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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , D . C . 20460 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENT I ON Memorandum DATE: April 17, 2015 SUBJECT: Transmission of Background Materials and Charge to the Pane l for the May 19 through 21, 2015 Session of the FIFRA Scientif i c Advisory Panel (SAP} Entitled "Research to Eva l uate the Potentia l for Juvenile Sensitivity to Pyrethroids" TO: FROM :

Fred Jenkins, Ph.D. Designated Federal Official FIFRA Scientific Advisory Panel Office of Science Coord i nation and Pol i cy

Dana Vogel, Acting**Directo .**  Health Effects Division Off i ce of Pesticide Programs Transmitted with this memo are copies of all background documents and the charge to the Panel for the May 19 through May 21 sess i on of the F I FRA Scientif i c Advisory Panel (SAP} rev i ewing the topic "Research to Eva l uate the Potential for Juven i le Sensitivity to Pyrethroids." These documents do not contain any information protected by copyright or protected under statute as Confidential Business Information (CB I }. All materials submitted by CAPHRA are considered proprietary (See CAPHRA cover letter} and are protected under FIFRA section 10(g}. The atta c hed documents include the following: - - Document Title Date May 2015 FIFRA SAP Charge Quest i ons April17, 2015 CAPHRA Cover letter Apri l 16, 2015 CAPHRA Program Overview Apri l 16, 2015 Appendix A1 - 1: list of Pyrethroids Represented by CAPHRA Members Apri l 16, 2015

Append i x A1 - 2: CAPHRA Member list April16, 2015 Appendix A1-3: Team of I nternationa ll y Recognized Scientists Apr i l16, 2015 Appendix A2-1: Effects of Pyrethroids on Human Sodium Channels Co - February 25, 2015 Expressed with 131 and 132 Subunits - Report Appendix A2-2: Effect of Pyrethro i ds on Rat Brain Neurolemma-Injected Apri l 14, 2015 1Document Title Date Oocytes: Examining Potential AgeRelated Pharmacodynamic Differences- Report Appendix A2-3: Evaluating Potential Age-Related Pharmacodynamic Apri l 16, 2015 Sensitivity in the Rat: Acute Neurotoxicity of Deltamethrin and Permethrin in Adult and Juvenile Rats - Summary Appendix A2-4: Studies ofthe Acute Neurotoxicity of Deltamethrin and February 18, 2015 Permethrin in Adult and Juvenile Rats - Report Appendix A3-1: In V ivo Pharmacokinetic Studies of Deltamethrin in April 16, 201 5 PND15, PND21, and PND90 Rats- Summary Appendix A3-2-1: Toxicokinetic Assessment of Blood and Tissue March 3, 2015 Deltamethrin Conc entrations Following the Administration of a Single Oral Dose to Mature Rats - Report Appendix A3-2-2: Toxicokinetic Assessment of Blood and Tissue March 3, 201 5 Deltamethrin Concentrations Following the Administration of a Single Oral Dose to PND 21 Rats- Report '"---·

Appendix A3-2-3: Toxicokinetic Assessment of Blood and Tissue March 3, 201 5 Deltamethrin Concentrations Following the Administration of a Single Oral Dose to PND

_ 15 Rats - Report Appendix A3-3: Partition Coefficients Study of Deltamethrin in PND15, April 16, 2015 PND21 , and PND90 Rats - Summary Appendix A3-4: Deltamethrin: Assessment of Partition Coefficients- March 3, 2015 Report Appendix A3-5: Binding of Pyrethroids to Human and Rat Plasma April16, 2015 Proteins - Summary Appendix A3-6-1: Deltamethrin: Assessment ofAge Differences in Rat March 6, 2015 Plasma Protein Binding - Report Appendix A3-6-2: Delt:amethrin: Assessment ofAge Differences in March 6, 2016 Human Plasma Protein Binding- Report Appendix A4-1 : Movement of Deltamethrin through the Human Blood April 16, 2015 Brain Barrier- Summary Appendix A4-2: Investigation of Blood Brain Barrier Transport of March 9, 2015 Deltamethrin - Report Appendix A4-3: Absorption by the Gastrointestinal Tract- Summary April 16, 2015 Appendix A4-4: Investigation ofthe Potential Role of Gastrointestinal March 9, 2015 Membrane Transporters in Deltamethrin Absorption - Report PBPK model code (Directory called : Model files Feb 2015 package) February 25, 2015 i

2Charge to FIFRA Scientific Advisory Panel May 19-21, 2015 Research To Evaluate The Potentia l For Juvenile Sensitivity To Pyrethroids The Fede r al I nsecticide, Fungic i de, and Rodenticide Act (FIFRA) authorizes EPA to register pesticides and the Federal Food, Drug, and Cosmetic Act (FFDCA) gives the agency the authority to establish tolerances for residues on food and/or feed resulting f rom use of a pesticide. The studies required to allow the agency to make the appropriate statutory safety findings under both of these acts are stipulated under 40 Code of Federal Regulations (CFR) Part 158. There is flexibility, however , in implementing Part 158. Additional data can be required (§158.75), alternative approaches can be accepted, and studies can be waived (§158.45}. The 2007 National Research Council (NRC) report from the National Academy of Sciences (NAS ) on Toxicity Testing in the 21st Century describes this new vision for toxicity testing. In response to the NRC report, EPA's Office of Pesticide Programs (OPP) developed a Strategic Direction for New Pesticide Testing and Assessment Approaches (http:ljwww.epa.gov/pesticides/science/testing assessment.htm l) which describes OPP's approach to implementing the NAS vision. One of the key components of OPP's Strategic Direction is imp roved approaches to more tradi tiona l toxicity tests to minimize the number of animals used while expanding the amount of infor mat io n obtained. OPP also has a recent document, Guiding Principles for Data Requirements ( http :1/www .epa .gov /pesticides/regulating/ data-require-guide-principle .pdf) which describes the principles fo r requiring toxicology data for pesticides, specifically to "only require data that adequately inform regulatory decision making and thereby avoid unnecessary use of time and resources, data generation costs, and animal testing." OPP is ac t ively working on a ree valuation of the human health effects of the pyrethroids and py re thrins under the OPP registration review program ( http://www.epa.gov/oppsrrdl/regi stration review /index.htm), required under FIFRA. Until late 2009, OPP requested developmental neurotoxicity (DNT) studies for pyrethroids. In 2009, the Agency reviewed the pyrethroid toxicological database of laboratory animal studies, carefuUy considering all the available reproductive and developmental studies, including the DNT study. From this examination, the

Agency determined that the DNT study did not provide toxicity data beyond that available from the developmental and reproductive guideline studies. Furthermore, the agency concluded that the DNT study did not identify increased juven i le sensitivity which had been reported in literature studies at high doses. The 2009 review of the pyrethroid toxicology database also concluded that the Agency did not

have data to adequately characterize post-natal juvenile susceptibility. To address this shortfall, the Agency requested proposals for study protocols which could 1) identify and 2) quantify potential j uvenile sensitivity. There was a single response from the Pyrethroid and Pyrethrin Technical Working Group (PPTWG)\ a conglomerate of pyrethroid registrants. In July 2010, the FIFRA Scientific Advisory Panel (SAP) reviewed a proposed research strategy from the PPTWG to assess the potential for j uvenile sensitivity consistent with the recommendations of the NAS in its report on Toxicity Test ing in the 21s t

Century using a combination of in vitro studies, targeted in vivo studies, and physiologically-based pharmacokinetic (PBPK) models . Based on feedba ck from the SAP and the agency, the industry research proposal was revised . The PPTWG was eventually expanded and became the Council for the Advancement of Pyreth roid Human Health Risk Assessment (CAPHRA). Since late 2010, the CAPHRA has worked wit h academic scientists to 1 The PPTWG was the predecessor to the CAPHRA 3develop assays and models to assess the potential forjuvenile post-natal sensitivity to pyrethroids. The on-going research effort is organized around the adverse outcome pathway (AOP) for pyrethroids: alterations with voltage-gated sodium chan nels (VGSC), leading to alterations in membra ne excitability and firing potentials and ultimately to 

### in vivo clinical syndromes. Specifica lly, the CAPHRA is evaluating the potency of pyrethroids to human sodium channels and transplantation of adult 

### & juvenile rat synaptic membrane into oocytes. In addition, the CAPHRA is cond uc t ing targeted 

### in vivo studies on behaviora l metrics and developing PBPK models
. The research , thus far, has focused on development ofthe overall approach using data for deltamethrin and permethrin (Type II and Type I pyrethroids, respectively). The CAPHRA research is at a point where feedback on extending t his research to the other pyrethroids would be constructive. The CAPHRA proposal is to use the knowledge gained with deltamethrin and permethrin to develop more targeted datasets with less data generation using read across and computational approaches for other pyrethroids
. As such, the agency 

### will be seeking the SAP's advice on the current state ofthe science with the CAPHRA research effort and proposals for next steps which include extension ofdata on deltamethrin and permethrin to other pyrethroids. 

### C

harge Questions to the Panel: 1. High-throughputscreening studies using human sodium channels expressed in mammalian cells with their regulatory Beta subunits (ChanTest Data): Human voltage-gated sodium channels (NaVs 1.1, 1.2, 1.3, 1.6) we re expressed in human embryonic kidney (HEK) cells and 9 pyrethroids were tested for their effects on channel conductance, as well as the effects of coexpressing the Beta 1 and 2 regulatory subunits. a. Please comment on the ChanTest studies conducted for nine pyrethroids. Please include in your comments a consideration oftheir robustness (i.e. repr oducibility, controls, statistics, background i nformation, NaV selection, etc)
. Please comment on the confidence and uncertaint ies in the ChanTest experi ments and re lated findings. b. CAPHRA thinks that additiona l data from t his line of evidence are unlikely to contribute useful information to characteri ze the potential forjuvenile sensitivity. Th us, the CAPH RA has proposed NOT to continue this line of research. Please com ment on this proposal and deg ree to which the ChanTest data inform the issue ofevaluating the potential forjuvenile sensitivity
. c. The ChanTest experiments focus on human sodium channels and generally show weak response. In contrast, 

### in vitro studies in rodents in the literature (e.g. C hoi and Soderlund, 2006; Meacham et al., 2008; Tan and Soderlund, 20092) show stronger responses to pyrethroids. Please comment on the extent to which the expressed

2 Choi JS and Soderlund DM
. 2006
. Structure-activity relationshi ps for the action of I I pyrethroid insecticides on rat Nav 1
.8 sodium channels expressed in Xenopus oocytes. Toxicol Appl Pharmacol. 2006 Mar 15:211(3):233-44. Epub 2005 Ju1 26 Meacham, C.A., et at., Developmentally-regulatedsodium channelsubunits are differentially sensitive to [alpha]-cyano containingpyrethraids. Toxicology and Applied Pharmacology, 2008. 231: p. 273-81. Tan, J. and O.M. Soderlund, Human and rat Nav1.3 voltage

- gatedsodium c hannels differ in inactivation properties andsensitivity to thepyrethroid insecticide tefluthrin. NeuroToxicology, 2009
. 30(1): p. 81-89
. 4human sodiu m channels can be used in co mbinat ion with these sources to infer relative pharmacodynamic se nsitiv ity between rats and humans. 2. Transplantation of adult & juvenile rat brain synaptic membrane into Xenopus oocytes: Purified neurolemma membranes from adu lt and juvenile rats were separate ly micro-injected into Xenopus oocytes. Patch clamp testing was performed on the oocyte membranes versus various doses of pyrethroids to determin e their ECso values for NaV channel activation. Inhibitors of competing channels were added to isolate the sodium channel conductance (i.e.

chloride and calcium chan nels ). a. Please comment on the synaptic membrane ("neurolemma") studies conducted for deltamethrin and permethrin. Please include in your comments a consideration of the

robustness of data from the synaptic membrane transplantation into oocytes ( r eproducibility, co ntrol compounds, c hann e l modulator reagents, statistics, use of Na blocker tricaine as an anesthetic, etc) and the degree to which these data inform the issue of eva luat ing the potential for juvenile sensitiv ity . Please comment on the confidence and uncertainties and associated findings in the oocyte experiments. b. In the context of your re sponse to 2a, the CAPHRA has proposed to co ll ect doseresponse data in adult and juvenile rat synaptic membrane transp la nted into oocytes for f i ve more pyrethroids (i ncludin g Type I, Type II, and mixed). CAPHRA's proposed path forward: If the additional five pyrethroids show similar patterns to deltamethrin and permethrin (i.e. no l ifes tage sensitivity observed), no additional pyrethroids will likely be tested in this system. Alternatively, if a different pattern is observe · d, add itional pyrethroids are likely to be tested. Please comme nt on the CAPHRA's proposed path forward. 3. Targeted in vivo studies in adult and juvenile rat: acoustic startle/detailed clinical observations: Preliminary experiments that measure acoustic startle and detailed clinical

observations have been conducted by the University of Cincinnati. In t h ese experiments, j uven ile and adult male Sprague-Dawley rats were treated with deltamethrin and permethrin as model Type II and Type I pyrethroids, respectively. In addition, brain and plasma concentrations

were measured in PND 15 and 90 rats exposed to deltamethrin. The preliminary resu lts suggest

that juvenile r ats a r e more sensitive to deltamethrin based on changes in detailed clinica l signs and, to less extent, acoustic startle. However, there was no greater sens i tivity in juvenile rats exposed to permethrin. Preliminary data from whole brain tissue samples with deltamethrin indicated that PND 15 rats had higher deltamethrin concentrations as compared to adult rats given the same dose. The brain concentration and toxicity data from the CAPHRA studies for 5deltamethrin are consistent to those previously published by the Bruckner lab (K im et al. , 20103) which showed that at a sim ilar dose (2 mg/kg deltamethrin) PND 10 rats had increased severity of clin ical signs and higher C m

ax of deltamethrin in the brain as compa red to PND 21, PND 40, and PND 90 rats.
a. The in vivo behavior studies reported thus far are preliminary eva luations
. Please comment on the study design in the preliminary in vivo studies for deltamethrin and permethrin conducted at the University ofCincinnati (Vorhees lab). b. In 2010, FIFRA SAP commented on some challenges that were anticipated with the use of auditory startle to compare the relative sensitivity ofjuveni les and adults. In the studies conducted by the CAPHRA, both auditory startle and detailed clinical observations were evaluated. Please comment on the degree to which t he auditory startle and detailed clinical observations provide useful data for evaluati ng the potential for juvenile sensitivity. Please include in your comments discussion ofthe dynamic range; the direction ofthe response varying between Type I and lis; and the type of data obtained (continuous, ranked)
. c. Please provide comments comparing the tempora l pattern and magnitude ofthe brain and plasma concentration data from the Vorhees 

### & Bruckner labs and utility of such data to aid in the interpretation ofthe auditory startle and detailed clinical observation data. d. Please comment on the use of a 5 ml/kg dosing volume in the CAPHRA studies and how any impact on pyrethroid kinetics affects correlations with behavioral effects.
4. Pharmacokinetic studies: A number of pharmacokinetic studies using deltamethrin were performed bythe CAPHRA in orderto further refine and validate the developing rat PBPK model and construct a developing human PBPK model. For refinement ofthe rat PBPK model, tissue: plasma partition coefficients (in vivo measurements in PND 21 and adult rats), age dependent plasma protein binding (in vivo measurements in PND 10, 15, 21, and 90 rats), and cytochrome P450 (CYP) and carboxylesterase (CES) metabolism (in v itro measurements using rat liver and plasma preparations from PND 15, 21, and 90 rats) were evaluated. Further in vivo pharmacokinetic studies in rats were conducted with single IV (PND 90), single oral (PN D 90) , and multiple oral (PND 15, 21, and 90) doses to generate plasma and tissue data to validate the developing rat PBPK model. Additional experiments with deltamethrin were conducted for c

onstruc ting the developing human PBPK model. Parameters evaluated in vitro included age dependent plasma protein binding derived in plasma from hu man donors aged from birth to adults, transport across the blood brain barrier using a human brain microvascular endot helia l cell line, estimates of gastrointestinal absorption in caco-2 cells, and ontogeny data forCES,

3 K im KB. Anand 

### s s

, Kim HJ, White CA. FisherJ W. Torn ero-Velez R, Bruckner JV. 2010. Age, dose, and time-dependency 

### of plasma and t is sue distri bution of deltamethrin in immature ra ts. Jun; 11 5(2):354-68
. doi : 10.1 093/toxsci/kfq074
. Epub 20 10 M ar S
. 6CYP1A2, and CYP2C8 determ ined in human l iver tissue from donors (age 1-18 years old). Further in vitro experiments are currently underway by the CAPHRA to determine human adult and

j uvenile liver metabolism of del ta methrin by CYP and CES enzymes using human liver p r eparat io ns or recombinant human enzymes . a. Please comment on the in vitro experiments to support the PBPK model development in the rat conducted thus far for deltamethrin. b. Please co mment on the in vivo experiments to support the PBPK model development in the rat conducted thus far. c. Please comment on the in vitro experiments in the human tissue conducted thus far. Please incl ude in your co mments a discussion of the ongoing in vitro experiments with recombinant enzymes for use in PBPK models and associated confidence and u n certainty with the use of such data . 5. Physiologically-based pharmacokinetic model in rat: Using data described i n Question 4, the CAPHRA has developed a PBPK model for deltamethrin using age - specific metabolism p arame ter s in rats to simulate plasma and brain internal exposures in young and adult rats. This PBPK model relies on in vitro to in vivo extrapolation (IVIVE) to use age-specific metabolic data collected in vitro to estimate hepatic metabolic clearance in vivo. The deltamethrin brain or p l asma concentrations estimated by the model in rats are then compared to measured concentrations from in vivo r a t studies to verify the model. Non - chemical specific physiological parameters for rats were obtained from the published literature , including body weight, cardiac output, hematocrit levels, tissue vo lumes , and tissue blood flows. As discussed in Question 4, recently generated data by the CAPHRA and published data were used for compound depende nt parameters, including partition coefficients, metaboli c rate constants, absorption r a tes, protein binding, compartments and tissue permeability. a. Please comment on th e robustness of the rat PBPK model for simul ating internal exposures i n the developing rat. I n your r es ponse, please include evaluation of the

structure and parameters used to build the model, as well as its ability to accommodate d iffere nt oral absorption scenarios (i.e. different vehicles used for in vivo studies) and discussion of confidence, accuracy and uncertainties associated with the deltamethrin developing rat model. Please also comment on the sensitivity analyses of parameters CAPHRA has completed thus far. b. In the context of your response to Sa, please comment on the extent to which additional data are/are not needed to ref ine the developing rat model. 6. Physiologicolly-based pharmacokinetic model for human: Similar to the rat PBPK model , the human PBPK model integrates n on-c hemical specific physiological parameters for humans from the literature . Compound-dependent parameters , such as partition coefficients and oral absorption parameters, were adapted from the rat PBPK model. Recently generated data by the 7CAPHRA were used for the remaining compound-dependent parameters, such as metabolic rate constants, protein binding and tissue permeabi lity. With respect to metabolic constants, CYP and CES enzymes involved in metabolism of a given pyrethroid will be identified and the in vitro metabolic constants for those enzymes will be determined for integration into the PBPK model. Intrinsic clearance for each active enzyme will be scaled to in vivo using scaling factor data collected by the CAPHRA and the SIMCYP database. The ontogeny of enzyme expression (also from the CAPHRA data and the SIMCYP database) will be incorporated

**i~to** the process of obtaining distributions of age-specific intrinsic clearance for each enzyme. Preliminary simulations have been conducted for deltamethrin to demonstrate the process used with the PBPK model.
a. Within the context of understanding potentialjuvenile sensitivity, characterize the robustness ofthe PBPK model for extrapolating age-specific internal tissue exposures for humans. In your response, please comment on the structure and parameters used to build the model and include discussion of confidence, reliability, and uncertainties associated with the deltamethrin human model. Please include in your comments a discussion oft he data from the McCarver/Hines Laboratory (submitted as part ofthe CAPHRA package) for providing ontogeny of CES enzymes. b
. Please comment on the proposed use of SIMCYP for providing enzyme ontogeny patterns and deriving population distributions for metabolic parameters in humans. Please i nclude in your comments whether or not other tools with similar capacity to SIMCYP are available. 

### 7. Physiologically-basedpharmacokinetic models for humans with otherpyrethroids: Thus far, the CAPHRA has focused its human PBPK efforts on deltamethrin and, to a lesser extent, permethrin. Soon laboratory efforts will turn to other pyrethroids
. CAPHRA's proposal is to conduct fewer studies and in l ieu of such data, use read across and computational approaches. Please comment on the appropriateness ofthe current human PBPK model to be used for otherType I, Type II, or mixed type pyrethroids. Please include in your response evaluation ofthe path forward provided by the CAPHRA regarding in vitro and in vivo studies for other Type I, Type II, or mixed type pyrethroids. 

### 8. Integration oflines ofevidence: a. The tissue dosimetry data from the rat suggest higher brain levels in juveniles compared to adults. In contrast, the preliminary PBPK modelling for human predicts slightly lower brain concentrations in young children compared to adults. Please comment on these differences, including comments on the key inputs that lead to this difference, human variability associated with the key parameters, and the confidence and uncertainties associated with the difference between the rat and human internal dosimetry
. b. The CAPHRA has proposed to continue in vivo behavioral testing for deltamethrin and permethrin in definitive dose-response evaluations. Pyrethroids have been studied for decades and thus there is a large body of evidence for these pesticides. Given your 8response to 8a along with 1) the extensive body of scientific literature on pyrethroid toxicity syndromes and high dose stud i es in juvenile rats (e.g., Sheets et al, 1994 4 ); 2) neurolemma studies supported by the CAPHRA (Question 2) along with additional in vitro studies (e.g. Meacham et al., 2008); and 3) recent in vivo stud i es from the Vorhees & Bruckner labs, please comment on the additional scientific value that would be provided in conduct i ng f u rther in vivo rat experiments to assess potentia l for P D sensitivity of human i n fants and chi l dre n . c. I f you believe there is add i t i ona l scientific va l ue to conducting additional in vivo experi m ents (8b), please comment on the CAPHRA's proposed experiments. In the context of your response to Question 3, p l ease include in your comments a discussion of dose leve l s and/or a dditiona l study design elements to improve existing preliminary evaluations. 4 Sheets , l.P . , et al., Age
- Dependent Differences in the Sus c eptibility of Rots to Deltomethrin . Toxi c ology and Applied Pharmacology, 1994 . 126(1) : p. 186 - 190 . 9