doses) might be needed. 

Whether the 10-fold higher levels seen within the first two hours after oral dosing with 

Type II pyrethroids are due totally to absorption and first pass metabolism is not clear. If 

it is, then gut absorption and renal excretion may be even more important than 

metabolism for initial Type II pyrethroid concentrations in the blood and brain. 

Consequently, more detail needs to be provided for modeling the absorption and 

excretion of Type II pyrethroids in neonates than now proposed. If significant age 

differences are found in blood and brain concentrations after either i.v. or s.c. 

administration then more consideration should be given to the vast differences in body 

and organ fat levels, gut absorption, and possible renal excretion in neonates versus 

adults that affect Type II pyrethroid concentrations. If the i.v. or s.c. studies were 

designed more elaborately and toxic effects were closely monitored during the first four

hours, then it would be easier to discern if there were significant differences in PND 10 

animals versus adults with respect to the Type II pyrethroid pharmacodynamic aspects of 

neurotoxicity and lethality than can be currently determined from available data from oral 

administration.

2) Another suggestion was to use a Cassette-Dosing approach to expedite the data gathering 

for the PBPK model. Cassette-Dosing is a high-throughput screening tool used in the 

drug discovery process for the rapid (although imprecise) assessment of the 

pharmacokinetics of a series of compounds. This approach involves the simultaneous 

administration of multiple compounds to a single animal. 

Pharmacokinetic Considerations

1) Mortality is a poor measure of age-dependent sensitivity. One panel member noted that 

the age-related difference in sensitivity with mortality as an endpoint reflects a saturation 

of detoxication ability which is lower in juvenile animals than in the adults. Therefore,

running an experiment at a level that causes mortality does not reflect the difference in 

sensitivity between juveniles and adults that would be operational at realistic exposure 

levels.

2) Beyond Michaelis-Menten (M-M) kinetics. As previously mentioned in the Panel’s 

response to Charge Question 2.2, the current data rely solely on M-M kinetics, and M-M 

kinetics may not always hold true either for single compounds or for mixtures of 

chemicals (specifically pyrethroids). One panelist suggested that the Agency reanalyze 

the available datasets using sigmoidal and/or inhibition approaches to modeling CYP and 

other enzymes. However, another panelist cautioned that trying to consider non-M-M 

kinetics, inhibition kinetics or activation kinetics would add levels of complexity to the 

modeling process when the model is not intended to give exact estimates.

3) Pharmacodynamic versus pharmacokinetic considerations. One panelist pointed out that 

pharmacodynamics, with respect to the toxic mode of action, should also be considered in 
30

addition to pharmacokinetics. Pyrethroid insecticides share the voltage-gated sodium 

channel (VGSC) as a common molecular target site. Pyrethroids modify the sodium

channel kinetics, resulting in a delayed channel closing and altered nerve cell 

transmission ultimately leading to fine tremors or choreoathetosis and salivation.

VGSCs exhibit a complex regional and temporal ontogeny, and embryonically expressed 

forms of these channels are replaced by expression of adult forms during 

neurodevelopment (Shafer et al. 2005). The complex ontogeny of VGSCs could result in 

altered sensitivity of the developing central nervous system to perturbations by 

pyrethroids (Shafer et al. 2005). Although toxicokinetic factors are thought to account 

for differences in susceptibility to pyrethroids between young and adult animals (Sheets 

et al. 1994), toxicodynamic factors have not been systematically studied (Shafer et al.

2005). A recent study (Mackenzie et al. 2009) links changes in VGSCs to auditory 

deficits which provide a possible rationale for the use of the ASR in determining toxicity, 

despite its short-comings.

On the other hand, one panelist stated that pharmacokinetics instead of 

pharmacodynamics should be the focus of attention because of concerns with the utility 

of the ASR (see Panel Response to charge question 1.1) as well as the utility of other tests 

of neurotoxicity for comparing very young rats to adult rats. This panelist suggested that 

protecting children be examined by pharmacokinetics instead of pharmacodynamics 

because the data from the former are straightforward and interpretable based on 

metabolism studies using the current state of the science; whereas, this might not be true 

for neurotoxic endpoints. 

Non-human primate studies 

One panelist proposed that pyrethroid studies could be conducted using monkeys as a means 

of model validation. Such studies could potentially yield valuable data that may be more 

interpretable for producing a better SVV model than the one being developed (i.e., TorneroVelez et al. family of PBPK models). The appropriate time span for testing the 

pharmacokinetics of pyrethroids to represent the newborn to 3-year old human is much 

longer in the monkey (being at least 4 months) compared to only a few days in the rat (e.g., 

PDN 10-17). In addition, there is the possibility of examining the effect of multiple 

exposures of pyrethroids on the same animal over some period of time. The relative rate of 

development of brain, gut, kidney and liver in monkey are more in synchrony with human 

than that of rat. Also, the body fat ratio during monkey development would be more like 

human. This panelist stressed the importance of having a suitable species to validate the final 

SVV model. 

Other comments concerning the PPTWG proposal

One panel member suggested that the Agency evaluate the PPTWG proposal in the context 

of answering questions similar to those used by the U.S. Army Research Laboratory's Human 

Research and Engineering Directorate (HRED) during the development and review of the 

detailed study protocols (see Appendix 2).
31

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Appendix 1: Expanded References and Suggestions for Conducting ASR Testing

References for Development of ASR:

1) Auditory acuity in the rat improves progressively between 16 and 20 days. During this 

time both sensitivity and range of frequencies to which the animal can respond increase

with postnatal age (Crowley and Hepp-Raymond 1966). 

2) The startle response can be elicited by a tactile stimulus and the sensitivity matures 

earlier than auditory sensitivity (Gottlieb 1971). However, modification of the startle 

response is progressively elaborated over a period of days during the 2nd and 3rd weeks of 

life. This also occurred with a tactile stimulus (Parisi and Ison 1977). Both serotonin and 

norepinephrine have been implicated in the modulation of the startle response in the adult 

rat. Both the noradrenergic and serotonergic systems are undergoing intense maturation 

during the 2-3rd weeks of life in the rat with regards to cellular differentiation and 

synthetic enzyme activity. 

3) Kungel et al. (1996) reported that exposure to cysteamine to reduce somatostatin levels in 

the developing rat brain between PND 10 and PND 17 animals resulted in ASRs that 

were similar between exposed and control animals at PND 13 while by PND 18, the ASR 

was lower in the cysteamine-dosed rats as compared to controls reflective of a delay in 

the maturation of the ASR between PND 13 and PND 18. 

Points to Consider in Conducting ASR to Decrease Variance in Data Sets:

[Note: These points are not arranged in any order of priority.]

1) The EPA DNT guideline requires a minimum sample size (n) of 10 male and female rats

at each dose level. One would assume, based upon the variability across a large 

majority of the data sets for ASR, that a sample size of 10 is not adequate to detect a 20-

30% difference. Based upon the available data, a power analysis would be required to 

determine the necessary sample size. This may be different across various test methods, 

commercial apparatus, and SOPs, but needs to be sufficient to detect a specific level of 

difference. An inadequate sample size and high level of variability found in individual 

test labs will not provide data sufficient for determining chemical-related effects.

2) In the current EPA DNT guideline, auditory startle response for habituation is performed 

on PND 60. The mean response amplitude is calculated for 10 sequential trials –

referred to as a block for a total of 5 blocks. There are no specific requirements for the 

parameters of the startle stimulus, i.e., dB level, Hz range, pure tone versus white noise, 

background noise levels, inter-trial intervals and fixed or random delivery. Each of 

these components has been demonstrated in the literature to modify the ASR and can 

alter the sensitivity of the test. 
38

3) Interpretation of an ASR is confounded if the hearing ability of the animal is altered. 

While both the EPA and WHO IPCS neurotoxicity risk assessment guidance documents 

discuss these features, the DNT guideline does not address this issue with regard to 

either data collection or within the SOP to confirm a relatively uniform hearing 

capability at the stimulus level. While hearing threshold evaluation requires auditory 

brainstem response recordings, a crude evaluation of the response of animals across a 

range of stimulus intensities can confirm no gross differences in hearing. 

4) Weight and size of the test animal can influence the measured ASR. This requires that 

each AS chamber be calibrated and sensitivities adjusted for testing of animals to ensure 

the responses are kept within the scale of the system. This can be required in adult 

animals between ages of 5 – 10 weeks and is definitely required for the testing of 

younger animals. In addition, within the SOP it should be required that the sound levels 

are measured in dBc using a sound level meter and calibrated and equalized across all 

chambers. An acclimation period within the chamber is required. For adult rats, this is 

often 5 minutes. This can vary in the younger rodent. 

5) In control rats, there is usually a high correlation between the peak amplitude and the 

average amplitude; however, this may vary with exposure and thus, both types of data 

should be analyzed. Standardize control animal response and any specific age with 

regards to response amplitude, lag-time to response, etc. Use uniform testing at an age 

that is considered “adult,” an age that must be post puberty.

6) Habituation to an auditory startle stimulus can vary as a function of age or exposure. 

Using the commercially available ASR systems, the generation of a habituation curve 

normally requires a test session of approximately 100 stimulus deliveries. However, 

other earlier data using a direct delivery of the stimulus upon visual confirmation of an 

absence of movement of the rat indicated that habituation occurs much earlier. Thus, 

examination of the individual startle responses made by the animal can provide a greater 

level of sensitivity. If a pre-pulse startle inhibition (PPI) protocol is employed, 

habituation occurring in the later portions of the test session represents a floor-effect and 

the ability to detect PPI can be diminished. Thus, PPI calculated during the test period 

of a non-habituated startle response will provide data less compromised. Prepulse 

paradigms can be used to record gap detection.

7) In combination with new commercial apparatus to measure ASR, data generated for 

evaluating the basic neuroscience of the ASR and the association to neurological 

diseases such as, schizophrenia, provide additional methods to assess components of the 

ASR. These new ways of evaluating the ASR waveform should be evaluated for 

inclusion into neurotoxicity testing to provide more sensitive and possibly less variable 

and more relevant response data. 

8) Using the commercial apparatus for ASR, a measurement can be obtained during a time 

of no-stimulus presentation and if sampled over the course of the test session can 

provide an indication of activity levels. 
39

9) The following elements needed for any neurobehavioral test need to be standardized: 

housing, husbandry, handling (i.e. bedding changes), litter sizes, strain and time of day 

when the test is performed. In immature rodents, additional efforts are required to 

address the temperature and humidity regulation of the environment and to minimize 

stress. Ultrasonic vocalizations between animals must be considered in the logistics of 

handling animals during the pre-weaning periods. 
40

Appendix 2: Protocol Review Criteria Provided by One Panelist Based on Those used by 

the U.S. Army Research Laboratory's Human Research and Engineering Directorate 

(HRED)

Review Criteria Yes No

Is the experimental design clearly stated?

Is the methodology described in sufficient detail to evaluate the statistical 

analyses possible with this experimental design?

Are the experimental conditions (treatments) to be compared well defined? 

Are the proposed statistical analyses appropriate to the design, the sample 

size, and the type of data that will be collected? (e.g., interval versus ordinal 

variables, fixed versus random effects models, assumptions for the use of the 

proposed analyses met, etc.)

Is the proposed sample size appropriate to the experimental design? (e.g., 

Could the sample size be smaller and still provide statistical power? Is the 

sample too small for the planned analyses? )

Is the process of assigning experimental units (subjects) to experimental 

conditions (treatments) appropriate and clearly detailed or defined?

Does the design make the best use of available statistical techniques? (e.g., 

Was blocking utilized correctly or a Latin Square design proposed correctly 

based on the needs and assumptions of the research?)

Does the design and methodology create any sources of bias or threats to the 

validity of the proposed analyses and interpretation of results that need to be 

addressed?

Based on a review of the entire proposal, are the research design and analyses 

proposed in this protocol sufficient to answer the primary research questions?

Will any results based on these data and analyses be generalizable?