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.

25 

### 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

S teven 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 Scienti fic 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 Uni versity Raleigh, NC

Carey N. Pope, Ph.D. Professor, Head & Sitlington Chair of Toxicology6 Department of Physiological Sciences

Oklaho ma 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 Saskatchewa n 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 Pathobiolog y Virginia

- Maryland Regional College of Veterinary Medicine Virginia Polytechnic Institute and State University Blacksburg, VA7 Penelope A. Fenner

- Crisp, Ph.D., DABT

Private Consultant North Garden, VA

Mari S. Golub, Ph.D., DABT Director, Murine Behavi or 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.

Asso ciate 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

Resear ch 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.

Professo r Emeritus Department of Statistics Kansas State University

Manhatt an, KS

Teresa L. Leavens, Ph.D.

Research Assistant Professor8 College of Veterinary Medicine Center for Chemical Toxicology Research and Pharmacokinetics North Carolina State University

Ra leigh, 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, DC9 INTRODUCTION

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) has completed its review of the Agency’ s a nalysis of Comparative Adult and Juvenile Sensitivity Toxicity Protocols for Pyrethroids

. Advance not ice 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 Pesticid e Programs (OPP) public docket or via Regulations.gov, D ocket 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 Technica l Working Group (PPTWG).

Synthetic pyrethroids and naturally occurring pyrethrins 1 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/pyrethroids

pyrethrins.html ). In additi on, 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 leadi ng 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 Gr ouping (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 delay ed 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 (t he 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 channe l 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 intermedia te effects and were considered “mixed” pyrethroids.

As part of the pesticide registration process, numerous guidelines studies 2

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 15 8 Toxicology Data Requirements for Conventional Pesticides. http://www.epa.gov/lawsregs/search/40cfr.html10 Among these regis tration 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 reg istered 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 stu dies ( i.e. , deltamethrin, bifenthrin, esfenvalerate, beta

and 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 s ignificantly 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 an d 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 proposals 4 from the pesticide registrants to eva luate 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 pyrethrin s and the synthetic pyrethroid insecticides. The Working Group proposal consisted of three areas or “study blo cks” to address the data gaps: Block 1

– Further studies of possible age

related 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 tiss ue. 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 mee ting 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.html11 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 prov ided by:

1. J. TerBush, private citizen

2. Thomas Osimitz, Ph.D.; Chair, PPTWG12 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 Sensitivit y 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 t he 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 ac ute dosing paradigm and the ASR as an endpoint to assess peak

- time of exposure effects and age

- dependent sensitivity to pyrethroids w as not
sufficient to address the concerns of age

- dependent vulnerability. The Panel raised concerns that a peak time for c hemical 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 pres ent 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 age

dependent 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 t he 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 map13 (Rice and Barone 2000

, Clancey et al. 2007)

. Based on the available published data and the expertise of the Panel mem bers

, the Panel agr eed 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

pa ttern of development across species, t he majority of P anel 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 woul d 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. (20 10) 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 P anel generally supported the pro posed efforts to further develop and refine the Tornero

Velez 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, c hemical

- 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 li mited 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 decrea se 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

. I n vitro to in vivo extrapolations of data using hepatocyt es 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

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

commented that the data from in vitro hepatocyte experiment s should be related to the corresponding whole liver function by considering he terogeneities 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 acti vities. These panelists stated that such pooling would obscure any information which may be obtained on human inter

- individual

variabi lity among the pooled samples.

Panel members noted that t here 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 microsoma l 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. S imcyp Limited and Simulation Plus, Inc. , for example, provide simulation and modeling software tha t 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 Ag e 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, particula rly with respect to differences in pharmacokinetic profiles, that could be used by the Agency?

The Panel did not recommend a ny specific approach to evaluat e the potential for post

- natal sensitivity, particularly with respect to differences in pharmacokine tic 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 curren t 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 a pproaches, 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 alternative s 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 Sensitiv ity 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 (D avis 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 dat a 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

st artle 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 p otentially sensitive measure to evaluate differences in neurobehavioral effects between adults and pups.

Since ASR is a behavioral measurement, it is important to consider development, dose

response and variability during interpretation of the results. I n 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; howev er, 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 t he 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

- to

tissue

- re covery) 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 (Raf faele 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 r esponse, variability of peak response, etc. ) and resulting

data.

Panel Response

The Panel concluded that t he acoustic startle response is not the best measure for juvenile sensitivity to pyrethroids because the response itself varies with age. Therefor e, 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 effec ts and age

- dependent sensitivity to pyrethroids w as 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 wo uld 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 two

generation 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 GLP s following

the current DNT guideline and submitted by the registrants demonstrate a high level of variability. This raise s the question of the experimental quality of the datasets and their utility for adequately ev aluat ing 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 highe r level of pyrethroid

exposure is observed in toddler s due primarily to in creased hand

- to

- mouth activity. The Panel commented that the Agency was justified in its concern for early childhood exposure which

led to its publically s oliciting proposal s that w ould evaluate the potential for differential sensitivity between juveniles and adults.

T he Panel discussed the level of documentation supporting the Agency’s request for proposals concerning study designs and protocols to evaluate potential differential sensitivity of17 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 deta ils 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 concl uded that t he ASR has many excellent traits for evaluating ne urotoxicity in the adult, the Panel was concerned about the high level of variability in the data submitted by the pesticide registrants to fulfill the developm ental neurotoxicity testing require ment. 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 Pan el 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 endorse ment 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 d isplaying 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 wit h 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 the ir selection of the four Type I, one Type II and one mixed Type pyrethroids included the observation that these wer e 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 answ ers 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

toxicit y in the young and adult rats. At peak

- time , changes in the ASR would be e valuated at

lower dose levels. A number of members of the Panel expressed concern that e xamination 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 a n 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.

Th e 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 differen ces 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 th e 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 sys tem 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 t wo

- generation reproductive toxicity studies submitted to the Agency

. A ccording to the Agency

, these studies have provided no indication of an

inc reased 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

eva luation 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 occurri ng 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 indicate d 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

- d ependent toxicity has been observed in rat studies following high doses (i.e., LD 50 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 AS R as a measure of toxicity, ED 50 (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 v ivo (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, parti cularly the carboxylesterases and cytochrome P450s. These clearance mechanisms are overwhelmed in younger animals given LD50 doses, leading to increased accumulation of19 the pyrethroids in nervous tissue and ultimately increased toxicity.

Carboxylesterase s 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; d e 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 co ntrast, 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 enzyme s 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 level s 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 enzym es 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 McKhan n 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 t hose 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 pyrethro ids 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, th e 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 age

dependent 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 Response20 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 sen sory 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 agr eed that at weaning, the 21

- day old rat would not serve as an equivalent model for determining the susceptibility of the huma n 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 func tional process prior to using data to assess age

- dependent toxicity. For the pyrethroids, this would include information on th e ontogeny of sodium channels. Extrapolation from rodent to human on any developmenta lly

- related biological endpoint require s a s pecific 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 a nd 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 o pening 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 re

evaluation of the available literature on pyreth ro id s to deter mine whether data exist s 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 decr eased 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 rat s 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, give n the neural circuitry involved in the ASR, changes detected i n an immature animal may potentially have a different basis than response s in the adult. The Panel concluded that t he inability to determine a consistent response among different age groups, an d 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 d eveloped 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 development21 and enhancement of pyrethroid PB PK 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 Torne ro

- 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 endo rsed 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

appr oach 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, th e 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 coef ficients using i n 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 P anel generally supported the proposed efforts to further develop and refine the Tornero

Velez et al. family of pyrethroid P BPK 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, el ectrostatic 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 Pane l

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 prospectiv e usefulness of the proposed U s sing 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 asses s, 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 accor ding 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

re garding 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 dif ferent pyrethroid

isomers. With recent improvements

, i.e.

, the 3

- day C aco

- 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 us ing 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 wi th 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 p anelists 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 o f 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 i s 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 perme ability (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 meas urements 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

anim al welfare perspective.

The P anel 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 n ot contribute appreciably if at all to the neurotoxicity. In particular, t he PBPK model discussed here is needed primarily for relating pyrethroid concentrations to differences between juvenile to adult sensitivit ies to the ir neurotoxic effects. The predi ction of expected and observed biomonitoring results and reverse dosimetry are secondary applications for the model. Some P anel members suggested23 that the priorities for measurements of individual compounds should be : 1) metabolism rates,

2 ) partition co efficients and 3) membrane transport across the intestinal barrier or the blood

brain

- barrier

. The Panel was in general agreement that partition coefficients should be

measured in vitro

. O ne panelist suggested that the model not be limited to those resul ts 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 contr ast , 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 p artition coeffic ient measurements should not be a pro blem 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 t he 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 examina tion of six pyrethroids in detail

, as proposed

, might not be fully representative of the whole group of p yr ethroids registered in the U.S

. This limited series of proposed experiments might not even be extensive enough to adequately explore differences betw een 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 metabol ism 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 nu mber 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 Response24 1) Hepatocytes

Panel members expressed considerable enthusiasm for the use of human hepatocyt es. 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

, P hase II enzymes, as well as directly oxidative Phase I microso mal 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 ( Guilb ouzo 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 consider ed 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 th e 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 tha t it would be desirable to focus efforts on relating data from in vitro hepatocyte experiments to corresponding whole liver function by considering he terogeneities in the liver ( e.g. , Andersen et al. 1998

, Allen et al. 2005). Some panelists w ere concerned with the PPTWG’s proposal to pool hepatocytes before measuring enzyme activiti es. These panelists stated that such pooling would obscure any information, which may be obtained on human inter

- individual variabi lity among the pooled samples. The Panel disc ussed 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 commercia l suppliers of these preparations

, e.g. , Xeno T ech , LLC. Puracyp, Inc.

, KaLy

- Cell , Cellz Direct, BD Biosciences 5

. Panelists believed that commercial availability of adult hepatocytes is fairly constant since additional samples are continuously being collec ted 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 availabili ty 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 microsom al 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 t here is considerable experience with the use o f 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 onl y.25 established. Enantiomers can also be easily evaluated with microsomal systems and hepatocyte systems

. “J uvenile ” 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, V

max 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. W ith regard to extrapolation from adult to juvenile or early life stages, another approach would be to model the infant response based on adult data.

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

software tha t can m odel 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 S

enantiomer 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 a nalytical 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 micro somal 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 primar ily responsible for the toxicity will be underestimated. This would be the

case although th e overall concentration of the four enanti o mers is meas ured. The Panel advised that both the advantages and disadvantages of the substrate depletion approach and

t he 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 Ph ase 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 con cluded 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 d iversity 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 P anel note d that microsomes from the collection of26 Dr. Ronald Hine s 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 quantific ation 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 kno wn regarding ontological studies of enzyme expression in the small intestine. The Panel

recommended that V
max and Km comparisons between CYP and carboxy l esterases be

evaluated in enterocytes of the small intestine

. For example, one panelist pointed out t hat
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 curr ent 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 s uggested 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 c omplex Phase I and Phase II

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

as well as u ridinediphosphate

- glucuronosyl transferases (UGT s ) and s ulfotransferases (SULTs ). However, at least some Panel members cautione d 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 o f a practical and useful

model. Added l evels of complexity to the model must be wei ghed carefully as these decrease model parsimony.

3) Some panelists recommended that specific stereoselective interactions be examined

further . I ncubations would be conducted with individual enantiomers

, diaster e omers ( e.g., cis and trans permethrin)

, and t he 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 lik ely 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 v ersus the blood for some p y re throid s 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 o f equilibrium partitioning in the brain v ersus 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 pote ntial 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 previous ly 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 techniqu es 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

- hu man 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 u sed 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 considera tions for in vitro studies, and 4)

non

- human primate (monkey) in vivo studies. Although these suggestions and alternative s 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 would28 be a critical and thorough review of the existing pyrethroid database pe rtinent to the age

specific acute toxicity question at hand ( e.g., pharmacok i netics, pharmacodynamics, acute toxicity, pre

- and post

- natal developmental toxicity). This new data

- mining effort would update the pre

- existing comprehensive review paper by Sha fer 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, o ne 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 relation ship s (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 neurob ehavioral 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 i nsights 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 cav ea t regarding additional in vitro studies : A ll future testing

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

between adults and juvenil es

. The Agency reminded the Panel that, currently, the mandatory default 10X safety factor is being retained in the a ssessment 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” f or 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 interspecie s 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/rel iable 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 been29 proposed to b e 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 ra nge 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 do sing with Type II pyrethroids are due totally to absorption and first pass metabolism is not cle ar. 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

excretio n 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 f at levels , gut absor ption, and possible renal excre tion 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

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

animals versus adult s with respect to the Type II pyrethroid pharmacodynamic aspects of neurotoxicity and lethality than can be currently determined from available data f rom 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. On e panel member noted that

t he 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 C harge Q uestion 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 sig moidal 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

p harmacodynamic s, with respect to the toxic mode of action, should also be considered in30 addition to pharmacokinetics. P yrethroid 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 t remors 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 ont ogeny 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 betwee n 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 VGSC s 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 Respons e 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 fro m 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 co uld be conducted using monkeys as a means of model validation . Such studies c ould potentially yield valuable data that may be more interpretable for producing a better SVV model than the one being developed ( i.e. , Tornero

Velez 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. , PD N 10

- 17). In addition, there is the possibility of examining the effect of multiple exposures of pyrethroi ds 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. Als o, 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 t he 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

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- 127.37 Appendix 1: Expanded References and Suggest ions fo r 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 p eriod of days during the 2 nd and 3 rd weeks of

life

. T his 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 noradrener gic and serotonergic systems are undergoing intense maturation during the 2

- 3 rd 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 somatost atin levels in the developing rat brain between PND 1 0 and PND 1 7 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 si ze ( 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 an d 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 amp litude 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 v ersus 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 anim al 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 h earing 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 u niform 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 ses sion 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 indi vidual 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 presenta tion 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, s train 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 consider ed 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 )

Revi ew 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 th e 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 corr ectly 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 general izable?