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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

Memorandum 

DATE: April 17, 2015 

SUBJECT: Transmission of Background Materials and Charge to the Panel for the May 19 

through 21, 2015 Session of the FIFRA Scientific Advisory Panel (SAP} Entitled 

"Research to Evaluate the Potential for Juvenile Sensitivity to Pyrethroids" 

TO: 

FROM: 

Fred Jenkins, Ph.D. 

Designated Federal Official 

FIFRA Scientific Advisory Panel 

Office of Science Coordination and Policy 

Dana Vogel, Acting Directo . 

Health Effects Division 

Office 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 session of the FIFRA Scientific Advisory Panel (SAP} 

reviewing the topic "Research to Evaluate the Potential for Juvenile Sensitivity to Pyrethroids." 

These documents do not contain any information protected by copyright or protected under 

statute as Confidential Business Information (CBI}. All materials submitted by CAPHRA are 

considered proprietary (See CAPHRA cover letter} and are protected under FIFRA section 10(g}. 

The attached documents include the following: 

- -

Document Title Date 

May 2015 FIFRA SAP Charge Questions April17, 2015 

CAPHRA Cover letter Apri l 16, 2015 

CAPHRA Program Overview April 16, 2015 

Appendix A1-1: list of Pyrethroids Represented by CAPHRA Members April 16, 2015 

Appendix A1-2: CAPHRA Member list April16, 2015 

Appendix A1-3: Team of Internationally Recognized Scientists April16, 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 Pyrethroids on Rat Brain Neurolemma-Injected April 14, 2015 

1 
Document Title Date 

Oocytes: Examining Potential AgeRelated Pharmacodynamic Differences- Report 

Appendix A2-3: Evaluating Potential Age-Related Pharmacodynamic April 16, 2015 

Sensitivity in the Rat: Acute Neurotoxicity 

of Deltamethrin and Permethrin in Adult and Juvenile Rats - Summary 

Appendix A2-4: Studies of the Acute Neurotoxicity of Deltamethrin and February 18, 2015 

Permethrin in Adult and Juvenile Rats -

Report 

Appendix A3-1: In Vivo Pharmacokinetic Studies of Deltamethrin in April 16, 2015 

PND15, PND21, and PND90 Rats- Summary 

Appendix A3-2-1: Toxicokinetic Assessment of Blood and Tissue March 3, 2015 

Deltamethrin Concentrations Following the 

Administration of a Single Oral Dose to Mature Rats - Report 

Appendix A3-2-2: Toxicokinetic Assessment of Blood and Tissue March 3, 2015 

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, 2015 

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 of Age Differences in Rat March 6, 2015 

Plasma Protein Binding - Report 

Appendix A3-6-2: Delt:amethrin: Assessment of Age 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 of the 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 

2 
Charge to FIFRA Scientific Advisory Panel 

May 19-21, 2015 

Research To Evaluate The Potential For Juvenile Sensitivity To Pyrethroids 

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

the Federal Food, Drug, and Cosmetic Act (FFDCA) gives the agency the authority to establish tolerances 

for residues on food and/or feed resulting from use of a pesticide. The studies required to allow the 

agency to make the appropriate statutory safety findings under both of these acts are stipulated under 

40 Code of Federal Regulations (CFR) Part 158. There is flexibility, however, in implementing Part 158. 

Additional data can be required (§158.75), alternative approaches can be accepted, and studies can be 

waived (§158.45}. The 2007 National Research Council (NRC) report from the National Academy of 

Sciences (NAS) on Toxicity Testing in the 21st Century describes this new vision for toxicity testing. In 

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

New Pesticide Testing and Assessment Approaches (http:ljwww.epa.gov/pesticides/science/testingassessment.html) which describes OPP's approach to implementing the NAS vision. One of the key 

components of OPP's Strategic Direction is improved approaches to more traditional toxicity tests to 

minimize the number of animals used while expanding the amount of information obtained. OPP also 

has a recent document, Guiding Principles for Data Requirements 

(http :1/www .epa .gov /pesticides/regulating/ data-require-guide-principle .pdf) which describes the 

principles for requiring toxicology data for pesticides, specifically to "only require data that adequately 

inform regulatory decision making and thereby avoid unnecessary use of time and resources, data 

generation costs, and animal testing." 

OPP is act ively working on a reevaluation of the human health effects of the pyrethroids and pyrethrins 

under the OPP registration review program 

(http://www.epa.gov/oppsrrdl/registration 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 juvenile 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 

juvenile 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 juvenile 

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

Century using a combination of in vitro studies, targeted in vivo studies, and physiologically-based 

pharmacokinetic (PBPK) models. 

Based on feedback 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 Pyrethroid Human 

Health Risk Assessment (CAPHRA). Since late 2010, the CAPHRA has worked with academic scientists to 

1 The PPTWG was the predecessor to the CAPHRA 

3 
develop assays and models to assess the potential for juvenile post-natal sensitivity to pyrethroids. The 

on-going research effort is organized around the adverse outcome pathway (AOP) for pyrethroids: 

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

and firing potentials and ultimately to in vivo clinical syndromes. 

Specifically, the CAPHRA is evaluating the potency of pyrethroids to human sodium channels and 

transplantation of adult & juvenile rat synaptic membrane into oocytes. In addition, the CAPHRA is 

conduct ing targeted in vivo studies on behavioral metrics and developing PBPK models. The research, 

thus far, has focused on development of the overall approach using data for deltamethrin and 

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

feedback on extending 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 of the science 

with the CAPHRA research effort and proposals for next steps which include extension of data on 

deltamethrin and permethrin to other pyrethroids. 

Charge Questions to the Panel: 

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

cells with their regulatory Beta subunits (ChanT est Data): Human voltage-gated sodium 

channels (NaVs 1.1, 1.2, 1.3, 1.6) were expressed in human embryonic kidney (HEK) cells and 9 

pyrethroids were tested for their effects on channel conductance, as well as the effects of 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 of their robustness (i.e. reproducibility, 

controls, statistics, background information, NaV selection, etc). Please comment on 

the confidence and uncertaint ies in the ChanT est experiments and related findings. 

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

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

CAPH RA has proposed NOT to continue this line of research. Please comment on this 

proposal and degree to which the ChanT est data inform the issue of evaluating the 

potential for juvenile sensitivity. 

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

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

Soderlund, 2006; Meacham et al., 2008; Tan and Soderlund, 20092

) show stronger 

responses to pyrethroids. Please comment on the extent to which the expressed 

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

sodium channels expressed in Xenopus oocytes. Toxicol Appl Pharmacol. 2006 Mar 15:211(3):233-44. Epub 2005 Ju1 26 

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

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

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

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

4 
human sodium channels can be used in combination with these sources to infer relative 

pharmacodynamic sensitivity between rats and humans. 

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

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

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

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

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

chloride and calcium channels). 

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

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

robustness of data from the synaptic membrane transplantation into oocytes 

(reproducibility, control compounds, channel modulator reagents, statistics, use of Na 

blocker tricaine as an anesthetic, etc) and the degree to which these data inform the 

issue of evaluating the potential for juvenile sensitivity. Please comment on the 

confidence and uncertainties and associated findings in the oocyte experiments. 

b. In the context of your response to 2a, the CAPHRA has proposed to collect doseresponse data in adult and juvenile rat synaptic membrane transplanted into oocytes for 

five more pyrethroids (including Type I, Type II, and mixed). CAPHRA's proposed path 

forward: If the additional five pyrethroids show similar patterns to deltamethrin and 

permethrin (i.e. no lifestage sensitivity observed), no additional pyrethroids will likely be 

tested in this system. Alternatively, if a different pattern is observe·d, additional 

pyrethroids are likely to be tested. Please comment 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 hese experiments, 

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

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

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

that juvenile rats are more sensitive to deltamethrin based on changes in detailed clinical signs 

and, to less extent, acoustic startle. However, there was no greater sensitivity in juvenile rats 

exposed to permethrin. Preliminary data from whole brain tissue samples with deltamethrin 

indicated that PND 15 rats had higher deltamethrin concentrations as compared to adult rats 

given the same dose. The brain concentration and toxicity data from the CAPHRA studies for 

5 
deltamethrin are consistent to those previously published by the Bruckner lab (Kim et al., 20103) 

which showed that at a similar dose (2 mg/kg deltamethrin) PND 10 rats had increased severity 

of clinical signs and higher Cmax of deltamethrin in the brain as compared to PND 21, PND 40, 

and PND 90 rats. 

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

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

permethrin conducted at the University of Cincinnati (Vorhees lab). 

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

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

studies conducted by the CAPHRA, both auditory startle and detailed clinical 

observations were evaluated. Please comment on the degree to which t he auditory 

startle and detailed clinical observations provide useful data for evaluating the potential 

for juvenile sensitivity. Please include in your comments discussion of the dynamic 

range; the direction of the response varying between Type I and lis; and the type of data 

obtained (continuous, ranked). 

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

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

data to aid in the interpretation of the auditory startle and detailed clinical observation 

data. 

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

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

plasma partition coefficients (in vivo measurements in PND 21 and adult rats), age dependent 

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

P450 (CYP) and carboxylesterase (CES) metabolism (in vitro measurements using rat liver and 

plasma preparations from PND 15, 21, and 90 rats) were evaluated. Further in vivo 

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

and multiple oral (PND 15, 21, and 90) doses to generate plasma and tissue data to validate the 

developing rat PBPK model. Additional experiments with deltamethrin were conducted for 

constructing the developing human PBPK model. Parameters evaluated in vitro included age 

dependent plasma protein binding derived in plasma from human donors aged from birth to 

adults, transport across the blood brain barrier using a human brain microvascular endot helial 

cell line, estimates of gastrointestinal absorption in caco-2 cells, and ontogeny data forCES, 

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

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

Mar S. 

6 
CYP1A2, and CYP2C8 determined in human liver tissue from donors (age 1-18 years old). Further 

in vitro experiments are currently underway by the CAPHRA to determine human adult and 

juvenile liver metabolism of deltamethrin by CYP and CES enzymes using human liver 

preparations or recombinant human enzymes. 

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

the rat conducted thus far for deltamethrin. 

b. Please comment 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 include in your comments a discussion of the ongoing in vitro experiments with 

recombinant enzymes for use in PBPK models and associated confidence and 

uncertainty with the use of such data. 

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

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

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

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

collected in vitro to estimate hepatic metabolic clearance in vivo. The deltamethrin brain or 

plasma concentrations estimated by the model in rats are then compared to measured 

concentrations from in vivo rat studies to verify the model. Non-chemical specific physiological 

parameters for rats were obtained from the published literature, including body weight, cardiac 

output, hematocrit levels, tissue volumes, and tissue blood flows. As discussed in Question 4, 

recently generated data by the CAPHRA and published data were used for compounddependent parameters, including partition coefficients, metabolic rate constants, absorption 

rates, protein binding, compartments and tissue permeability. 

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

exposures in the developing rat. In your response, please include evaluation of the 

structure and parameters used to build the model, as well as its ability to accommodate 

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

discussion of confidence, accuracy and uncertainties associated with the deltamethrin 

developing rat model. Please also comment on the sensitivity analyses of parameters 

CAPHRA has completed thus far. 

b. In the context of your response to Sa, please comment on the extent to which 

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

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

human PBPK model integrates non-chemical specific physiological parameters for humans from 

the literature. Compound-dependent parameters, such as partition coefficients and oral 

absorption parameters, were adapted from the rat PBPK model. Recently generated data by the 

7 
CAPHRA were used for the remaining compound-dependent parameters, such as metabolic rate 

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

and CES enzymes involved in metabolism of a given pyrethroid will be identified and the in vitro 

metabolic constants for those enzymes will be determined for integration into the PBPK model. 

Intrinsic clearance for each active enzyme will be scaled to in vivo using scaling factor data 

collected by the CAPHRA and the SIMCYP database. The ontogeny of enzyme expression (also 

from the CAPHRA data and the SIMCYP database) will be incorporated 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 potential juvenile sensitivity, characterize the 

robustness of the PBPK model for extrapolating age-specific internal tissue exposures for 

humans. In your response, please comment on the structure and parameters used to build 

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

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

from the McCarver/Hines Laboratory (submitted as part of the 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 include 

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

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

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

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

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

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

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

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

Type I, Type II, or mixed type pyrethroids. 

8. Integration of lines of evidence: 

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

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

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

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

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

associated with the difference between the rat and human internal dosimetry. 

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 

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

syndromes and high dose studies in juvenile rats (e.g., Sheets et al, 19944

); 2) neurolemma 

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

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

please comment on the additional scientific value that would be provided in conducting 

further in vivo rat experiments to assess potential for PD sensitivity of human infants and 

children. 

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

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

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

additional study design elements to improve existing preliminary evaluations. 

4 Sheets, l.P ., et al., Age-Dependent Differences in the Susceptibility of Rots to Deltomethrin. Toxicology and Applied 

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

9