Draft, Do Not Cite

Draft Page 2 of 58 Chemical PC Code CAS No. Allethrin 004001 584

- 79

- 2 Bioallethrin 004003 28057

- 48

- 9 S

- Bioallethrin 004004 28434

- 00

- 6 D

- Allethrin 004005 84030

- 86

- 4 Esbiothrin 004007 84030

- 86

- 4 Bifenthrin 128825 82657

- 04

- 3 Cyclethrin 004052 97

- 11

- 0 Cyfluthrin 128831 68359

- 37

- 5 Cyfluthrin, beta

- 118831 68359

- 37

- 5 Cyhalothrin 128867 68085

- 85

- 8 Cyhalothin, lambda

- 128897 91465

- 08

- 6 Cyhalothrin, gamma

- 128807 76703

- 62

- 3 Cypermethrin 109702 52315

- 07

- 8 Cypermethrin, alpha

- 209600 67375

- 30

- 8 Cypermethrin, beta

- 118831 65731

- 84

- 2 Cypermethrin, zeta

- 129064 52315

- 07

- 8 Cyphenothrin 129013 39515

- 40

- 7 Deltamethrin 097805 52918

- 63

- 5 Esfenvalerate 109303 66230

- 04

- 4 Fenvalerate 109302 51630

- 58

- 1 Fenfluthrin 109705 69409

- 94

- 5 Fenpropathrin 127901 39515

- 41

- 8 Imiprothrin 004006 72963

- 72

- 5 Metofluthrin 109709 240494

- 70

- 6 Permethrin 109701 52645

- 53

- 1 Prallethrin 128722 23031

- 36

- 9 Pyrethrins (not synthetic) 069001 8003

- 34

- 7 Resmethrin 097801 10453

- 86

- 8 Sum i thrin ( d

- phenothrin) 069005 26002

- 80

- 2 Tau

- fluvalinate 109302 102851

- 06

- 9 Tefluthrin 128912 79538

- 32

- 2 Tetramethrin 069003 7696

- 12

- 0 Tralomethrin 121501 66841

- 25

- 6Draft Page 3 of 58 

# Draft Science Policy Paper Proposed Common Mechanism Grouping for the Pyrethrins and Synthetic Pyrethroids
May 19, 2009Draft, Do Not Cite

Draft Page 4 of 58 

### TABLE OF CONTENTS

1.0 Background

................................ ................................ ................................ ........... 5 2.0 Key Concepts

................................ ................................ ................................ ........ 6 3.0 Chemical Structure

................................ ................................ ............................... 7

3.1 Chirality of pyrethroids

................................ ................................ ................. 9 3.2 Quantitative Structure Activity Relationships (QSAR)

............................. 11 4.0 Toxicology of Pyrethroids

................................ ................................ .................. 12

4.1 Overview

................................ ................................ ................................ ...... 12 4.2 Voltage
- Gated Sodium Channels (VGSC)

................................ ................. 13 4.2.1. Protein structure of the VGSC

................................ .............................. 13 4.2.2. Channel modulation by pyrethroids

................................ ..................... 14 4.2.3. Relationship between pyrethroid
- induced alterations in VGSC function & disrupted membrane excitability

................................ ....... 16 4.2.4. Potential differences in tissue sensitivity to pyrethroids

................... 16

4.2.4.1. Voltage
- clamp measurements

................................ ......................... 16 4.2.4.2. Sodium channel cloning

................................ ................................ .. 17 4.2.5. Other molecular targets for pyrethroids

................................ .............. 18 4.3 **In vivo** profile

................................ ................................ ............................... 23 4.3.1. Pharmacokinetics

................................ ................................ .................. 23 4.3.2. Toxicity syndromes in rodents

................................ ............................. 25 4.3.3. Information in humans

................................ ................................ .......... 30 4.4 Data in mixtures of pyrethroids

................................ ................................ . 31 5.0 Proposed Common Mechanism Grouping under the FQPA

........................... 32

5.1 Weight of the Evidence

................................ ................................ ............... 32 5.2 Summary

................................ ................................ ................................ ...... 37 6.0 Proposal to Sub
- Group the Pyrethroids by Type I & Type II

........................... 37

6.1. Pyrethroid Effects on Cloned VGSC Na v 1.8

................................ .............. 38 6.2. Micro electrode arrays (MEA) Studies

................................ ....................... 39 6.3. **In Vivo** Rodent Studies

................................ ................................ ............... 40 6.4. Proposed Subgrouping

................................ ................................ .............. 41 7.0 References

................................ ................................ ................................ ........... 43 Attachment 1.0. Classification (as proposed by the Agency) and Structure of pyrethroids registered for use with the US EPA.

........................... 56Draft, Do Not Cite

Draft Page 5 of 58 1.0 Background The Food Quality Protection Act (USEPA,1996) requires EPA to take into account “available evidence concerning the cumulati ve effects on infants and children of such residues and other substances that have a common mechanism of toxicity.” As described in detail in the cumulative risk assessment (CRA) guidance (USEPA, 2002) , developed by the Office of Pesticide Pro grams (OPP), a cumulative risk assessment begins with the identification of a group of chemicals referred to as a common mechanism group (CMG), that induce a common tox ic effect by a common mechanism of toxicity. To date, OPP has conducted four cumulative risk assessments on the organophosphates (OPs), N

- methyl carbamates (NMCs), chloracetanilides, and triazines. Pyrethroids are a class of synthetic insecticides which are structurally based on the pyrethrins, botanical insecticides extracted from Chrysanthemum cinerariaefolium (Soderlund et al. 2002) . Pyrethroid exposure c an occur from food, water, or non

- occupational settings. Due, in large part, to changes in usage trends resulting from risk mitigation, residential use of OPs and NMCs has decreased. Consequently, potential exposure of the general public to pyrethroid p esticides has increased over the past decade. There is an extensive published literature concerning the toxicity of pyrethroids and potential modes of action of these compounds. In addition, in the last few years, EPA’s Office of Research and Development (ORD) and the Pyrethroid Working Group 1 (PWG) have conducted independent, large

- scale research efforts to evaluate aspects of pyrethroid exposure, pharmacokinetics (PK), mode/mechanism of action, and toxicity profiles. These research efforts have added t o the scientific literature and also led to a meeting of The Federal Insecticide, Fungicide, and Rodenticide Act Science Advisory Panel (FIFRA SAP) in August, 2007, on a proposed approach to developing physiologically

- based pharmacokinetic (PBPK) models fo r multiple pyrethroids (USEPA, 20 07) . The research by both EPA and the PWG was conducted, in large part, in anticipation of a future cumulative risk assessment on the pyrethroids. The purpose of this draft science policy document is to describe this scientific evidence which support s a proposal to designate the pyrethroids as a CMG under the FQPA. OPP is soliciting comments from the FIFRA SAP and the public on this document in June, 2009. This document follows OPP’s guidance on forming CMGs (2002) and also considers the concepts contained in the Mode of Action and Human Relevance Frameworks developed by EPA, WHO’s International Programme for Chemical Safety and the International Life Sciences Institute (USEPA, 1999; Sonich

- Mullin et al. 2001; Meek et al. 2003; Seed et al. 2005; Boobis et al. 2006) . Based on the current state of the science, OPP is proposi ng to designate the naturally occurring pyrethrins and synthetic pyrethroids form a CMG based on 1) shared structural characteristics; and 2) shared ability to interact with voltage

- gated sodium channels (VGSC), resulting in disruption of membrane excitabi lity in the nervous system, and ultimately neurotoxicity characterized by two different toxicity syndromes. As described in more detail below, OPP is further proposing to subgroup the pyrethroid CMG into two subgroups representing Type I and II pyrethroid s. This subgrouping is consistent with historical categorization of pyrethroids and is supported by i n vitro and in vivo studies from both older studies and new research. Pyrethroids considered for the CMG are listed in Appendix 1.

1 The Pyrethroid Working Group is a group of pesticide companies who own technical registrations for pyrethrins and some synthetic pyrethroids.Draft, Do Not Cite

Draft Page 6 of 58 The designation of Type I and Type II pyrethroids is based , in large part, upon differences in structure; the α

- cyano moiety is absent in Type I pyrethroids and present in Type II. Type I members of the proposed CMG include allethrin, bifenthrin, permethrin, resmethrin, te fluthrin and tetramethrin, Type II members of the proposed CMG include cyfluthrin, cyhalothrin, cypermethrin, and deltamethrin. The absence/presence of the α

- cyano group is correlated with length of time the sodium channel is inactivated (

- CN=shorter; +C N=longer) which in turn corresponds with the two distinct toxicity syndromes (

- CN=T syndrome; +CN=CS syndrome). The nature of these effects is discussed in more detail in section 4.0. Two pyrethroids, esfenvalerate and fenpropathrin, though structurally similar to Type II pyrethroids, are considered m ixed Type I/II because they demonstrate properties of both groups during In vitro and in vivo evaluations. The Agency’s plans for future classification of the additional pyrethroids shown in Attachment 1 is described in Section 6.4 below.

This document also describes some uncertainties considered including i) other endogenous channels (e.g., calcium and chloride) which have been proposed by some (Soderlund et al. 2002; Ray and Fry 2006; Clark and Symington 2007 ) as potential other target sites for pyrethroids; ii); sodium channel structural heterogeneity among tissue and across life stages with respect to alpha

- and beta

- subunits; and iii) uncertainties associated with lack of an in vivo biomarker for pyrethroid

- VGSC interaction.

2.0 Key Concepts OPP’s has guidance documents for designating CMGs (USEPA 1999) and for developing cumulative risk ass essments (USEPA 2002)

. These documents provide definitions for some key concepts and provide the guiding principles for the analysis contained in this propos al. OPP’s guidance documents and approaches to grouping chemicals for common mechanism and to developing cumulative risk assessments have been through extensive peer review with many reviews by the FIFRA SAP. Some key concepts included in the current ana lysis include:

**•** Mechanism of toxicity is defined as the major steps leading to a toxic effect following interaction of a pesticide with biological targets. All steps leading to an effect do not need to be specifically understood. Rather, it is the identi fication of the crucial events following chemical interaction that are required in order to describe a mechanism of toxicity. Generally, the more that is understood about the various steps in the pathway leading to an adverse effect, the more confident on e is about the mechanism of toxicity. For instance, a mechanism of toxicity may be described by knowing the cascade of effects such as the following: a chemical binds to a given biological target i n vitro , and causes the receptor

- related molecular respon se; in vivo it also leads to the molecular response and causes a number of intervening biological and morphological steps that result in an adverse effect.

**•** A pesticide and another substance that are known to cause the same toxic effect in or at the same anatomical or physiological site or locus (e.g., same organ or tissue) are said to
cause a common toxic effect

.

**•** A c ommon mechanism of toxicity pertains to two or more pesticide chemicals or other substances that cause a common toxic effect to human health by the same, or essentiallyDraft, Do Not Cite

Draft Page 7 of 58 the same, sequence of major biochemical events. Hence, the underlying basis of the toxicity is the same, or essen tially the same, for each chemical. Such that a common mechanism group (CMG) is a group of pesticides which share a common mechanism of toxicity
.

**•** Under FQPA (1996) , cumulative risk is defined as the risk associated with exposure to a group of chemicals that are toxic by a common mechanis m from all pathways. Thus, cumulative risk assessments include multiple chemicals and multiple pathways (e.g., food, drinking water, consumer uses).

**•** A common mechanism endpoint(s) is/are those common toxic effect(s) which are pertinent and sensitive e ndpoints associated with the common mechanism which will provide a scientifically sound basis for determining relative potency of chemicals in a cumulative risk assessment.

3.0 Chemical Structure The basic pyrethroid structure consists of an acid and alcohol moiety (Fig. 1). Pyrethroids, the synthetic derivatives of the pyrethrins, have evolved structurally over the past several decades. However, the basic components of pyrethrins, a chrysanthamic acid linked to an aromatic alcohol through an ester l inkage have been conserved (Fig. 1). Structural modifications such as the addition of halogens to the chrysanthamic acid and aromatic alcohol moieties and the addition of the α

- cyano group have increased photostability, insecticidal potency, and in some i ncidences, stereoisomerism of the pyrethroids.Draft, Do Not Cite

Draft Page 8 of 58 

### Pyrethrin I (Type I)

O O

H O unsaturated side-chain cyclopropyl ring planar spacer methylene or equivalent bridge group non-coplanar center of unsaturation acid moiety alcohol moiety central ester bond

* *



### Resmethrin (Type II)

O

O O

* *



### Cypermethrin (Type II)

O

O Cl Cl

N O * * *

* = chiral centers

Figure 1. Representative s tructures for pyrethrins (Pyrethrin I), Type I ( r esmethrin) and Type II ( c ypermethrin) pyrethroids.Draft, Do Not Cite

Draft Page 9 of 58 3.1 Chirality of pyrethroids Pyrethroids can possess one to three chiral centers, resulting in two to eight enantiomers. For example, in the Type I pyrethroids, the presence of two chiral carbons, C1 and C2, in the in the cyclopropane ring of the chysthanthamic acid moiety produces t wo pairs of diastereomers which are designated trans and cis (Fig 2) . The cis and trans structural confirmations are further divided into R and S confirmations based on the relationship of the cyclopropane ring to the central ester bond, for a total of 4 stereoisomers. The addition of a third chiral carbon in Type II pyrethroids (e.g. cypermethrin) results in 8 stereoisomers (Fig. 1).

Stereoiso merism of the pyrethroids is an important consideration in the toxicity profile of pyrethroids. For example, the orientation of the C1 and C3 constituents in the cyclopropane ring of the chrysanthamic acid moiety determine the cis/trans structural confirm ation, which can in turn impact potential toxicity and metabolism. Furthermore, the 1R configurations are insecticidal, whereas the enantiomeric 1 S compounds, though physically identical, are without insecticidal activity (Elliot et al. 1974)

. Effects of pyrethroids on VGSC function have also been demonstrated to be stereo

- isomer dependent (L und and Narahashi 1982; Narahashi 1982)

. As will be discussed in Section 4.0, the insecticidal efficacy correlates with the ability of isomers to modify sodium channel kinetics. Furthermore, isomerism influences the metabolism of these compounds (See PK section 4.3.1 for more details). Ideally, there would be sufficient data to distinguish the pharmacodynamics (PD) , PK, and environmental exposure of the active stereoisomer(s). For some, such as bioresmethrin, deltamethrin, and esfenvalerate, the re gistered products are enrichments of the more insecticidally active isomers. As such, for these, there is comparatively little uncertainty with respect to assessing the hazard and exposure. For permethrin, residue data and some limited toxicity informati on are reported as cis and trans. However, toxicity and exposure information available for the majority of pyrethroids are for the isomeric mixture. Specifically animal toxicity studies submitted for pesticide registration are performed with the isomeric mixture. In addition, residue data used to evaluate food, water, and residential exposure are typically reported as isomeric mixtures. Thus, although the Agency acknowledges the stereoisomerism as a consideration for pyrethroids, the ability of the Agen cy to quantitatively account for these in risk assessment is limited. Instead of assessing the active stereoisomer(s), the Agency typically assesses the risk to pyrethroids by evaluating the risk to the isomeric mixture. The resources required to collect such toxicity and exposure data on the isomers would be substantial. Although there is some uncertainty associated with evaluating the mixture instead of the active stereoisomer, the lack of such data does not prevent the Agency from developing risk
asse ssments which are both health protective and scientifically sound 2

.

2 With respect to the common mechanism grouping, pending the outcome of the SAP review and comments from the publ ic, the Agency expects to identify the registered technical products (i.e., the isomeric mixture) as a potential member of the CMG, not the active stereoisomer(s).Draft, Do Not Cite

Draft Page 10 of 58 Figure 2. Stereochemistry of the pyrethroid acid moietyDraft, Do Not Cite

Draft Page 11 of 58 3.2 Quantitative Structure Activity Relationships (QSAR) As stated above, one of the common features of pyrethroids is their common structural characteristics. With respect to similarity in chemical structure, OPP’s CMG guidance (2002) states that:
“Use of structural similarity as a starting point for grouping chemicals relies on the assumption that substances that are structurally analogous could contain a common toxophore 3 (or may yield a common toxophore upon metabolism) and may interact analogou sly with cellular biomolecular sites to cause a common toxic effect (p. 8).” In other words, for purposes of determining a CMG, identification of a toxophore is important for evaluating a common PD interaction at the molecular site of action.

Some au thors have argued for the lack of a toxophore among pyrethroid pesticides based on the contention that the appropriate fit of the entire molecule at the site of action is required for interaction (Byberg et al. 1987; Soderlund et al. 2000)

. However, Ford et al. (2002) has performed a three

- dimensional quantitative structure activity relationship (QSAR) analysis of pyrethroids. Conformational sampling of active pyrethroids using molecular dynamics and cluster analysis, taking into account the multitude of bond angles possible, reveal ed a common conformation in 39 of the 41 structures examined (Fig. 2). Type I and Type II pyrethroids both kill insects. Type I pyrethroids possess an effective ‘knockdown’ ability, debilitating insects within minutes. However, they may take days to kill , allowing the opportunity for recovery. Conversely, Type II pyrethroids lack the knockdown ability of Type I pyrethroids but are generally more potent at killing. The ‘knockdown’ and kill responses are also differentiated by their temperature dependenc e, negative for kill and positive for ‘knockdown’. Ford et al (2002) related the putative toxophore to the killing potential, inherent to both Types I and II. The authors postulate that restriction about the ether linkage by an α

- cyano group in Type II pyrethroids precludes the knockdown capacit y associated with Type I pyrethroids. The unrestricted movement about the ether linkage requisite for ‘knockdown’ is not the most stable conformation, as reflected by its positive temperature dependence.

3 The CMG guidance defines t he term “toxophore” with respect to toxic substances, as is aki n to the term “pharmacophore” with respect to drug substances: the pharmacophore is that structural moiety of a drug substance or substances which imparts a desired pharmacological property.Draft, Do Not Cite

Draft Page 12 of 58 Figure 3. Conformational structure of p utative pyrethroid toxophore As detailed in the following section, vertebrate VGSC structures are significantly more complex than insect channels. However, there is homology in structure and function between insect and mammalian sodium channels

. Insect and mammalian VGSC are both composed of pore

- forming α subunits with modifying proteins (mammalian β units and insect tipE proteins) and Type I and Type II pyrethroids cause similar modifications in channel kinetics in both species (Type I results in repet it ive firing and Type II pyrethroids result in depolarization

dependent block of the action potential). It is therefore reasonable to assume the toxicophore identified in insects would be similar to that in mammals. As such, the Agency further assumes th at a shared toxophore and chemical structure of pyrethroids provides part of the basis for grouping the pyrethroids as a CMG.

4.0 Toxicology of Pyrethroids

4.1 Overview It is well established that pyrethroids modify the kinetics of the VGSCs in insect and mammalian neurons. The Agency has identified multiple lines of evidence which support the shared ability of the synthetic pyrethroids and pyrethrins to interact with th e Na channels leading to depolarization of membranes in the nervous system, and ultimately neurotoxicity characterized by two different toxicity syndromes. This evidence is summarized below starting with the VGSC followed by mechanistic information from v oltage clamp/patch clamp studies, use of neuronal networks (e.g., micro electrode arrays (MEAs)), in vivo toxicity studies, and PK. This paper does not provide an exhaustive review of this body of work but highlights and summarizes critical data relevant to common mechanism grouping findings. For extensive reviews, see Soderlund and Bloomquist (1989) , Vijverberg and van den Berken (1990) , Narahashi (1992) , Clark (1995) , Soderlund (1995) , Bloomquist (1996) , Narahashi (1996) , Shafer et al (2005) , Wolansky and Harrill (2008) , Nemec (2006)

.Draft, Do Not Cite

Draft Page 13 of 58 4.2 Voltage

- Gated Sodium Channels (VGSC)

4

. 2 .1. Protein structure of the VGSC Briefly, the voltage

- gated sodium channel α subunits consists of four homologous domains, each domain is comprised of six transmembrane helical protein segments forming the ion channel pore (Fig. 4). Expression of the α subunit alone is minimally sufficient to form a functioning channel. A voltage

- sensing element located in the S4 transmembrane segment detects changes in membrane potential resulting in a conformation change of the channel leading to opening and rapid closing of the voltage dependent gating mechanism. In vertebrates, smaller auxiliary β subunits modulate α subunit gating, as well as insertion and distribution of the channel complex into the membrane (Isom et al. 1992; Isom et al. 1995; Isom 2001; Smith and Soderlund 2001) . Insects have a single isoform of the α

- subunit, expressed exclusively in the nervous system (Hong and Ganetzky 1994) and insects lack β subunits, per se. However, tipE protein has been suggested as a homologous, though structurally unrelated, protein to the vertebrate β subunit (Warmke et al. 1997)

.

Figure 4: Schematic of the Transmembrane Voltage

- Gated Sodium and Calcium Channel

In contrast to the simplicity of the insect sodium channel, mammalian VGSC are much more complex, and this complexity creates uncertainty in correlating effects on specific VGSC isoforms i n vitro to in vivo symptomology caused by pyrethroid toxicity in mammals. There are at least 10 distinct genes that encode sodium channel α subunit proteins (Na v 1.1

- Na v 1.9; Na v 1.X ) in humans and mice (Plummer and Meisler 1999; Goldin 2001) . In addition, four β subunits (β1

- β4) (Yu et al. 2003) have been identified to date. The heterogeneity of the sodium channel can also be enhanced by the alternative mRNA splicing of some isoforms (Sarao et al. 1991; Schaller et al. 1992; Gustafson et al. 1993) . Finally, the functional and pharmacological diversity of sodium channel expression varies by tissue and stage of development (reviewed in Meacham etDraft, Do Not Cite

Draft Page 14 of 58 al (2008) and Mandel (1992) ). The implications of this diversity on uncertainty related to actions of pyrethroids will be discussed further in the following sections.

4

. 2.2

. Channel modulation by pyrethroids The effects of pyrethroid exposure on neuronal tissue (both insects and mammals) have been extensively reviewed (Soderlund and Bloomquist 1989; Vijverberg and Van den Bercken 1990; Narahashi 1992; Bloomquist 1993a; Clark 1995; S oderlund 1995; Bloomquist 1996; Narahashi 1996)

. Figure 5 depicts generalized pyrethroid effects on individual channels, whole

- cell sodium currents, and action potentials (extracted from Shafer et al (2005) ). Depolarization opens VGSCs (top left) allowing sodium to enter the cell. To limit sodium entry and depolarization length, VGSCs inactivate and must return to a “resting” state before reopening. Pyrethroids inhibit the function of two different “gates” that control sodium flux through VGSCs (top right), delaying inactivation (i ndicated by double arrows between states) of the channel and allowing continued sodium flux (Open*). If sodium current through an entire cell is measured, depolarization leads to a rapidly inactivating current under normal circumstances (bottom left, Sodiu m Current). Pyrethroid

- modified VGSCs remain open when depolarization ends (bottom right, Sodium Current), resulting in a “tail” current (the notch at the end of example currents). If membrane voltage is examined, depolarization under normal circumstances generates a single action potential (bottom left). VGSCs modified by type I compounds (bottom right, Action Potential) depolarize the cell membrane above the threshold for action potential generation, resulting in a series of action potentials (repetitive firing). Type II compounds cause greater membrane depolarization, diminishing the sodium electrochemical gradient and subsequent action potential amplitude. Eventually, membrane potential becomes depolarized above the threshold for action potential generat ion (depolarization

- dependent block) (Shafer et al. 2005)

. Modification of the Na channels in mammals has been shown to vary by Type I and II pyrethroids (Bottom right, Fig. 5). Natural pyrethrins and Type I synthetic pyrethroids are typically characterized by slight prolongations for the sodium current tails (20 ms) , resulting in trains of action potentials accompanied with little or no effect on resting membrane potential. In contrast, pyrethroids containing the α

- cyano moiety typically do no t induce repetitive firing. Instead, Type II pyrethroids produce long

- lasting sodium current tails ( >> 200ms ) that ultimately result in action potential block due to depolarization of resting membrane potential above the threshold for action potential gener ation

. It has been estimated that a s few as 1% of the sodium channels need be modified to render a cell hyperexcitable and induce repetitive firing (Song and Narahashi 1996; Motomura and Narahashi 2001)

.Draft, Do Not Cite

Draft Page 15 of 58 Figure 5. Effects of pyrethroids on Na channels, currents, and action potentials (From Shafer et al., 2005)Draft, Do Not Cite

Draft Page 16 of 58 4

. 2.3

. R elationship between pyrethroid

- induced alterations in VGSC function & disrupted membrane excitability The relationship between pyrethroid

- induced alterations in VGSC function and disrupted membrane excitability (membrane depolarization and/or changes in firing of action potentials) is well established and has been demonstrated in a number of species i n vit ro . Using crayfish giant axon, Salgado and co

- workers (1989) demonstrated temperature

- dependent actions of fenvalerate on VGSC function as well as membrane excitability. In addition, voltage

- d ependent potassium currents were not altered by fenvalerate . Lund and Narahashi (1981) demonstrated that cyphenothrin disrupts sodium channel function, depolarized the membrane, and upon stimulation of the nerve, induces repetitive firing that is blocked by tetrodotox in (TTX). These same authors demonstrated that tetramethrin altered VGSC function and induced repetitive firing in squid axon (Lund and Narahashi 1982) . Similarly pyrethroids alter sodium current at the node of Ranvier and depolarization and/or induce repetitive in peripheral nerves from (Vijverberg and Bercken 1979) , reviewed in Vijverberg and van den Berken (1982) . Similar findings have also been reported in mammalian neurons i n vitro , wherein pyrethroids modify VGSC function and produce changes in membrane excitability in dorsal root ganglion neurons (Tabarean and Narahashi 1998) and cerebe llar Purkinje neurons (Song and Narahashi 1996) . These alterations in neuronal excitability are consistent with reports from in vivo measurements of modified neurophysiological endpoints. Following deltamethrin administration the typical period of supranormal nerve excitability of compound action potentials in rat tail nerve was increased from ~30 msec to ~400 msec (Parkin and Le Quesne 1982) . Similar changes in CAP were reported following administration of fenvalerate or allethrin (Nozaki et al. 1995) . When dorsal root potentials were recorded from urethane anesthetized rats, cismethri n increased the amplitude of the potential by up to 142% (Smith 1980) . Finally EEG measurements demonstrated that administration of cypermethrin produced bursts of epileptic activity following the fir st administration, and on subsequent days of administration, these were accompanied by shaking, myoclonus, and tonic

- clonic seizures (Condés

- Lara et al. 1999) . Thus, data from i n vitro studies indicate that pyrethroid modification of VGSC leads to changes in membrane excitability. In vivo , changes in neurophysiological meas ures occur that are consistent with alterations in action potentials, and correlate with behavioral changes.

4

. 2.4

. Potential differences in tissue sensitivity to pyrethroids

4

. 2.4.1

. Voltage

- clamp measurements The earliest evidence of the ability of pyrethroids to disrupt sodium channel kinetics was achieved through voltage clamp techniques. Voltage clamp techniques allow measurement of voltage

- gated membrane channel function by “clamping” the voltage differenc e across the membrane by electronic feedback (current injection), thus allowing controlled changes in membrane voltage. Changes in current injected to hold voltage at a constant level reflect changes in conductances across the membrane as a result of ion c hannel openings and closings. Thus, the membrane can be clamped to different voltages and resulting currents measured. Using solutions of different ionic composition as well as pharmacological compounds to block specific channels, it is possible to exami ne the behavior of different kinds of ion channels through which theseDraft, Do Not Cite

Draft Page 17 of 58 currents pass (Eckert et al. 1988) . However, it should be pointed out that pharmacological antagonists to specific VGSC subunit combinations are not available; this precludes iden tification of the exact composition of VGSC subunits mediating pyrethroid sensitive currents in native mammalian neurons. A number of voltage

– clamp studies have demonstrated the selective ability of pyrethroids to perturb sodium channels based on chemical structure. Dosing with tetramethrin (Type I) caused the mean open time of Na channels to increase resulting in repetitive firing (Chinn and Narahashi 1986; Holloway et al. 1989) . Deltamethrin an d fenvalerate ( Yu et al. 2003) increased mean open time 200 fold greater than noted for tetramethrin. These results are characteristic of the distinction commonly noted between Type I and Type II pyrethroids. Since individual neurons are known to contain multiple α and β subunits, it is not possible to determine the effect of the pyrethroids on individual channel subunit combinations using voltage

- clamp techniques. However, some tissue types have demonstrated structure

- based sensitivity to pyrethroids, indicating channel isoform populations may differ with tissue type. Dorsal root ganglion (DRG) cells contain tetrodotoxin

- sensitive (TTX

- S) and tetrodotoxin

resistant (TTX

- R) sodium channels. TTX

- R channels are susceptible to perturbation by both Type I and Type II pyrethroids while TTX

- S channels are relatively less sensitive (Ginsburg and Narahashi 1993; Tatebayashi and Narahashi 1994; Song and Narahashi 1996; Narahashi et al. 1998) . These examples demonstrate the different ial activity of Type I and Type II pyrethroids in multiple neuronal tissues. These differences in TTX

- S and TTX

- R VGSC sensitivity to pyrethroids are consistent with the sensitivity of the Na v 1.8 channels (which belong to TTX

- R VGSC) expressed in oocytes t o pyrethroids (Choi and Soderlund 2006)

. 4

. 2.4.2

. Sodium channel cloning As discussed above, mammalian sodium channels are heterogeneous and expressed in a tissue, time and region specific manner. Further, there appear to be differences in pyrethroid effects on mammalian neurons that are dependen t on the composition of the channel under study. Advances in molecular techniques have allowed researchers to express specific combinations of VGSC subunits in exogenous expression systems such as Chinese hamster ovary (CHO) cells and unfertilized Xenopus laevis oocytes. The isolation and expression of specific channel isoforms has allowed researchers to examine the direct impact of pyrethroids on these channels. For example, the expression of pore

- forming α subunits Na v 1.2 and Na v 1.8 in CHO cells in th e absence of β subunits produced characteristic tail currents following exposure to Type I and Type II pyrethroids (Trainer et al. 1997; Smith and Soderlund 1998, 2001) . Co

- expression with β subunits increased sensitivity but was not essential for sodium channel perturbation, confirming their roles as modulators of pyrethroid activity on sodium channels (Smith and Soderlund 200 1) . When expressed in these systems, α subunits have demonstrated differential structure

- dependent sensitivity to pyrethroids. Na v 1.2 is sensitive to Type II pyrethroids, but insensitive to Type I pyrethroids (Smith and Soderlun d 1998) . Na v 1.8, a principle carrier of TTX

- R current (Smith and Soderlund 2001) in DRG neurons discussed in the preceding section, is sensitive to perturbation by both T ype I and II pyrethroids resulting in characteristic modification of sodium currents (Choi and Soderlund 2006)

. These findings are supported by similar results in the voltage

- clamp experiments previously reviewed.Draft, Do Not Cite

Draft Page 18 of 58 Young rats (post

- natal day 8) have been shown to be more sensitive to the acute toxicity of deltamethrin (Sheets et al. 1994) and cisme thrin (Cantalamessa 1993) than adults but not to p ermethrin and cismethrin (Sh eets 2000) . PK differences between the immature and adult rats have been demonstrated (Cantalamessa 1993; Sheets et al. 1994; Anand et al. 2006)

. Meacham et al., (2008) explored the PD possibility of age

- dependent pyrethroid toxicity using combinations of Na v 1.2 or Na v 1.3 and β 1 o r β 3 subunit expressed in oocytes . Na v 1.3/β 3 proved to be the most sensitive combination. Na v 1.3/β 3 is expressed embrionically while Na v 1.2/β 1 predominates in adults, s upporting the hypothesis of developmentally

- dependent PD differences

. Although these exogenous expression systems are useful in evaluating tissue and life stage differences in the Na channel populations (Trainer et al. 1997; Smith and Soderlund 1998, 200 1; Choi and Soderlund 2006; Meacham et al. 2008) , such studies must be interpreted cautiously. High pyrethroid sensitivity of VGSC in neurons has been difficult to reproduce to date in channels expressed in oocytes or other systems, although effects of pyrethroids on all combinations of alpha and beta subunits have not been examined to date. Specifically, many of these In vitro studies have been conducted in non

- mammalian systems, non

- neuronal systems, and/or at temperatures lower than 37° C. Such differences may contribute to differences in sensitiv ity of the channel to pyrethroid modification, for example due to differences in post

- translational modification, membrane composition or phosphorylation status . Even extrapolation from non

- mammalian to mammalian exogenous expression systems is difficult. Studies examining pyrethroid effects on VGSC subunits expressed by transfection into mammalian cells often do not include co

- expression of β subunits or concentration

- response evaluations (Wang et al. 2001; Vickery et al. 2004; Dekker et al. 2005)

. Moreover, due to differences in techniques (electrophysiology vs. fluorescence measurements), studies with transfected mammalian cells are difficult to compare directly to studies utilizing oocytes. For example, Vickery et al (2004) showed that non

- cyano pyrethroids (allethrin, permethrin or tetramethrin) were without effect on rat Na v 1.2 expressed in Chinese hamster ovary cells, even when veratridine was present to activate VGSC. However, in the same study, deltamethrin and fenvalerate were also without effect individually and produced a small response in the presence of veratridine (Vickery et al. 2004)

. From a r isk assessment perspective, sodium channel cloning studies are interesting for purposes of evaluating species differences, potential population PD variability, and life stage differences. With respect to the proposal to form a CMG, t his research highlight s the importance of sodium channel interaction as a key event in pyrethroid toxicity . Specifically, although there is heterogeneity among the subunit combinations, the pathway of toxicity remains the same

--- namely that sodium channel interaction as a crit ical and common key event in toxicity of pyrethroids. All the pyrethroids examined to date have the ability to modify VGSC kinetics in
neurons. It is not necessary to incorporate this high level of specificity and/or variability with respect to the VGSC subunits into a risk assessment for human health and t his level of detailed understanding is not necessary for forming a CMG.

4

. 2.5

. Other molecular targets for pyrethroidsDraft, Do Not Cite

Draft Page 19 of 58 In the CMG guidance, OPP acknowledges that individual chemicals can exhibit multiple mechanisms of action and can have multiple molecular targets (USEPA, 1 999) . Specifically, the CMG guidance states:

“ Many substances can cause more than one toxic effect, depending upon level of exposure, and do so by different mechanisms of toxicity that take place at different sites of toxic action. However, a chemic al may also cause multiple toxic effects at multiple sites from a single mechanism of toxicity taking place at a single site of toxic action, provided that the function initially altered at the site of toxic action normally controls oth er functions at dist ant sites... The Agency will group substances that cause multiple toxic effects by a common mechanism from a common site of toxic action (e.g., the multiple effects caused by certain endocrine disruptors) for purposes of cumulative risk assessment, provided a t least one of the toxic effects is common among the substances (p. 7 )

. ” For the pyrethroids, the Agency has proposed interaction with VGSCs as the common initial key event for determining the CMG. Pyrethroids , however, also have been reported to inhibit other types of ion channels, including chloride

- permeable ion channels, voltage

- gated calcium channels (Vijverberg and Bercken 1990; Narahashi 1996; Narahashi et al. 1998; Ray and Forshaw 2000; Ray 2001; Soderlund et al. 2002; Burr and Ray 2004; Shafe r and Meyer 2004) and ligand

- ga ted chloride channels. It has been suggested that pyrethroid actions at these separate target sites combine to prolong neuronal membrane depolarization, resulting in massive neurotransmitter release (Clark and Brooks 1989) . While pyrethroid actions on VGSCs and neuronal excitability are well understood, very little is known about how effects on other target sites contribute to pyrethroid mammalian neurotoxicity (Ray 2001; Soderlund et al. 2002)

.Draft, Do Not Cite

Draft Page 20 of 58 A class of chloride

- permeable channels has been proposed as potential targets for pyrethroid actions in the mammalian central nervous system (CNS) (Forshaw et al. 1993; Ray et al. 1997; Forshaw et al. 2000; Burr and Ray 2004)

. However, limitations of the supporting studies justify cautious interpretation of the results with respect to the role of effects on this target in pyrethroid toxicity. A large conductance, chloride

- permeable ion channel (denoted as ‘maxi

- chloride’) isolated using the ‘inside out patch’ elect rophysiological technique is inhibited by various pyrethroid compounds. Pyrethroids inhibit these channels by reducing the probability that the channel will open. Information regarding concentration

- response relationships is lacking, but several pyrethro ids have been tested for their ability to modulate maxi

- chloride channel opening In vitro . Deltamethrin, cypermethrin, cyfluthrin, fenpropathrin, and bioallethrin
were effective between 10 - 5 to 10 - 6 M. Cismethrin, bifenthrin, cis

- permethrin, cyfluthrin i somers, cyhalothrin, esfenvalerate, and tefluthrin had no significant effects a t 10 - 5 M. A distinct pattern for the Type I and II pyrethroids was not evident.

The maxi

- chloride channel has been detected only by utilizing the ‘inside

- out patch’ electro physiological technique (Walz 2002) , thus, it is difficult to associate pyrethroid induced changes in maxi chloride channel function with any othe r alterations in cellular function or excitability. The electrophysiological parameters of the pyrethroid

- sensitive maxi

- chloride channel are homologous to those of the voltage

- dependent anion channel (VDAC) family associated with the mitochondrial outer membrane (Dermietzel et al. 1994; Buettner et al. 2000)

. VDAC channels are non

- specific pores that allow passage of anions and cations as well as small diameter molecules such as ATP and, possibly, cytochrome C across the mitochondrial outer membrane (Dermietzel et al. 1994; Sampson et al. 1997; Buettner et al. 2000) . A plasma membrane associated form of the VDAC channel is normally present only in glial cells (Dermietzel et al. 1994; Buettner et al. 2000)

. It is still not known whether plasma membrane associated VDAC channels function in the regulation of membrane excitability or if they are expressed in neurons (Walz 2002)

. Available i n vitro studies evaluating the possible interaction between pyrethroids and the chloride channels are limited in that the studies have been conducted utilizing only the N1E

- 115 neuroblastoma cell line and large

- conductance chloride channels. Published data indicate N1E

- 115 cells are a glia X neuron hybrid that possesses characteristics of both cell types (Moolenaar and Spe ctor 1977, 1978)

. Thus, these cells also contain glial characteristics and are not true neurons. Therefore, given the characteristics listed above, the maxi

- chloride channels expressed in N1E

- 115 cells are likely to be related to the VDAC family of channels. Reports that pyrethroids inhibit other volume sensitive anion channels (Culliford et al. 2004) support this conclusion.

In vivo studies in rats tested the ability of non

- specific chloride channel agonists ivermectin and pentobarbitone to alleviate deltamethrin (2 mg/kg, iv)

- induced neurotoxic signs. Some antagonism of behavioral measures resulting from deltamethrin exposure was noted with ivermectin and pentobarbitone. Neither compound was effective at reducing the in vivo signs of
c ismethrin neurotoxicity. There are also several limitations related to the in vivo data as well. Ivermectin is a competitive agonist of GABA

- gated (GABA A ) and glycine

- gated chloride ion channel receptors present in the central and peripheral nervous sys tems (Abalis et al. 1986) (Dawson et al. 2000) (Mascia et al. 1996; Huang and Casida 1997; Lu and Xu 2002; Hadipour

- Jahromy and Daniels 2003) (Shan et al. 2001; Steinbach and Akk 2001) . At the doses used for in vivo studies (Forshaw et al. 2000) , the possibility that the in vivo signs of pyrethroidDraft, Do Not Cite

Draft Page 21 of 58 neurotoxicity were alleviated by the actions of ivermectin on GABA A and glycine receptors, rather than on maxi

- chloride channels, cannot be eliminated . Pentobarbitone is a sedative that also binds to, and activates, GABA A and glycine receptors (Hadipour

- Jahromy and Daniels 2003) (Mascia et al. 1996; Steinbach and Akk 2001; Lu and Xu 2002) . At the doses used (Forshaw et al. 2000) , the possibility cannot be eliminated that the decreased in vivo signs of pyrethroid neurotoxic ity in the presence of pentobarbitone were the result of its action on GABA A and/or glycine receptors. The ability of ivermectin or pentobarbitone to competitively antagonize the pyrethroid dependent modulation of the maxi

- chloride channel has not been te sted. Given the lack of knowledge regarding the identity of the neuroblastoma maxi

- chloride channel, and the questionable role of the VDAC channel, which is the most likely candidate for the ‘maxi

- chloride’ channel, in regulation of membrane excitability, the results of the in vivo studies must be interpreted with caution. The lack of specificity of ivermectin and pentobarbitone in these experiments precludes definitive conclusions regarding the target of pyrethroid action. In summary, the overall weight of scientific evidence for maxi

- chloride channel effects as a basis for designating a CMG is not conclusive. In addition to the voltage

- gated sodium and chloride channels, In vitro evidence suggests that function of voltage

- gated calcium channels (VGCCs) is altered by pyrethroids. VGCC α 1 subunits are structurally similar to the α subunit of VGSCs, and like mammalian VGSCs, mammalian VGCCs are comprised of pore

- forming α 1 and auxil iary (β, λ, α 2 δ) subunits. Multiple isoforms of the VGCC α 1 subunit are present in the mammalian genome and expressed in a tissue, region and temporal manner. Pyrethroid actions on various types of VGCC have been examined using a variety of direct (e.g. vo ltage

- clamp) and indirect (e.g. neurotransmitter release, Ca influx) approaches. Rather than provide an extensive review, (see Shafer and Meyer (2004) , s everal findings are highlighted here. Type II pyrethroids have been shown to increase Ca 2+

- dependent norepinehphrine release from rat brain synaptosomes that were depolar ized by high K + . Type I pyrethroids were much less potent and efficacious in evoking release (Brooks and Clark 1987; Clark and Symington 2007) . Allethrin , a Type I pyreth roid, has been reported to inhibit different isoforms of VGCC expressed in HEK293 cells, and to shift the kinetics of channel inactivation, but not activation (Hildebrand et al. 2004) . More recently, electro physiological studies with rat brain Ca v 2.2 expressed in Xenopus laevis ooctyes substantiated that deltamethrin modified Ca v 2.2 i n vitro (Symington and Clark 2005) . Using Ba 2+ as a carrier for current through calcium channels, deltamethrin reduced peak current, caused a hyperpolarizing shift in potential activation and slowed activation and inactivation rates. The in hibition of peak current by deltamethrin appears inconsistent with 1) increased Ca 2+ influx and enhanced neurotransmitter release reported using functional synaptosomal preparations (Symington and Clark 2005) ; 2) the lack of deltamethrin effects on spontaneous glutamate release at intact synapses in hippocampal neurons i n vitro (Meyer and Shafer 2006) ; and 3) in vivo observations (Aldridge et al. 1978; Hossain et al. 2004) of increased neurotransmitter release following deltamethrin. Such differences suggest that other regulatory components of modulation Ca 2+ influx and subsequent neurotransmitter release are necessary to elicit a neurophysiologic response that is consistent with in vivo symptoms. Overall, these and other uncertainties indicate that the potential role of direct effects of pyrethroids on VGCC i n toxicological responses is presently not well enough understood to serve as the basis for a CMG.Draft, Do Not Cite

Draft Page 22 of 58 Finally , there is evidence that pyre throids may interfere with ligand

- ga ted chloride channel current s . Briefly, when the inhibitory neurotransmitter γ

- ami nobutyric acid (GABA A ) is released from the presynaptic nerve terminal, it binds to a postsynaptic receptor protein containing an intrinsic chloride ion channel (i.e. GABA A - activated chloride channel). When GABA A binds to its receptor, the chloride chann el is opened and chloride ions flow into the cell resulting in an inhi bitory post

- synaptic potential which causes membrane hyperpolarization leading to a dampening affect on neuronal excitation, making it less likely that a post

- synaptic action potential w ill occur (Costa 1998)

. GABA A antagonists, such as the chlor inated cyclodiene insecticides (e.g. toxaphene and lindane) and picrotoxinin (Lawrence et al. 1985) , have an excitatory effect by blocking GABA A - mediated chloride current, resulting in hyperexcitability convulsions, and death (Bloomquist 1993b)

. R eceptor agonists like diazepam (central) and RO5

- 48 64 (peripheral) increase the affinity of GABA A for its receptor causing an increased frequency of chloride channel opening and increased inhibition. Deltamethrin and fenvalerate, Type II pyrethroids, have shown the ability to weakly antagonize GABA A bindi ng in rodent and cockroach tissues at high concentrations. Allethrin and permethrin, Type I pyrethroids, had no effect (Gammon and Casida 1983; Lawrence et al. 1985; Bloomquist et al. 1986) . Conversely, deltameth rin had no effect on the amplitude of GABA A - induced chloride current in cultured newborn DRG cells at a concentration that produced modifications of VGSC current (Ogata et al. 1988) . In an in vivo study, Crofton and Reiter (1987) evaluated the combinatorial effects of cismethrin (Type I) and deltamethrin (Type II) on neurobehavioral function (i.e., acoustic startle response (ASR) and motor activity) in conjunction with controls for GABA A receptor activated chlori de current, picrotoxin, muscimol, or chlordiazepoxide. Only deltamethrin and picrotoxin showed a dose

- additive effect indicating an interaction at the picrotoxin binding site of the GABA A receptor

- ionophore complex. Conversely, other in vivo rodent studie s using anesthetized rats, failed to show any impact of GABA antagonists on GABA

- mediated inhibition of hippocampal granule cells. These authors concluded that pyrethroid effects on GABA A receptors were unlikely to account for pyrethroid induced changes in hippocampal synaptic transmission (Gilbert et al. 1989; Joy et al. 1990) or dorsal root ganglion potentials (Smith 1980) . While i n vitro ligand

- mediated chloride channel studies indicate that GABA A receptors may be a potential site of interaction for Type II pyrethroids, the conflicting in vivo studies make it difficult to draw definitive conclu sions for the class based on the lack of confirmation from in vivo studies and the limited number of compounds examined. Thus, the basis for a CMG group centered on actions of pyrethroids on GABA A receptors is insufficient. In summary, chloride channels have demonstrated susceptibility to some pyrethroids, however, interpretation of the i n vitro results with respect to the purpose of the CRA groupings are confounded by the potential with VDAC channels and choice of cell line; in vivo results are compromised due to the potential antagonism of ivermectin and pentobarbitone on voltage

- gated chloride channels and GABA A and glycine receptors. Voltage

- gated calcium channels are potential sites of action of pyrethroids ; however the inconsis tency of data from different types of preparations and compounds limits the utility of using this information for the purposes of determining a mechanism of action. Type II Pyrethroids have also demonstrated the ability to disrupt the ligand

mediated chlo ride current by antagonizing GABA A receptors In vitro and a potential interaction at the picr o toxin binding site of the GABA A receptor

- ionophore complex i n vitro . While these pathways may be susceptible to perturbation by pyrethroids In vitro ,Draft, Do Not Cite

Draft Page 23 of 58 evidence fr om other studies indicate that GABA A receptors are less sensitive to pyrethroids than VGSC, and not all in vivo pyrethroid effects are clearly not mediated by actions on GABA A receptors . Furthermore, the limited number of chemicals examined does not provi de a sufficiently broad represent ation of the pyrethroid class of pesticides very well. Therefore, while these mechanisms may be involved in the overall neurotoxicity inherent with pyrethroids, there is not enough evidence supporting their inclusion into the finding of a common mechanism.

4.3 In vivo profile The previous sections focus on effects on pyrethroids on the VGSC and membrane excitability. The following text summarizes data from in vivo studies, primarily in rodents.

4

. 3.1

. Pharmacokinetics Pyrethroid pharmacokinetics in rats have been described for a limited number of compounds. However, as a class, pyrethroids are lipophilic compounds with relatively short mammalian in vivo half

- lives, being absorbed and eliminated relat ively quickly. Focusing on plasma (as a measure of bioavailability) and brain (as a primary target tissue) compartments, half

- life estimates are described herein for deltamethrin, lambda

- cyhalothrin, and permethrin. Kim et al. (2008) administered deltamethrin IV (2 mg/kg) and orally (0.4, 2, and 10 mg/kg). Following a 2 mg/kg dose, the oral and IV plasma half

- lives were similar, 15.8 and 13.3 hours, respectively. Following 2 and 10 mg/kg oral dose, brain half

- li ves were 12.5 and 19.5 hours, respectively, similar to plasma. Anadón et al. (1996) estimated a slightly longer plasma half

- life, 38.5 and 33.0 hours following oral (26 mg/kg) and IV (1.2 mg/kg) administration. In contrast, estimated half

- lives for several sections of the brain ranged from 18 to 33 hours, similar to Kim et al. (2008) estimates. Estimates for lambda

- cyhalothrin half

- lives f ollowing oral (20 mg/kg) and IV (3 mg/kg) dosing were 9.9 and 8.2hours (plasma) and 15

- 35 hours (brain) (Anadón et al. 2006) . Following high doses of permethrin, 460 mg/kg oral and 46 mg/kg IV, plasma and brain half

- life estimates were similar to the preceding examples; 12.4 (oral) and 8.7 hours (IV) for plasma and 13.9

- 23 hours in brain (Anadón et al. 1991) . Maximum plasma were achieved under 4 hours (deltamethrin <2; lambda

- cyhalothrin 2.9; and permethrin 3.5 hours) and maximum brain concentrations under 8 hours (deltamethrin 1

- 7.3; and lambda

- cyhalo thrin 2.2

- 3.1)were generally achieved between under 2 hours. Finally, the oral bioavailability of deltamethrin in the Kim et al. (2008) and Anadón (1996) studies were similar at 18 and 14.4 %, respectively. Oral absorption rates for lambda

- cyhalothrin (67.4%) and permethrin (60.7%) were greater. Pyrethroids do not require metabolic activation to result in neurotoxicity. Indeed, metabolic de toxification in mammals leads to very short half

- lives of the parent compound . Furthermore, most data available to date suggests that the major esterase metabolites of pyrethroid lack neural activity. Thus, an important aspect of pharmacokinetics is deto xification and clearance. In vitro, mammalian clearance half

- lives of pyrethroids vary considerably by compound (Table 1, (Godin et al. 2007; Scollon et al. 2009) . Generally, the pyrethrins and first

generation p yrethroids tested (e.g. allethrin and resmethrin) have considerably shorter half

- lives than the Type II pyrethroids (e.g. deltamethrin and β

- cypermethrin), often differing by orders of magnitude (Wolansky and Harrill 2008) . Furthermore, the relative clearance rates are structure dependent. Cis

- isomers are subject to oxidation through numerous liver enzymes in mammalsDraft, Do Not Cite

Draft Page 24 of 58 (Anand et al. 2006; Godin et al. 2007; Scollon et al. 2009) while the trans

- isomers are metabolized by the liver enzymes as well as hepatic carboxylesterases (Ross et al. 2006)

. Oxidative clearance is a relatively slow mechanism compared to hydrolysis by carboxylesterases. Therefore, the half

- lives of trans

- isomers are generally shorter by 2 to 10 fold than their cis

counterparts. It has been postulated that since the cis

- isomers are more persistent in vivo , they are inherently more toxic from a pharmacokinetic perspective.

Table 1. Intrinsic clearance rates of selected pyrethroids from rat hepatic macrodomes
Pyrethroid Intrinsic clearance (ml/min/kg bw ± SD) Allethrin; S

- Bioallethrin 1218 ± 106
Bifenthrin a 224 ± 20 Cyfluthrin a 260 ± 27 Cypermethrin a 104 ± 63
Deltamethrin b 89±23.7
Esfenvalerate b 108.2± 2.2

Permethrin a 529 ± 19(cis); 438 ± 46 (trans) Resmethrin a 1515 ± 367 (trans)

a (Scollon et al. 2009) ; b (Godin et al. 2007) Pyrethroids share many physical chemical and PK properties as a class (i.e., molecular weight, liphophilicity, common metabolic pathways, and disposition). The Agency is taking advantage of these similarities by using a “family modeling approach”. Conceptually, “family modeling” begins with developing a hypothesis based model for a single representative chemical. In this approach, a single model structure may be applied to all pyrethroids with similarities in the number of compartments and the physiological processes that determine the absorption, disposition, metabolism and elimination are described in those com partments. For individual pyrethroids, the model incorporates chemical specific parameters, such as, hepatic clearance rates, partition coefficients, coefficients of permeability. The initial models are developed and evaluated in rodents. T hrough PBPK m odeling evaluations in the rodent, constants that are largely invariant across pyrethroids tested are identified , calibrated a nd retained in the model structure. Likewise, parameters that are determined to be compound

dependent through relevant in vivo an d i n vitro studies are corrected accordingly. For example, while the enzymes involved in pyrethroid metabolism are the same across chemicals, the metabolic rates are not. Thus the mathematical description of metabolism remains the same across chemicals, but the rate constants are chemical specific. Once the ability to properly scale and incorporate the derived constants in the PBPK model in rodents is evaluated, the models can be scaled to humans by incorporating the appropriate species

- specific physiolo gical, chemical and biochemical parameters. Deltamethrin is used by EPA as the representative chemical as th e pharmacokinetics and metabolism of this pyrethroid are w ell characterized and isomer ic composition is not an issu e (Godin et al. 2007) . Based upon this version , PBPK model s for several other pyrethroids identified by their exposure and haz ard potential are being considered for development.Draft, Do Not Cite

Draft Page 25 of 58 4

. 3.2

. Toxicity syndromes in rodents Pyrethroids have historically been classified according to the acute intoxication syndromes they exhibit in vivo . Initial studies examined “high

- dose” pyrethroid exposures, doses resulting in near

- death or death (Table 2). Verscholyle and Aldridge (1980) investigated toxicity following rat IV tail injection of 36 pyrethroids. Signs of intoxication generally fell into one of two categories; the T

- syndrome characterized by aggressive sparring, increased sensitivity to external stimuli and fine whole body tremors, and the CS

- syndrome characterized by initial pawing a nd burrowing, salivation, and course whole body tremors (choreoathetosis). Eighteen pyrethroids were classified as T

type, 12 as CS

- type and 2 compounds resulted in intermediate symptoms. It was also noted that pyrethroids containing the α

- cyano group wer e associated with the CS symptoms while those lacking the α

- cyano generally resulted in T symptoms. The authors did not make any absolute conclusions regarding the relationship between cyan

- and non

- cyano

- containing pyrethroids, however, linking the assoc iation between structure and function was highly significant.

Lawrence and Casida (Lawrence and Casida 1982) administered 29 pyrethroids to mice via intracerebral (ICV) injections of 29 pyrethroids. The pyrethroids were classified as Type I or Type II, equivalent to th e T

- and CS

- syndromes described by Verscholyle and Aldridge (Verschoyle and Aldridge 1980) . The ICV injections were used to bypass primary metabolism and correlate intoxication with pyrethroid co ncentration in neuronal tissue directly. Study findings indicate α

- cyano containing pyrethroids (Type II) produced choreoathetosis, convulsions and salivation, whereas compounds lacking the α

- cyano group (Type I) produced tremors and convulsions, supporti ng the division of pyrethroids based on structure and intoxication syndromes. Furthermore, Gammon et al. (Gammon et al. 1981) classified pyrethroids as either Type I or Type II based on their effects on cercal sensory nerve recordings in vivo and In vitro and on symptomolog y produced in dosed cockroaches. Type I pyrethroids produced repetitive firing in the cercal sensory nerve i n vitro and trains of cercal sensory spikes accompanied by restlessness, lack of coordination, hyperactivity, prostration, and paralysis in vivo

. Type II pyrethroids did not induce repetitive firing in the cercal sensory nerve. The prolonged discharges in the CNS were longer and of lower frequency than the Type I pyrethroids. I n vivo symptomology also distinguished the Type II pyrethroids. Cockro aches became ataxic and lack of coordination with periods of convulsions and intense hyperactivity, sometimes associated with salivation. Periods of sustained contractions with the extension of metathoracic legs were particularly noticeable. Fenpropathri n and an oxime O

- α

cyanophenoxybenzyl ether were classified as Type I based on their action on the cercal sensory nerve but resulted in symptoms associated with Type II pyrethroids. It is therefore clear from these early studies that pyrethroids toxicity can be separated into two subgroups, Type I and Type II, based on structure and in vivo symptomology.Draft, Do Not Cite

Draft Page 26 of 58 Table 2. Behavioral characterization of pyrethroid toxicity of pyrethroids included in the Proposed CMG from peer

- reviewed literature
Pyrethroid Vers choyle and Aldridge (1980 ) Lawrence and Casida (1982 ) Gammon et al ( 1981 ) Allethrin; S

- Bioallethrin T I I Permethrin T I I Resmethrin T I I Fenpropathrin CS/ T I/II I/II Esfenvalerate CS II II Cypermethrin CS II II Deltamethrin CS II II

As recently reviewed (Wolansky and Harrill 2008) , more contemporary studies were designed to detect behavioral changes at a broader range of doses includin g both lower doses as well as some “high

- dose” ranges. Some of these studies include evaluation of motor activity, coordination, neuromuscular response, tremors, acoustic startle response, learning and memory, sematosensory response, social/sexual interac tions and behavior to handling, and the functional observational battery (FOB). Although the number of pyrethroids with available data for coordination, neuromuscular response, social/sexual interactions, and cognitive response (Wolansky and Harrill 2008) is limited, these studies support a similar finding, that pyrethroids can be separated into two major groups based on neurological signs. Table 3 below pr ovides an overview of behavioral changes observed in rodent studies (extracted from Wolansky and Harrill 2008)

. Motor activity is an apical measurement of nervous system function commonly measured in acute and sub

- chronic neurotoxicity guideline studies ( OPPTS Guidelines 870.6200, 870.6200b)

. Motor activity measured as changes in ambulatory movement of rodents is currentl y the endpoint with the most extensive dose

- response data for a large number of both Type I and II pyrethroids. Pyrethroids have been repeatedly shown dose

- dependent decreases in motor activity in rodents following acute exposures. Past work (Crofton and Reiter 1984; Crofton and Reiter 1987; Crofton and Reiter 1988; Gilbert et al. 1990; McDaniel and Moser 1993; Crofton et al. 1995) has demonstrated decreased motor activity for eleven pyrethroids using the same test method (i.e., figure

- eight mazes). In addition, decreases in motor activity have been demonstrated in other testing devices after acute or short

- term exposure, including permethrin ( Hoy et al. 2000) , fenvalerate (Spinosa et al. 1999) , cyhalothrin ( Righi and Palermo

- Neto 2003) , cyhalothrin (Hornychova et al. 1995; Ratnasooriya et al. 2002) , and cypermethrin (Hornychova et al. 1995) . The acute motor depressant effect has been also obse rved in mice after oral exposure to fenvalerate ( Mandhane and Chopde 1997) and deltamethrin (Chanh et al. 1984) . A limited number of studies have observed increases or no change in motor activity (Mitchell et al. 1988; Husain and Husain 1996; Dayal 2003) . These reports of increased activity are difficult to interpret due to the use of commercial formulations rather than a technical product. Commercial formulations routinely contain very low concentrations of pyrethroids (e.g., 2

- 5%) and contain solvents and other ‘inerts’. Overall, the available studies on the effects of pyrethroids on motor activity suggest a common effect, i.e., decreased activity, on this apical neurobehavioral endpoint.Draft, Do Not Cite

Draft Page 27 of 58 Table 3. Comparison of the signs of pyrethroid poisoning across rodent species and different routes of exposure (Extracted from Wolansky and Harrill, 2008)

Mice Rat

via i.c. 1,2,6 via i.v. 3,4,5,6,7 via oral gavage 6,,8,9

Type I Type II Type I Type II Type I Type II

Mild poisoning

signs Hyperactivity Circling (mostly ipsilateral direction) Hyper

excitability Aggressive

sparring Restlessness Chewing; nosing; pawing; burrowing Episodes of restlessness Chewing; nosing; pawing burrowing; head bobbing; whole body shakes

↑ Sensitivity Salivation ↑ Responsivity Salivation ↑ Responsivity Salivation ↑↓ responsivity
Aggressive sparring

Moderate to
severe poisoning

signs Whole body tremors Tremors followed by CA Fine tremors progressing to

whole body Impaired movements followed by CA

(tail, limbs) Fine tremors progressing to

whole body Tremor followed by CA

Prostration Sinuous writhing Un

- coordinated twitches Hind limb extensor rigidity; rolling gait Spontaneous muscular contractions Motor incoordination;

↑ hindlimb extensor tone
Hyperthermia Normothermia or hypothermia Hyperthermia Normothermia or hypothermia

Nearly lethal

syndrome Clonic seizures Tonic seizures Rigor occasionally just

before death Loss of Consciousness; Tonic seizures Clonic seizures Loss of consciousness; severe writhing spasms; tonic

seizures Death, at 10

- 60 min Death, at 0.5

- 2 h Death, at 2

- 8 h Table 3 is a compendium of clinical effects observed in studies of rodents following acute exposure to Type I and Type II pyrethroids. Different stages of the poisoning syndromes are noted at the far

- left of the table. Note that the clinical landmarks pro duced under different routes of exposure are similar despite differences in body size and species. 1 Gammon et al. (1982), 2 Lawrence & Casida (1982), 3 Ray (1982), 4 Verschoyle & Aldridge (1980), 5 Wright et al., 1988, 6 Souyri & Hoellinger (1983), 7 Cremer (198 3), 8 Soderlund et al (2002), 9 McDaniel & Moser (1993). † Deltamethrin and other cyano compounds induces little or no salivation with icv dosing (Gray & Soderlund, 1985).Draft, Do Not Cite

Draft Page 28 of 58 Data from studies on auditory startle and FOB support the conclusion that pyrethroi ds historically categorized as Type I and Type II cause different toxicity profiles in rodents. Auditory startle reflex is a short

- latency behavior (6

- 8 ms) elicited by sudden and intense acoustic stimuli. The proposed reflex path is short, consisting of auditory nerve, posteroventral cochlear nucleus, the nucleus reticularis pontis caudalis, and motor neurons in the spinal cord (Davis et al. 1982) , and is sensitive to a variety of drugs and toxicants making the reflex a useful animal model of sensorimot or reactivity (Lee et al. 1996) . Audito ry startle data have demonstrated divergent patterns related to pyrethroid structure (Crofton and Reiter 1984; Crofton and Reiter 1988) . Possible groupings include Type I compounds producing an increase in startle amplitude (e.g., permethrin, bifenthrin, cism ethrin); Type II compounds that produce a decrease in startle amplitude (e.g., deltamethrin, cypermethrin, cyfluthrin); and compounds that produce alterations in the auditory startle reflex parameters not predicted by the absence or presence of the α

- cyano group (fenvalerate) (Wolansky and Harrill 2008) . With respect to the proposed common mechanism grouping, the auditory startle studies support the conclus ion that Type I and Type II pyrethroids exhibit different in vivo toxicity outcomes.

The FOB consists of a series of behavioral measurements from standardized tests such as posture, convulsions, tremors, salivation, mobility, rearing, and an array of se nsory observations

- touch, startle, and pain to name a few. In the case of pyrethroids, the FOB is designed to evaluate potential neurotoxicity by examining behaviors such as open field, home cage, sensory observations, physiological, and neuromuscular o bservations. McDaniel and Moser (1993) described the neurobehavioral syndromes produced by high acute doses of permethrin (Type I) and cypermethrin (Type II) in rats using a FOB. Permethrin produced whole body tremors, ataxia, greater resistance to removal from home cage, and increases in arousal, handling reactivity, and the response to touch or experimenter approach. Cypermethrin caused salivation and urination, splayed limbs, choreoathet osis, increased foot splay, resistance to removal from home cage, decreased arousal, and decreased response to touch or tail pinch. There were also a number of neuromuscular effects that were affected by both chemicals including grip strength, neuromuscul ar tone, and gait changes. This work along with other data from FOB studies (Soderlund et al. 2002; Wolansky et al. 2007) , and observations of clinical signs from high dose acute toxicity studies (Verschoyle and Aldridge 1980; Lawrence and Casida 1982) , demonstrate that that methods for observation of clinical signs (e.g., FOB) can be used to systematicall y evaluate and potentially quantify changes in behaviors in rats exposed to pyrethroids. Moreover, McDaniel and Moser (1993) demonstrated that the FOB can be tailored to evaluate neurobehavio ral syndromes specific to pyrethroids and can thus be used as a tool to compare the toxicity profile of different pyrethroids.Draft, Do Not Cite

Draft Page 29 of 58 OPP has received acute neurotoxicity data on more than 15 registered pesticides, most of these include FOB observation in rats post

- dosing, typically 2 to 8 hours after exposure. As part of the on

- going work to re

- evaluate the pyrethroids as part of registration review ( http://www.epa.go v/oppsrrd1/registration_ review/), OPP has performed a preliminary review of the available regulatory acute neurotoxicity studies. Typically, neurotoxic signs are observed at or near peak time of effect in these studies. As a result, these studies are often the most sensitive st udy in the available database and are therefore are often used by OPP to derive points of departure 4 for acute, single day risk assessments of individual pyrethroids. However, for purposes of evaluating the available data for a common toxic effect and f or comparing potency and responses across chemicals and doses as required in a cumulative risk assessment, the guideline acute neurotoxicity studies suffer from several limitations. There is a lack of consistency between i) laboratory techniques and equip ment; ii) the quality, duration and temporal spacing of observations; iii) lack of specificity in reporting (e.g., fine vs. whole body tremors; iv) route of administration; v) dosing vehicle and volume; vi) isomer concentration; vii) formulation; viii) a nd isomer composition. These experimental variables are known to impact the ability to detect and estimate potencies of pyrethroids (i.e., dosing vehicle (Crofton et al. 1995; WHO 2005) , route of exposure (Nishimura et al. 1984; Crofton et al. 1995) , stereoisomer composition (Verschoyle and Barnes 1972; Glickman and Casida 1982; Soderlund et al. 2002) , and commercial formulation (Mitchell et al. 1988; E.G. Williamson 1989; Lepeshkin et al. 1992; Villarini et al. 1995; Axelrad et al. 2002)

.
T he PWG conducted a special FOB study designed to characterize effects classically associated with T and CS pyrethroid toxicity for 12 test chem icals (1 pyrethrin, 5 Type I and 6 Type II pyrethroids) (Nemec 2006) . The FOB consisted of 56 observations covering home cage, sensory, handling, physiological, open field, and neuromuscular parameters. The special FOB study offers two principle advantages lacking amongst the literature studies and registrant submitted data; 1) specific neurological behaviors that addr ess the issue of common toxic effect and 2) consistency among testing and observational parameters. Each chemical was tested was at 2 or 3 doses, minimally and moderately effective dose s were used to establish a spectrum of neurotoxicological effects.

4 Point of departure (PoD)

— the value from a dose

- response cur ve that is at the low end of the observable data and that is the toxic dose that serves as the ‘starting point’ in extrapolating a risk to the human population.Draft, Do Not Cite

Draft Page 30 of 58 In the evaluation of pyrethroid toxicity profiles, t he PWG used principal component analysis and dissimilarity indices to evaluate the clustering of four independent data sets around common dimensions (Breckenridge and Holden 2007) . These datasets included:

1. 56 behavioral and physiological parameters from the special FOB study (Nemec 2006) ; 2. 8 electrophysiological parameters from patch clamp experiments conducted on five different sodium channe ls constitutively expressed in Xenopus oocyte (Choi and Soderlund 2006; Soderlund 2006) ; 3. Indices of efficacy (maximum response), potency (log EC 50 ) and binding (slope of the logistic dose

- response function) were calculated fo r calcium ion influx across neuronal membrane, membrane depolarization and neurotransmitter (glutamate) released from rat brain synaptosomes (Clark and Symington 2006)

. 4. One measure of the change in the open chloride ion channel probability using reverse patch clamp techniques for voltage

- gated chloride channels in neuronal membranes from mouse n euroblastoma cells (Burr and Ray 2004)

. This analysis identified four primary patterns of variation in the FOB data. Two of these patterns described behaviors associated with CS behaviors (lower temperature, excessive salivation, impaired mobility) and two described behaviors associated with the T syndrome (elevated temperature, tremors, myoclonus). Non

- cyano pyrethroids tended to have a high loading on the T factors whereas cyano

- containing pyrethroids tended to have high loading on the CS factors. Pyreth roids with the strongest CS and T responses were deltamethrin/ λ

cyhalothrin and resmethrin/bifenthrin, respectively. Fenpropathrin and pyrethrin had weak responses for either the C or TS characteristics. The consistency of the special FOB study with t hose of Verschoyle and Aldridge (1980) and Lawrence and Casida (1982) is remarkable

. Notably g roupings of Type I and Type II pyrethroids based on symptoms of in vivo toxicity ar e similar in studies conducted four decades apart. Based upon the FOB, sodium channel, calcium channel, and chloride channel studies, the PWG proposed that the pyrethroids separate into Type I and Type II subgroups based on the absence or presence of the α

- cyano group.
4

. 3.3

. Information in humans Pyrethroids are generally thought be less hazardous than OPs and NMCs and generally human incidences with synthetic pyrethroids and pyrethrins are low (Power and Sudakin 2007)

. Recent studies have, however, shown an increase in reported incidences following exposure to pyrethroids (Power and Sudakin 2007; Walters et al. 2009)

. The clinical manifestations of pyrethroid exposure have been recently reviewed by Bradberry et al, (2005)

. Dermal exposure can result in transient paraesthesias wi th many pyrethroids. I nhalation exposure can result in upper airway irritation . Ing estion can result in systemic intoxication that includes nausea, vomiting, abdominal pain, increased secretions, and in rare, severe cases, coma and seizures (Power an d Sudakin 2007)

.Draft, Do Not Cite

Draft Page 31 of 58 There are few epidemiology studies reporting effects of pyrethroids. Permethrin has been evaluated the Agricultural Health Study (AHS; Alavanja et al. (2003) , Rusiecki, et al (2009))

. Kamel et al (2005, 2007) have evaluated statistical associations between self

- reported pesticide use with neurological symptoms with cumulative use of insecticides classified by chemical type among licensed pesticide applicators enrolled in the AHS from 1 993 to 1997 (n =18, 782). The investigators found increased risk associated with use of OPs among applicators who had never experienced doctor

- diagnosed pesticide poisoning or high exposure events (OR: 1.6_1.7 for highest category of use) and a small incr ease in risk associated with use of carbamates, but there was little effect of permethrin.

4.4 Data in mixtures of pyrethroids EPA’s default assumption when evaluating multi

- chemical risk is to assume dose additivity. This assumption is thought to be pa rticularly appropriate when evaluating chemicals which share a common mechanism of action. Chemicals which share a mechanism of toxicity will share a common molecular target. As such mixture studies which support the dose additivity hypothesis also indir ectly support the common mechanism findings since dose additivity suggests that the chemicals of different potencies act as dilutions of each other. To date, there has been limited research conducted on the potential effects of exposure to pyrethroid mi xtures. Two i n vitro studies have shown that initial exposure of sodium channels to fenvalerate (Song and Narahashi 1996) and deltamethrin (Tabarean and Narahashi 1998; Motomura and Narah ashi 2001) caused use

- dependent VGSC modification, typical of α

- cyano containing pyrethroids. Subsequent exposure to tetramethrin (Type I) resulted in the slow tail decay being replaced by a more rapidly decaying current, characteristic of pyrethroids lacking the α

- cyano moiety. No channels exhibiting intermediate modifications were observed. Song et al

. (1996) concluded that tetramethrin may displace fenvalerate or interact allosterically from sodium channel protein binding sites in an ex vivo rat dorsal root ganglion preparation. While these reports suggest that pyrethroids do not act in an effect

- additive manner, there are a number of possible confounds in making definitive conclusions from these reports. First, the electr ophysiological work (Ray and Fry 2006) used dose levels that exceeded known LD 50 dosages. In addition, all of these previous reports lacked either by study design or statistical approach, the ability to test the hypothesis of additivity. The use of rigorous statistical models is critical for testing hypotheses of effect

- or dose

- addi tion, and determining whether antagonism or synergism exists (Feron and Groten 2002; Hertzberg and Teuschler 2002; Gennings 2004; LeBlanc and Olmstead 2004; Teuschler 2007) . Overall, these limited in vitro data suggest there may be complicated outcomes from in vitro mixtures at the molecular or cellular level.

In an in vivo assessment of the dose

- addition of pyrethroids , Wolansky and colleagues , tested a mixture of low doses of both Type I and II pyrethroids (Wolansky and Harrill 2008; Wolansky et al. 2009) . With a mixture composed of individual chemical at doses 30% lower that the thresho ld for neurobehavioral effects, that the effects of this mixture pyrethroids on motor activity was accurately predicted by a single chemical required dose additivity model (Wolanksy et al. 2009)

. While the data of Wolansky et al 2009 demonstrate dose

- addition of low dose effects for both Type I and Type II pyre throids, these data need to be interpreted with caution. Motor activity is an apical measure of the disruption of nervous system function (Reiter andDraft, Do Not Cite

Draft Page 32 of 58 McPhail 1979) , that is decreased by a wide variety of pesticides. Thus the specificity for a causative mode

- of

- action based linking of effects on VGSCs to apic al measures of neural function like motor activity are extremely difficult to demonstrate. Because a mechanistic understanding is not available for pyrethroid induced decreases in motor activity, these data on a
non

- specific endpoint have limited utility in supporting a dose

- additivity assumption for a CMG as established per the requirements of the FQPA.

5.0 Proposed Common Mechanism Grouping under the FQPA

5.1 Weight of the Evidence The above sections provide a survey of key studies which evalua te the mechanism of action and toxicity profile of pyrethroids. These sections summarize the current state of the science with respect to understanding of how pyrethroids modify the kinetics of the sodium channels, disrupting neuronal firing patterns and ultimately resulting in the signs of pyrethroid toxicity. Specifically, evidence of a common toxicity pathway and common toxic effects for pyrethroids can be found from studies evaluating effects on 1) the sodium channel from both mammalian and non

- mammal ian i n vitro studies, 2) intact neurons, 3) non

- mammalian in vivo studies, and 4) rodent in vivo studies. Although there is an extensive body of literature on pyrethroids, not all biological events between modification of VGSC, alterations in membrane
exc itability, and clinical signs and symptoms are completely understood, particularly in mammals. Data in non

- mammalian species provide support for the proposed mechanism where mammalian data are lacking. Moreover, there is a strong correlation between the pyrethroid structure, kinetic modulation of the sodium channel, and phenotypical clinical syndrome in rodents. Pyrethroids lacking an α

- cyano moiety are found to have a delayed deactivation of sodium channels resulting in extended tail currents of approxi mately 20 ms and are associated with clinical effects defined as the T syndrome. On the other hand, pyrethroids containing an α

cyano moiety delay deactivation of sodium channels resulting in prolonged tail currents of approximately 500 ms to minutes and are associated with clinical effects defined as the CS syndrome.Draft, Do Not Cite

Draft Page 33 of 58 Based on this review OPP is proposing that the naturally occurring pyrethrins and synthetic pyrethroids form a CMG based on 1) shared structural characteristics; and 2) shared ability to interact with voltage

- gated sodium channels (VGSC), resulting in disruption of membrane excitability in the nervous system, and ultimately neurotoxicity characterized by two different toxicity syndromes. The proposed pathway to toxicity is illustrated in the Figure 6. The presence of pyrethroids at the target tissue causes a disruption in the VGSC, leading to an alteration of action potentials, and ultimately resolving as clinical neurotoxicity characteristic of pyrethroid poisoning.

Figure 6

. Proposed common toxic pathway of the pyrethroids

Although there is an extensive body of literature on pyrethroids mechanism of toxicity and toxicity syndromes, the Agency acknowledges that some science issues remain. 

### Target Tissue Dose VGSC Alterations In Vivo Clinical

Signs Altered Neuronal Excitability

All pyrethroids modify the kinetics of VGSC activation and inactivation in mammalian neu r ons Changes in VGSC kinetics produce alterations in neuronal
exc itabilit y. It has been estimated that modification of <1% of VGSC alters neuronal excitability Changes in neuronal excitability underlie the clinical signs of pyrethroid toxicityDraft, Do Not Cite

Draft Page 34 of 58 a) Other endogenous channels (e.g., calcium, chloride, GABA) which have been propose d by
some (Soderlund et al. 2002; Ray and Fry 2006; Clark and Symington 2007) as potential other target sites for some pyrethroid

. A key task in forming a CMG is to distinguish those molecular target(s ) and pathways of toxicity which are common among the group members and those which are not. OPP’s CMG guidance notes that some chemicals may exhibit multiple mechanisms of toxicity but that OPP will group substances that cause multiple toxic effects by a common mechanism “ provided at least one of the toxic effects is common among the substances .”
Calcium, chloride, and GABA A channels have all shown the potential to interact with pyrethroids (Section 4.2.5). However, as described in more detail above, the data are not sufficiently robust to support a common mechanism grouping for these. In contrast, each member of the proposed CMG is believed to interact with the VGSC. The Agency is not aware of any data demonstrating that any pyrethroids lack an inte raction with VGSCs. In other words, all pyrethroids tested in mammalian neurons have shown interaction with VGSCs. As such, interaction with the VGSCs provides the common molecular target for pyrethroids. The Agency does acknowledge that interaction bet ween pyrethroids and these other channels may mediate the toxicity of specific pyrethroids but this does not preclude the formation of the CMG. b) Na channel structural heterogeneity among tissue and across life stages with respect to alpha

- and beta

- s ubunits. Another important issue when evaluating mechanistic information for a CMG finding is the concept that multiple tissues and/or sites can exhibit the same key events leading to toxicity but have different dose

- response relationships or different tissue sensitivit ies . This issue is applies to VGSC structural heterogeneity. As described in Section 4.2, mammalian sodium channels are comprised of α and β subunits that exist in multiple isoforms, giving rise to tissue, regional and life sta ge heterogeneity in sodium channel expression (Plummer and Meisler 1999; Goldin 2001) . Mammalian neurons typically express mult iple isoforms of both α and β subunits, making it difficult to determine the composition of subunits comprising sodium channel currents in native neurons. Specific combinations of VGSC subunits can be expressed exogenously in systems such as unfertilized f rog oocytes or non

- neuronal human embryonic kidney (HEK) cells. With respect to the current proposal to designate the pyrethroids as a CMG, data from this approach has provided evidence to support modification of sodium channel function by pyrethroids as a key event in neurotoxicity (Vais et al. 2001)

. Data from these i n vitro s ystems have also shown differences in pyrethroid sensitivity of various sodium channel isoforms that may influence differences in pyrethroid sensitivity based on tissue, region (Choi and Soderlund 2006) , life stage (Meacham et al. 2008) or species, including differences between rodents and humans (Vais et a l. 2000; Tan and Soderlund 2009)

. The pyrethroid sensitivity of all possible combinations of mammalian VGSC subunit s ha s not been tested using exogenous expression in oocytes or mammalian cells , and doing so is impractical. Even if such data were available, extrapolation of data from expression studies to intact, mammalian neurons is confounded by at least two significant uncertainties; 1) exogenous expression of VGSC subunits for these studies requires the use of non

- mammalian and/or non

-Draft, Do Not Cite

Draft Page 35 of 58 neu ronal cell types, and differences in post

- translational modification of subunits, their assembly and insertion into the membrane, or modification by other cellular processes (e.g. phosphorylation, glycosylation, etc) may contribute to differences in sensit ivity of the channels to pyrethroid modification, and 2) the subunit composition of VGSC when recording sodium currents in mammalian neurons is unknown and difficult to determine due to the expression of multiple VGSC subunits within a neuron. Thus, it is currently not possible to associate pyrethroid effects with specific combinations of VGSC subunits. Despite this, data from this line of scientific investigation support the hypothesis that effects on VGSC are an initial and common key event in pyrethroi d toxicity. A detailed understanding of pyrethroid effects on individual VGSC subunit combinations would contribute to a more complete mechanistic understanding for pyrethroids than is currently discernable. However, this level of detailed understandi ng is not necessary for purposes of designating a CMG. Instead, for purposes of defining the CMG, the “major steps leading to a toxic effect” need to be considered and that “All steps leading to an effect do not need to be specifically understood (1999) .” Thus, although differences in the sensitivity of different VGSC subunit combinations may influence region, tissue or life stage differences in sensitivity, or may influence th e types of toxic signs, the common key event in all cases, and the basis for the common mechanism grouping, is disruption of VGSC function. c) Uncertainty associated with a lack of an in vivo biomarker for pyrethroid

- VGSC interaction.

In the cumulative risk assessments for the OPs and NMCs, the common mechanism groupings were based on the ability of these pesticides to inhibit the enzyme acetylcholinesterase (AChE) 5 leading to accumulation of acetylcholine and ultimately to neurotoxicity. As such, AChE inhibition provides the first key event in the pathway to neurotoxicity for these pesticides. AChE inhibition (for example, blood AChE inhibition) is in an easily measurable biomarker in multiple species, tissues, and life stages. Thus, AChE i nhibition provides a scientifically robust and health protective endpoint for use as the common mechanism endpoint in the cumulative risk assessment. Such a readily quantifiable in vivo biomarker for pyrethroid

- modified VGSCs is not available for the pyrethroids.

5 OPs inhibit AChE via phosphorylation; NMCs inhibit AChE via carbamaylation. These different chemical reactions result in different patterns of toxicity with respect to time to peak effect and time to recovery.Draft, Do Not Cite

Draft Page 36 of 58 T here is a large body of evidence on the i n vitro effects of pyrethroids on sodium channels. Similarly there are multiple studies performed in rodents evaluating the neurob ehavioral changes associated with pyrethroid exposure in vivo , particularly at high doses. In newer studies performed by ORD and the PWG, the same pyrethroids were tested in three testing systems at three different levels of biological organization: chan nel, cells, and whole animal. The older literature and newer studies support common findings, namely that pyrethroids share the ability to interact with VGSC leading to disruptions of membrane excitability , and that pyrethroids share the ability to cause neurobehavioral sequel e in rodents. The relationship between pyrethroid

- induced alterations in VGSC function and disrupted membrane excitability (membrane depolarization and/or changes in firing of action potentials) is well established and has been demo nstrated in a number of species i n vitro

. Across multiple invertebrate and vertebrate species, including mammals, pyrethroid

- induced modification of VGSC function results in alterations in neuronal membrane excitability that are readily measured using neur ophysiological approaches. The observed in vivo changes in more apical neurophysiological measures such as CAP and EEG are consistent with the alterations in VGSC function at the cellular level. These apical neurophysiological changes either co

- occur with, or are the underpinnings of (depending on the measures), observed behavioral changes.

The current state of the science can not provide a quantitative link between alterations in sodium channels in vivo , and changes in the function of the nervous system a s evidenced by neurobehavioral effects in mammals . A quantitative link between the molecular target and downstream adverse outcomes is hampered by the lack of an easily accessible in vivo /in situ/ex vivo measure of ( biomarker for ) pyrethroid modified VGSC and a lack of a complete understanding of how subsequent changes in sodium channel function result in specific neurological outcomes. Despite this, the scientific evidence correlating pyrethroid

- induced changes in VGSC function with neurotoxicity is subs tantial. Given the availability of extensive studies on the mechanisms and toxic effects of pyrethroids, the lack of an in vivo biomarker does not preclude the common mechanism grouping.Draft, Do Not Cite

Draft Page 37 of 58 5.2 Summary In summary, although some uncertainties exist, OPP believes that there is a convincing amount of data from multiple lines of evidence and from studies spanning several decades which support a common mechanism grouping for the naturally occurring pyrethrins and synthetic pyrethroids under the FQPA. This co nclusion is based on the following characteristics:

**•** Common structure

- Pyrethrins and pyrethroids share a common structure; acid and alcohol moieties joined through an ether linkage. A putative toxophore based on methodology using molecular dynamics and cluster analysis has been described.

**•** Sodium channel disruption

- In vitro studies demonstrate the ability of pyrethroids to modify mammalian sodium channel kinetics, leading to alterations in membrane excitability and firing potentials.

**•** Neurotoxic Effects

- Pyrethroid toxicity is manifested through neurological syndromes described as either T (fine tremors), CS (choreoathetosis and salivation), or some combination thereof, depending on the structure. Open literature supports a correlation between the modif ication in sodium channel kinetics and the resulting syndrome.

6.0 Proposal to Sub

- Group the Pyrethroids by Type I & Type II As described in more detail above there are many i n vitro and in vivo studies which suggest that pyrethroid toxicity in manifested by two profiles. The following text will not repeat the above review. Instead, the following focuses on several studies conducted by the PWG or EPA’s ORD (Table 4). The following studies (Choi and Soderlund 2006; Nemec 2006; Wolansky et al. 2006; Shafer et al. 2008; Losa et al. 2009) are unique in that they were conducted on the same pyrethroids with the purpose of evaluating the common toxic effects of pyrethroids. The studies represent three different leve ls of biological complexity (sodium channel, nerve cell, whole animal) and provide a remarkably similar separation of the 11 chemicals.Draft, Do Not Cite

Draft Page 38 of 58 Figure 7
. Differences in Type I and Type II pyrethroids which support forming two sub

- groups within the Proposed CMG

6.1. Py r ethroid Effects on Cloned VGSC Na v 1.8

As part of the studies sponsored by the PWG, Choi and Soderlund (2006) examined the interactions of 11 pyrethroid compounds on function of the Na v 1.8 VGSC expressed in Xenopus oocytes. The Na v 1.8 VGSC is a highly responsive channel to all tested pyrethroids. All 11 compounds interacted with this chann el and produced characteristic changes in channel function, including alterations in activation (channel opening) and inactivation (channel closing) rates as well as induction of tail currents upon repolarization of the membrane. Based on the alterations i n the activation kinetics, as well as the time constants for tail current decay (tau 1 ,tau 2 ; Table 6), the 11 pyrethroid compounds could be clearly separated into 2 groups. The first group is comprised of the Type I compounds that produced modest increases in activation rate (tau act <12) and tail current decay described by a single exponential, while the second group is comprised of the Type II compounds, which produced large increases in the channel activation rate (tau act > 15) and tail current decay descr ibed by two exponential (one fast and one slow) components. Even considering the uncertainties detailed above regarding exogenous expression systems such as oocytes, these results demonstrate at the most basic level, two distinct groups of compounds based on actions at the level of the VGSC. Type II Sodium Current Less potent, generally, in mammalian neural

networks in vitro

(e.g., MEAs) T syndrome

in vivo

Action Potential Sodium Current

Action Potential Type I

More potent, generally, in mammalian neural

networks in vitro

(e.g., MEAs) CS syndrome in vivoDraft, Do Not Cite

Draft Page 39 of 58 6

. 2

. Micro electrode arrays (MEA) Studies Newer technologies have allowed researchers to examine how pyrethroids affect mammalian neural networks i n vitro . Primary neuronal cell cultures can be grown on microelectr ode arrays (MEAs), Figure 8, allowing the measurement of spontaneous electrophysiological activity. While voltage

- clamp techniques allow measurement of channel function in an individual neuron, MEAs measure function of networks of neuronal cells, and are thus a “summed” measure of effects of compounds on all ion channel constituents (e.g. Na, Ca) in the cells and network. Thus, multiple VGSC as well as VGCC isoforms contribute to the overall responses measured with MEAs. While this precludes direct examina tion of individual isoform interaction with pyrethroids, pharmacological approaches may be used to gain inferences regarding the relative importance of different ion channel types to the pyrethroid response. Glutamate is a major excitatory neurotransmitte r in the mammalian CNS and effects of pyrethroids on glutamatergic networks have been characterized using MEA techniques. Effects of pyrethroids on glutamatergic networks using spontaneously active cortical (Losa et al. 2009) and hippocampal (Shafer et al. 2008) cultures have re cently been examined. In hippocampal cultures, pharmacological approaches indicated that pyrethroid

- induced changes could be minimally explained via effects on Na channels. These data are consistent with voltage

- clamp measurements of permethrin and deltam ethrin effects on miniature excitatory post

- synaptic currents (mEPSCs) in hippocampal neurons (Meyer and Shafer 2006)

. Thus, these data support the modulation of VGSC as a key event in pyrethroid induced changes in neuronal excitability.

Figure 8 . Photograph of neurons on a MEA. There are 59 electrodes on each MEA “chip” and each electrode can record firing rates and patterns of any neurons in “contact” with it. Losa and colleagues determined IC 50 values for inhibition of spontaneous activity in glutamate networks for 11 pyrethroids (Losa et al. 2009) using primary cultures of cortical neurons. The values ranged from 25 to 1685 nM (Table 4); Type I and II pyrethroids resulted inDraft, Do Not Cite

Draft Page 40 of 58 higher and lower IC 50 values, respectively. Esfenvalerate and fenpropathrin resulted in intermediate values.

Table 4. IC 50 values for glu tamate network inhibition

Pyrethroid Type

- based on structure MEA IC50s
Cyhaloth r in, lambda

- II 25 Deltamethrin II 175 Cypermethrin II 181 Cyfluthrin II 305 Bifenthrin I 439 Tefluthrin I 499 Permethrin I 719 Esfenvalerate II 809 Fenpropathrin II 1518 Allethrin; S

- Bioallethrin I 1525 Resmethrin I 1685

6

. 3

. In V ivo Rodent Studies In an effort to determine the relative potency of pyrethroids, Wolansky et al (2006) determined acute dose

- response functions for 11 pyrethroids in rats using motor activity. Activity was measured for 1 hour in figure 8 mazes during the time of peak effect. It is difficult establish a common toxic effect using motor activity because the specificity for a causative mode

- of

- action based linking of effects on VGSCs to apical measures of neural function like motor activity are extremely difficult to demonstrate. However, the determined ED 30 s, that is the effective dose which causes a 30% decrease in motor activity, generally demonstrate a separation of Type I and Type I I pyrethroids based on potency with some exceptions.

T he PWG conducted a special FOB study designed to characterize effects classically associated with T and CS pyrethroid toxicity for 12 test chemicals (1 pyrethrin, 5 Type I and 6 Type II pyrethroids) (Nemec 2006) . The FOB consisted of 56 observations covering home cage, sensory, handling, physiological, open field, and neuromuscular parameters. Four primary patterns of variation were observed in the FOB data. Two of these patterns described behaviors associated with CS behaviors (lower temperature, excessive salivation, impaired mobility) and two described behavior s associated with the T syndrome (elevated temperature, tremors, myoclonus). Non

- cyano pyrethroids tended to have a high loading on the T factors whereas cyano

- containing pyrethroids tended to have high loading on the CS factors. Pyrethroids with the str ongest CS and T responses were deltamethrin/ λ

- cyhalothrin and resmethrin/bifenthrin, respectively. Fenpropathrin and pyrethrin had weak responses for either the C or TS characteristics.Draft, Do Not Cite

Draft Page 41 of 58 Table 5. Behavioral characterization of pyrethroid toxicity by PWG
Pyrethroid WIL* Allethrin; S

- Bioallethrin T Bifenthrin T Cyfluthrin CS Cyhaloth r in, lambda

- CS Cypermethrin CS Deltamethrin CS Esfenvalerate MIXED Fenpropathrin T

Permethrin T Resmethrin T Tefluthrin T

6

. 4

. Proposed Subgrouping

Table 6 below combines the information described in Sections 6.1

- 6.3 and thus summarizes data at the channel level (Nav 1.8 kinetics), the tissue level (MEA IC 50 s) and the behavioral level (WIL FOB data). Examination of the table indicates a clear pattern that t he 11 pyrethroids separate into 2 groups based on structure, ion channel gating activity, and neurobehavioral effects, with two chemicals, esfenvalerate and fenpropathrin, representing mixed Type I/II. OPP is proposing to subgroup the pyrethrins and py rethroids into Type I (lacking alpha

cyano moiety) and Type II (bearing alpha

- cyano moiety) based upon differences in structure, channel gating activity, and neurobehavioral effects. This conclusion is based on the following characteristics:

**•** Structure

– based on the absence or presence of an alpha

- cyano moiety

**•** Sodium channel kinetics

– Type I pyrethroids cause slight prolongations of the sodium current tails (20 ms), often resulting in long trains of action potentials. Type II pyrethroids significantly p rolong sodium current tails (200ms to minutes) typically resulting in increased resting membrane potential and can induce action potential block.

**•** T oxicity syndromes

– Type I and Type II pyrethroids generally elicit C and TS syndromes, respectively. Ex ceptions, including esfenvalerate and fenpropathrin, elicit symptoms that are a combination of the T and CS syndromes.Draft, Do Not Cite

Draft Page 42 of 58 Table 6. Summary of key data supporting the proposed subgrouping into Type I and Type II

Pyrethroid Nav 1.8 Kinetics a MEA IC 50 s d
WIL e
Activation b tau1 c tau2 c
Allethrin; S

- Bioallethrin 3.9 1.9 NA 1525 T Bifenthrin 3.9 4 NA 439 T Permethrin 2.5 2.3 NA 719 T Resmethrin 3.3 2.6 NA 1685 T Tefluthrin 3.8 4.3 NA 499 T Esfenvalerate 10.7 13.4 NA 809 MIXED Fenpropathrin 10.8 12.4 NA 1518 T Cyfluthrin 23.6 7.1 95.9 305 CS Cyhalothrin, lambda

- 22.3 9.4 84.7 25 CS Cypermethrin 12.6 6.9 55.8 181 CS Deltamethrin 15.7 5.3 52.1 175 CS

a Gating kinetics of the rat Na v 1.8 sodium channel isoform expressed in Xenopus laevis oocytes (Choi and Soderlund, 2006) b time constants (ms)for the activation of pyrethroid

- modified channels

c time constants (ms) for the fast (tau1) and slow (tau2) components of tail current delay d MEA IC50 values exhibited by XX tissue (Shafer SOT poster??)

e Neurotoxic syndrome expressed by rats following high doses of pyrethroids as defined by FOB observations (WIL Study) Attachment 1 provides the structures of the preliminary list of pyrethrins and synthetic pyrethroids being considered in the CM G. Attachment 1 also provides assignments of potential sub

- grouping (i.e., Type I or II) to 11 pyrethroids and the pyrethrins. These assignments were made based on the information in Table 6 and the results of the special FOB for pyrethrins. As of May, 2009, five additional pyrethroids (cyphenothrin, imiprothrin, prallethrin, tetramethrin, and d

- phenothrin) wer e being tested in a special FOB study similar to the study sponsored by the PWG. OPP will assign the sub

- grouping for these five following receip t and review of the on

going studies. For the remaining pyrethroids, OPP will first evaluate existing data from In vitro and/or in vivo studies for effects consistent with Type I or II effects. In the absence specific data to inform a particular sub

- group, OPP plans to use structure (i.e., presence/absence of an α

- cyano group) as the determining factor for assigning individual pyrethroi ds to a sub

- group.Draft, Do Not Cite

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Draft Page 56 of 58 Attachment 1.0. Classificati on (as proposed by the Agency) and Structure of pyrethroids registered for use with the US EPA.

Name Type I/II Structure

Allethrin (s

- Bioallethrin, d

allethrin) I

Bifenthrin I

Cyfluthrin (β

- cyfluthrin) II

Cyhalothrin (γ/λ

cyhalothrin) II

Cypermethrin ( α / β / θ / ζ

cypermethrin) II

Deltamethrin II

Fenpropathrin Mixed I/II

Fenvalerate (esfenvalerate) Mixed I/IIDraft, Do Not Cite

Draft Page 57 of 58 Name Type I/II Structure

Permethrin I

Resmethrin (bioresmethrin, cismethrin) I

Tefluthrin I

Tetramethrin Pending Agency Decision ; Special FOB Study On

- going

Cyphenothrin Pending Agency Decision ; Special FOB Study On

- going

Fluvalinate (τ

- fluvalinate) Pending Agency Decision

Imiprothrin Pending Agency Decision ; Special FOB Study On

- going

Metofluthrin Pending Agency DecisionDraft, Do Not Cite

Draft Page 58 of 58 Name Type I/II Structure

Phenothrin (d

- phenothrin, sumithrin) Pending Agency Decision ; Special FOB Study On

- going

Prallethrin Pending Agency Decision ; Special FOB Study On

- going

Tralomethrin Pending Agency Decision

Pyrethrins I

Cinerin I Pending Agency Decision

Cinerin II Pending Agency Decision

Jasmolin I Pending Agency Decision

Jasmolin II Pending Agency Decision