UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

MEMORANDUM

Date: August 28, 2013 OFFICE OF
CHEMICAL SAFETY AND
POLLUTION PREVENTION

SUBJECT: Common Mechanism Grouping for the Pyrethrins and Synthetic Pyrethroids

PC Code: See Below
Decision No.: 455436
Petition No.: None

. Risk Assessment Type: None
TXR No.: 0056776

, MRID No.: None DP Barcode: 394576 Registration No.: None Regulatory Action: None

Case No.: None
CAS No.: See Below

40 CFR: None

- ·

FROM: Edward Scallon, Ph.D., Toxicologist

Anna Lowit, Ph

. D

., Senior Scientist

Health Effects Division (7509P)

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THROUGH:

TO: Dana Friedman, Team Leader
Risk Management and Implementation Branch 2

Pesticide Re-Evaluation Division (7508P) • ,_

The Office of Pesticide Programs has determined that the pyrethrins and the synthetic pyrethroids cause toxicity through the same mode of action, interaction with the voltage-gated

sodium channels. The Agency's proposal for placing the pyrethrins and pyrethroids in a

common mechanism group was presented and supported by a FIFRA Scientific Advisory Panel (June 2009). Following the meeting and the consideration of public comments, the draft science

paper was duly posted in the Pyrethrins/Pyrethroid Cumulative Risk Assessment docket ( http : // www.regulations

. gov;EPA-HQ-OPP-2011-0746 ) on November 9, 2011. Since the
November 9 posting of the draft science doc ument, the Ag ency has updated the draft version

. This memo is accompanied by the final version of Science Policy Paper: Common Mechanism Grouping for the Pyrethrins and Synthetic Pyrethroids. The attached document is fmal.

Page 1 of2Page 2 of 2 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

Flumethrin 036007 69770-45-2

Fluvalinate 109302 69409-94-5 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 Sumithrin (phenothrin) 069005 26002-80-2

Tau-fluvalinate 109302 102851-06-9

Tefluthrin 128912 79538-32-2

Tetramethrin 069003 7696-12-0

Tralomethrin 121501 66841-25-6

# Science Policy Paper

Common Mechanism Grouping for the

Pyrethrins and Synthetic Pyrethroids

August 28, 2013Page 2 of 58 

### TABLE OF CONTENTS

1.0 Backgrou nd ........................................................................................................... 3
2.0 Key Concep ts ........................................................................................................ 4
3.0 Chemical St ructure ............................................................................................... 5

3.1 Chirality of pyrethroids ................................................................................. 7
3.2 Quantitative Structure Acti vity Relationships (QSAR) ............................... 9
4.0 Toxicology of Py rethroid s .................................................................................. 10

4.1 Overview ...................................................................................................... 10
4.2 Voltage-Gated Sodium Channels (VGSC) ................................................. 11
4.2.1. Protein structure of the VGSC .............................................................. 11
4.2.2. Channel modulation by pyrethroids ..................................................... 12
4.2.3. Relationship between pyrethroid-induced alterations in VGSC function & disrupted membrane excitability ....................................... 14
4.2.4. Potential differences in tissue sensitivity to pyrethroids ................... 14

4.2.4.1. Voltage-clamp measurements ......................................................... 14
4.2.4.2. Sodium channel cloning .................................................................. 15
4.2.5. Other molecular targets for pyrethroids .............................................. 17
4.3 In vivo profile ............................................................................................... 21
4.3.1. Pharmacokinetics .................................................................................. 21
4.3.2. Toxicity syndromes in rodents ............................................................. 22
4.3.3. Information in humans .......................................................................... 28
4.4 Data in mixtures of pyrethroids ................................................................. 28
5.0 Common Mechanism Grouping under the FQPA ............................................. 30

5.1 Weight of the Evidence ............................................................................... 30
5.2 Summary ...................................................................................................... 35
6.0 Potential to Sub-Group the Pyrethro ids by Type I & Type II ........................... 35

6.1. Pyrethroid Effects on Cloned VGSC Na v 1.8 .............................................. 36
6.2. Micro electrode arrays (MEA) Studies ....................................................... 37
6.3. In Vivo Rodent Studies ............................................................................... 38
6.4. Potential to Subgroup ................................................................................. 39
7.0 Referenc es ........................................................................................................... 41
Attachment 1.0. ........................................................................................................... 55Page 3 of 58 1.0 Background

The Food Quality Protection Act (USEPA,1996) requires EPA to take into account “available evidence concerning the cumulative 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 Pr ograms (OPP), a cumulative risk assessment begins with the identificat ion of a group of chemical s referred to as a common mechanism group (CMG), that induce a comm on toxic effect by a common mechanism of toxicity. To date, OPP has con ducted cumulative risk assessments on the organophosphates (OPs), N -methyl carbamates (NMCs), ch loracetanilides, triazines, and most recently, pyrethroids (Oct. 4, 2011) . 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 can occur from food, water, or non- occupational settings. Due, in large part, to changes in usage trends resulting from ri sk mitigation, residential use of OPs and NMCs has decreased. Consequently, potentia l exposure of the general public to pyrethroid pesticides has increased over the past decade.

There is an extensive body of published literature concerning the toxicity of pyrethroids and potential modes of action of these compounds. In addition, over the last decade, EPA’s Office of Research and Development (ORD) and the Pyrethroid Working Group 1 (PWG) have conducted independent, lar ge-scale research efforts to evaluate aspects of pyrethroid exposure, pharmacokinetics (PK), mode/mechanism of action, and toxicity profiles. These res earch efforts have added to the scientific
literature and also led to a meeting of The Federal Inse cticide, Fungicide, and Rodenticide Act Science Advisory Panel (FIFRA SAP) in August, 2007, on a proposed approach to developing physiologically-bas ed pharmacokinetic (PBPK) models for multiple pyrethroids (USEPA, 2007). T he research by both EPA and the PWG was conducted, in large part, in anticipation of a future cumu lative risk assessment on the pyrethroids. The purpose of this science po licy document is to describe this scientific evidence which supports the des ignation of pyrethroids as a CMG under the FQPA.

This document follows OPP’s guidance on fo rming CMGs (2002) and also considers the concepts contained in the Mode of Action and Human Relevance Frameworks developed by EPA, WHO’s International Pr ogramme 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. 200 6). Based on the current state of the science, OPP has determined that the natur ally occurring pyrethrins and synthetic pyrethroids form a CMG based on 1) shared st ructural 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. As des cribed in more detail below, the pyrethroid CMG may be divided into two subgroups r epresenting Type I and Type II pyrethroids, however, all pyrethroids were considered in a single group for the CRA. This

1 The Pyrethroid Working Group is a group of pesticide companies who own technical registrations for pyrethrins and some synthetic pyrethroids.Page 4 of 58 subgrouping is consistent with historical ca tegorization of pyrethroids and is supported by in vitro and in vivo information from both older studies and new research. Pyrethroids considered for the CM G are listed in Appendix 1.

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 CMG incl ude allethrin, bifenthrin, imiprothrin, metofluthrin, permethrin, resmethrin, phenothr in, tefluthrin, tetramethrin, sumithrin, prallethrin, and pyrethrins; Type II me mbers include cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin, delta methrin, flucythrinate, flum ethrin, and fluvalinate. The absence/presence of the α -cyano group is correlated with length of time the sodium channel is inactivated (-CN=s horter; +CN=longer) which in turn corresponds with the two distinct toxicity syndromes (-CN=T syndr ome; +CN=CS syndrome). The nature of
these effects is discussed in more detail in se ction 4.0. Two pyreth roids, esfenvalerate and fenpropathrin, though structura lly similar to Type II pyrethroids, are considered mixed Type I/II because they illicit effects intermediary between Type I and Type II pyrethroids during in vitro and in vivo evaluations. Additional discussion on the classification of pyrethroids c an be found in Section 6.4.

This document also describes some uncer tainties considered including i) other endogenous channels (e.g., calcium and ch loride) which have been proposed as potential target sites for pyrethroids; ii) s odium channel structural heterogeneity among tissue and across life stages with respec t 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 has guidance documents for desi gnating CMGs (USEPA 1999) and for developing cumulative risk assessments (USEPA 2002). These documents provide definitions for some key concepts and provid e the guiding principles for the analysis. 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 review s by the FIFRA SAP. Some key concepts
included in the curr ent analysis are:

 Mechanism of toxicity is defined as the major steps leading to a toxic effect following interaction of a pesticide with biol ogical targets. All steps leading to an effect do not need to be specific ally understood. Rather, it is the identification of the crucial events following chemical inte raction that are r equired in order to describe a mechanism of toxicity. Gener ally, the more that is understood about the various steps in the pathway leading to an adverse effect, the more confident one 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 in vitro, and causes the receptorrelated molecular response; in vivo it also leads to the molecular response andPage 5 of 58 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 lo cus (e.g., same organ or tissue) are said to cause a common toxic effect

.

 A common mechanism of toxicity pertains to two or more pesticide chemicals
or other substances that cause a comm on toxic effect to human health by the same, or essentially the same, sequence of major biochemical events. Hence, the underlying basis of the toxicity is t he same, or essentially 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 ar e toxic by a common mechanism from all pathways. Thus, cumulative risk asse ssments include multiple chemicals and multiple pathways (e.g., food, drinki ng water, consumer uses).

 A common mechanism endpoint(s) is/are those common toxic effect(s) which are pertinent and sensitive endpoints a ssociated with the common mechanism and 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 t he pyrethrins, have evolved structurally over the past several decades. However, the basic components of pyrethrins, a chrysanthamic acid linked to an aromatic al cohol through an ester linkage, have been conserved (Fig. 1). Structural modifications such as i) the addition of halogens to the chrysanthamic acid and arom atic alcohol moieties and ii) the addition of the α -cyano group, have increased photostability, insectic idal potency, and in some incidences, stereoisomerism of the pyrethroids.Page 6 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 I)

O

O O

* *



### Cypermethrin (Type II)

O

O Cl Cl

N O * * *

* = chiral centers

Figure 1. Representative structures for pyrethrins (Pyrethrin I), Type I (resmethrin) and Type II (cypermethrin) pyrethroids.Page 7 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 py rethroids, the presence of two chiral carbons, C1 and C2, in the in the cyclopropa ne ring of the chysthanthamic acid moiety produces two 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 additi on of a third chiral carbon in Type II pyrethroids (e.g. cypermethrin) results in 8 stereoisomers (Fig. 1).

Stereoisomerism of the pyrethroids is an important consideration in the toxicity profile of pyrethroids. For ex ample, the orientation of the C1 and C3 constituents in the cyclopropane ring of the chrysanthamic acid mo iety determine the cis/trans structural confirmation, which can in turn impact potent ial toxicity and metabolism. Furthermore, the 1R configurations are insect icidal, whereas the enantiomeric 1 S compounds, though physically identical, are without insecticidal activity (Elliot et al. 1974). Furthermore, interaction with the (Lund and Narahashi 1 982; Narahashi 1982), ability to modify sodium channel kinetics (Section 4.0), and metabolism (PK secti on 4.3.1) are also isomer dependent.

Ideally, there would be sufficient data to distinguish the pharmacodynamics (PD), PK, and environmental exposure of the acti ve stereoisomer(s). For some, such as bioresmethrin, deltamethrin, and esfenvalerat e, the registered produc ts are enrichments of the more insecticidally acti ve isomers. As such, for thes e, there is comparatively little uncertainty with respect to assessing the haz ard and exposure. For permethrin, residue data and some limited toxicity information are reported as cis and trans. However, toxicity and exposure information available for the majority of pyre throids are for the isomeric mixture. Specifical ly, animal toxicity studies subm itted for pesticide registration are performed with the isomeric mixture. In addition, residue data used to evaluate food, water, and residential exposure are typica lly reported as isomeric mixtures. Thus, although the Agency acknowledg es the stereoisomerism as a consideration for pyrethroids, the ability of the Agency to quantitatively account for these in risk
assessment is limited. Inst ead of assessing the active stereoisomer(s), the Agency typically assesses the risk to pyrethroids by ev aluating the risk to the isomeric mixture. The resources required to collect such toxi city and exposure data on the isomers would be substantial. Although ther e is some uncertainty a ssociated with evaluating the
mixture instead of the active stereoisomer, the lack of such data does not prevent the Agency from developing risk assessments wh ich are both health protective and scientifically sound.Page 8 of 58 Figure 2. Stereochemistry of the pyrethroid acid moietyPage 9 of 58 3.2 Quantitative Structure Acti vity Relationships (QSAR)

As stated above, one of the f eatures of pyrethroids is their common structural characteristics. With respec t 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 2 (or may yield a common toxophore upon metabolism) and may interact analogously with cellular biomolecular sites to cause a common toxic effect (p. 8).”

In other words, for purposes of determining a CMG, identi fication of a toxophore is important for evaluating a common PD interact ion at the molecular site of action.

Some authors have argued for the lack of a toxophore among pyrethroid pesticides based on the contention that the appropriate fit of t he entire molecule at the site of action is required for interaction (B yberg et al. 1987; Soderlund et al. 2000). However, Ford et al. (2002) has performed a three-dimensional qua ntitative structure activity relationship (QSAR) analysis of pyreth roids. Conformational sampling of active pyrethroids using molecular dynamics and cl uster analysis, taking into account the multitude of bond angles possible, revealed a common conformation in 39 of the 41 structures examined (Fig. 3) . 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 abilit y of Type I pyrethroids but are generally more potent at killing. The ‘knockdown’ and kill responses are also differentiated by their temperature dependence, negative for kill and positive for ‘knockdown’. Ford et al. (2002) related the putative tox ophore to the killing potential, inherent to both Types I and II. The authors postulate that rest riction about the ether linkage by an α -cyano group in Type II pyrethroids precludes the k nockdown capacity associated with Type I pyrethroids. The unrestricted movem ent about the ether linkage requisite for
‘knockdown’ is not the most stable conformati on, as reflected by its positive temperature dependence.

2 The CMG guidance defines the term “toxophore” with respect to toxic substances, as is akin to the term “pharmacophore” with respect to drug substances: th e pharmacophore is that structural moiety of a drug substance or substances which imparts a desired pharmacological property.Page 10 of 58 Figure 3. Conformational structure of putative pyrethroid toxophore

As detailed in the following section, vert ebrate VGSC structures are significantly more complex than insect channels. Howeve r, there is homology in structure and
function between insect and mammalian sodi um channels. Insect and mammalian VGSC are both composed of pore-forming α subunits with modifying proteins (mammalian β units and insect tipE proteins). Type I and Type II pyrethroids cause
similar modifications in channel kinetics in both species (Type I results in repetitive firing and Type II pyrethroids result in depolarizatio n-dependent block of the action potential).
It is therefore reasonable to assume the to xicophore identified in insects would be
similar to that in mammals. As such , the Agency further assumes that a shared toxophore and chemical structure of pyrethro ids 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 modi fy the kinetics of the VGSCs in insect and mammalian neurons. The Agency has ident ified multiple lines of evidence which support the shared ability of the synthetic pyrethroids and pyrethrins to interact with the Na channels leading to depolarization of me mbranes in the nervous system, ultimately leading to neurotoxicity characterized by two different toxicity syndromes. This evidence is summarized below starting wit h the VGSC followed by mechanistic information from voltage clamp/patch clamp studies, use of neuronal networks (e.g., micro electrode arrays (MEAs)), and in vivo toxicity studies. 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), Nar ahashi (1996), Shafer et al (2005), Wolansky and Harr ill (2008), Nemec (2006).Page 11 of 58 4.2 Voltage-Gated Sodium Channels (VGSC)

4.2.1. Protein stru cture of the VGSC

Briefly, the volt age-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 ve rtebrates, smaller auxiliary β subunits modulate α subunit gating, as well as insertion and dist ribution 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 lack β subunits, per se. However, tipE protein has been suggested as a homologous protein, though structurally unrelated, 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 cr eates uncertainty in correlating effects on specific VGSC isoforms in vitro to in vivo symptomology caused by pyrethroid toxicity in
mammals. There are at least 10 dist inct 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 ( β 1- β 4) (Yu et al. 2003) have been identified to date. The heterogeneity of the sodi um 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 functi onal and pharmacological diversity of sodiumPage 12 of 58 channel expression varies by tissue and st age of development (reviewed in Meacham et al. (2008) and Mandel (1992)). The implications of this diversity on uncertainty related to actions of pyrethroids will be discussed further in t he following sections.

4.2.2. Channel modulation by pyrethroids

The effects of pyrethroid exposure on neuronal tissue (both insects and mammals) have been extensively revi ewed (Soderlund and Bloomquist 1989; Vijverberg and Bercken 1990; Narahashi 1992; Bloomquist 1993a; Clark 1995; Soderlund 1995; Bloomquist 1996; Narahashi 1996).

Figure 5 depicts generalized pyrethroid e ffects on individual channels, whole-cell sodium currents, and action potentials (extracted from Shafer et al. (2005)). Depolarization opens VGSCs (top left) allowing s odium to enter the ce ll. To limit sodium entry and depolarization length, VGSCs inactivate and must re turn to a “resting” state before reopening. Pyrethroids inhi bit the function of two diffe rent “gates” that control sodium flux through VGSCs (top right), delayi ng inactivation (indicated 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, Sodium Current). Pyrethroid-modified VGSCs remain open when depolariz ation ends (bottom right, So dium Current), resulting in a “tail” current (the notch at the end of example curr ents). 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 ac tion potentials (repetitive firing). Type II compounds cause greater me mbrane depolarization, diminishing the sodium electrochemical gradient and subsequent ac tion potential amplitude. Eventually, membrane potential becomes depolarized abov e the threshold for action potential generation (depolarizati on-dependent block) (Shaf er et al. 2005).

Modification of the Na channels in ma mmals 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 tr ains of action potentials accom panied with little or no effect on resting membrane potential. In c ontrast, pyrethroids containing the α -cyano moiety typically do not induce repetitive firing. Inst ead, Type II pyrethroids produce long-lasting sodium current tails (>>200ms) that ultimately result in action potential block due to depolarization of resting memb rane potential above the threshold for action potential generation. It has been estimated that as few as 1% of the sodium channels need be modified to render a cell hyperexcitabl e and induce repetitive firing (Song and Narahashi 1996; Motomura and Narahashi 2001).Page 13 of 58Figure 5. Effects of pyrethroids on Na channels, currents, and action potentials (From Shafer et al., 2005)Page 14 of 58 4.2.3. Relationship between pyreth roid-induced alterations in VGSC function & disrupted me mbrane excitability

The relationship between pyrethroid-induc ed alterations in VGSC function and disrupted membrane excitabilit y (membrane depolarization and/or changes in firing of action potentials) is well established and has been demonstrated in a number of species
in vitro . Using crayfish giant axon, Salgad o and co-workers (1989) demonstrated temperature-dependent actions of fenvalerate on VGSC function as well as membrane excitability. In addition, voltage-dependent potassium curr ents were not altered by fenvalerate. Lund and Narahashi (1981) dem onstrated that cyphenothrin disrupts sodium channel function, depolarized the memb rane, and upon stimulation of the nerve, induces repetitive firing that is blocked by tetrodotoxin (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 causing depolarization a nd/or induce repetitive in peripheral nerves (Vijverberg and Bercken 1979, 1982). Similar findings have also been reported in
mammalian neurons in vitro , wherein pyrethroids modify VGSC function and produce changes in membrane excitability in dor sal root ganglion neurons (Tabarean and Narahashi 1998) and cerebellar Purkinje neurons (Song and Narahashi 1996). These alterations in neuronal excitability are consistent with reports from in vivo measurements of modified neurophysiological endpoints. Fo llowing 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 fenv alerate or allethrin (Nozaki et al. 1995). When dorsal root potentials were recorded from urethane anesthetized rats, cismethrin increased t he amplitude of the pot ential by up to 142% (Smith 1980). Finally EEG measurements demonstrated that administration of cypermethrin produced bursts of epileptic acti vity following the firs t administration, and on subsequent days of administr ation, these were accompanied by shaking, myoclonus, and tonic-clonic seizures (Condés-Lar a et al. 1999). Thus, data from in vitro studies indicate that pyrethroid modification of VGSC leads to changes in membrane excitability. In vivo , changes in neurophysiological measur es occur that are consistent with alterations in action potentials, and correlate with behavioral changes.

4.2.4. Potential differences in ti ssue 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 difference across the membrane by elec tronic 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 channel openings and closings. Thus, the membrane can be clamped toPage 15 of 58 different voltages and resulting currents measured. Using so lutions of different ionic composition as well as pharmacological com pounds to block specific channels, it is possible to examine the behavior of different kinds of ion channels through which these currents pass (Eckert et al. 1988). Howeve r, it should be pointed out that pharmacological antagonists to specific VGSC subunit combinations are not available; this precludes identification of the exact composition of VGSC subunits mediating pyrethroid sensitive currents in native mammalian neurons.

A number of voltage–clamp studies have dem onstrated the selective ability of pyrethroids to perturb sodium channels bas ed 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 and fenvalerate (Yu et al. 2003) increased m ean open time 200 fold greater than noted for
tetramethrin. These results are characte ristic of the dist inction 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 py rethroids on individual channel subunit combinations using voltage-clamp techniqu es. However, some tissue types have
demonstrated structure-based se nsitivity to pyrethroids, indicating channel isoform populations may differ with tissue type. Do rsal root ganglion (DRG) cells contain tetrodotoxin-sensitive (TTX-S) and tetrodotoxin -resistant (TTX-R) sodium channels. TTX-R channels are susceptible to perturbat ion by both Type I and Type II pyrethroids while TTX-S channels are relatively le ss sensitive (Ginsburg and Narahashi 1993; Tatebayashi and Narahashi 1994; Song and Narahashi 1996; Narahashi et al. 1998). These examples demonstrate the differential ac tivity 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 wit h the sensitivity of the Na v 1.8 channels (which belong to TTX-R VGSC) expressed in oocytes to 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 spec ific manner. Further, there appear to be differences in pyrethroid effects on mammalian neurons that are dependent on the composition of the channel un der study. Advances in molecular techniques have allowed researchers to express specific co mbinations of VGSC subunits in exogenous expression systems such as Chinese hamst er ovary (CHO) cells and unfertilized Xenopus laevis oocytes. The isolation and expressi on of specific channel isoforms has
allowed researchers to examine the direct im pact 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
the 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, c onfirming their roles as modulator s of pyrethroid activity on sodium channels (Smith and Soderlund 2001). When expressed in these systems, αPage 16 of 58 subunits have demonstrated differential struct ure-dependent sensitivity to pyrethroids.
Na v 1.2 is sensitive to Type II pyrethroids, but insensitive to Type I pyrethroids (Smith and Soderlund 1998). Na v 1.8, a principle carrier of TTX-R current (Smith and Soderlund 2001) in DRG neurons discussed in t he preceding section, is sensitive to perturbation by both Type I and II pyrethroids re sulting in characteristic modification of sodium currents (Choi and Soderlund 2006). T hese findings are supported by similar results in the voltage-clamp exper iments previously reviewed.

Young rats (post-natal day 8) have been show n to be more sensitive to the acute toxicity of deltamethrin (Sheets et al. 1994) and cismethr in (Cantalamessa 1993) than adults but not to permethrin and cismethrin (Sheets 2000). PK differences between the
immature and adult rats have been demonstrat ed (Cantalamessa 1993; Sheets et al. 1994; Anand et al. 2006). Meacham et al. (2008) explored the PD possibility of agedependent pyrethroid toxicity using combinations of Na v 1.2 or Na v 1.3 and β 1 or 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 embr ionically while Na v 1.2/ β 1 predominates in adults, supporting the hypothesis of developmenta lly-dependent PD differences.

Although these exogenous expression systems are useful in evaluating tissue and life stage differences in the Na channel popul ations (Trainer et al. 1997; Smith and Soderlund 1998, 2001; Choi and Soderlund 2006; Meacham et al. 2008), such studies must be interpreted cautiously. High pyreth roid 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 combin ations 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-neur onal systems, and/or at temperatures lower than 37°C. Such artificial settings may alter post-transla tional modification, membrane composition, or phos phorylation status, leading to differences in channel sensitivity to pyrethroid modification.

Even extrapolation from non-mammalian to mammalian exogenous expression systems is difficult. Studies examining pyre throid 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 tec hniques (electrophysiol ogy vs. fluorescence measurements), studies with transfected ma mmalian cells are difficult to compare directly to studies utilizing oocytes. For exam ple, Vickery et al. (2004) showed that noncyano pyrethroids (allethrin, permethrin or tetramethrin) were without effect on rat
Na v 1.2 expressed in Chinese hamster ovary ce lls, even when veratridine was present to activate VGSC. However, in the same st udy, deltamethrin and fenvalerate were also without effect individually and produced a small response in the presence of veratridine (Vickery et al. 2004).

From a risk assessment perspective , sodium channel cloning studies are interesting for purposes of evaluating s pecies differences, potential population PD variability, and life stage differences. With re spect to the CMG, this research highlightsPage 17 of 58 the importance of sodium channel interaction as a key event in pyrethroid toxicity. Specifically, although ther e is heterogeneity among the subunit combinations, the pathway of toxicity remains the same---nam ely that sodium channel interaction as a critical and common key event in toxicity of py rethroids. All the pyrethroids examined to date have the ability to modify VGSC kinet ics 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 this level of detailed understanding is not necessary for forming a CMG.

4.2.5. Other molecular targets for pyrethroids

In the CMG guidance, OPP acknowledges t hat individual chemicals can exhibit multiple mechanisms of action and can have multiple molecular targets (USEPA, 1999). Specifically, the CM G 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 chemical 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 other functions at distant 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 at least one of the toxic effects is common among the substances (p. 7 ).”

For the pyrethroids, the Agency has det ermined interaction with VGSCs is the common initial key event for determining t he CMG. Pyrethroids, however, also have been reported to inhibit other types of ion channels, including chloride-permeable ion channels, voltage-gated calcium channels (V ijverberg and Bercken 1990; Narahashi 1996; Narahashi et al. 1998; Ray and Forsha w 2000; Ray 2001; Soderlund et al. 2002; Burr and Ray 2004; Shafer and Meyer 2004) and lig and-gated 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) or are secondar y to perturbation of the VGSC (Cao et al. 2011b). While pyrethroid actions on VG SCs and neuronal excitability are well understood, very little is known about how ef fects on other target sites contribute to pyrethroid mammalian neurotoxicity (Ray 2001; Soderlund et al. 2002).Page 18 of 58 A class of chloride-permeable channels has been proposed as potential targets for pyrethroid actions in the mammalian c entral 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 interp retation of the results with respect to the role of effects on this target in pyrethro id toxicity. A large conductance, chloridepermeable ion channel (denoted as ‘maxi-chloride’) isolated using the ‘inside out patch’ electrophysiological technique is inhibi ted by various pyrethroid compounds. Pyrethroids inhibit these channels by reducing the probability that t he channel will open. Information regarding concentration-response relationships is lacking, but several pyrethroids have been tested fo r their ability to modulate maxi-chloride channel opening
in vitro . Deltamethrin, cypermethrin, cyfluth rin, fenpropathrin, and bioallethrin were
effective between 10 -5 to 10 -6 M. Cismethrin, bifenthrin , cis-permethrin, cyfluthrin isomers, cyhalothrin, esfenvalerate, and te fluthrin had no significant effects at 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’ electrophysiological technique (Walz 2002), thus, it is difficult to associate pyrethroid induced changes in maxi chloride- channel function with any other alterations
in cellular function or excit ability. The electrophysiological parameters of the pyrethroidsensitive maxi-chloride channel are homol ogous 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 we ll as small diameter molecules such as ATP and, possibly, cytochrome C across the mitochondrial outer membrane (Dermietzel et al. 1994; Sampson et al. 1997; Buett ner et al. 2000). A plasma membrane
associated form of the VDAC ch annel 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 functi on in the regulation of membr ane excitability or if they are expressed in neurons (Walz 2002). Available in vitro studies evaluating the possible interaction between pyrethroids and the chlori de channels are limited in that the studies have been conducted utilizing only the N1E- 115 neuroblastoma cell line and largeconductance chloride channels. Published data indicate N1E-115 cells are a glia X neuron hybrid that possesses characteristi cs of both cell types (Moolenaar and Spector 1977, 1978). Thus, these cells also contai n glial characteristics and are not true neurons. Therefore, given the characterist ics 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 de ltamethrin-induced neur otoxicity. Some antagonism of behavioral measures followi ng deltamethrin exposure was noted. Neither compound was effective at reducing the in vivo signs of cismethrin 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 ch loride ion channel receptors present in the central and peri pheral nervous systems (Abalis et al. 1986;Page 19 of 58 Mascia et al. 1996; Huang and Casida 1997; Dawson et al. 2000; Shan et al. 2001; Steinbach and Akk 2001; Lu and Xu 2002; Hadi pour-Jahromy and Daniels 2003). At
the doses used for in vivo studies (Forshaw et al. 2000) , the possibility that the in vivo signs of pyrethroid neurotoxicity were alle viated by the actions of ivermectin on GABA A and glycine receptors, rather than on maxi-chloride channel s, cannot be eliminated.
Pentobarbitone is a sedat ive that also binds to, and activates, GABA A and glycine receptors (Mascia et al. 1996; Steinbac h and Akk 2001; Lu and Xu 2002; HadipourJahromy and Daniels 2003). The ability of ivermectin or pentobarbitone to competitively antagonize the pyrethroid dependent modulati on of the maxi-chloride channel has not been tested. Given the lack of knowledge r egarding the identity of the neuroblastoma maxi-chloride channel, and the questionable role of the VD AC channel, which is the most likely candidate for the ‘maxi-chlo ride’ channel in regul ation of membrane excitability, the results of the in vivo studies must be interpret ed with caution. The lack of specificity of ivermectin and pentobarbitone in these experiments precludes definitive conclusions regarding the target of pyrethroid action. In su mmary, the overall weight of
scientific evidence for maxi-chloride channel e ffects as a basis for designating a CMG is
not conclusive.

In addition to the voltage-gat ed 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 VG CCs are comprised of pore-forming α 1 and auxiliary ( 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. voltage-clamp) and indirect (e .g. neurotransmitter re lease, Ca influx) approaches. Rather than provid e an extensive review (see Shafer and Meyer (2004)), several findings are highlighted here. Type II pyrethroids have been shown to increase
Ca 2+ -dependent norepinehphrin e release from rat brain synaptosomes that were depolarized 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 pyrethroid, has been reported to inhibit di fferent isoforms of VGCC expressed in HEK293 cells, and to shift the kinetics of channel inactivation, but not activation (Hildebrand et al. 2004). More recently, el ectrophysiological studies with rat brain
Ca v 2.2 expressed in Xenopus laevis ooctyes substantiated that deltamethrin modified
Ca v 2.2 in vitro (Symington and Clark 2005). Using Ba 2+ as a carrier fo r current through calcium channels, deltamethrin reduced peak cu rrent, caused a hyperpolarizing shift in potential activation and slowed activation and inac tivation rates. The inhibition of peak current by deltamethrin appears in consistent with 1) increased Ca 2+ influx and enhanced neurotransmitter release repor ted using functional synaptosomal preparations (Symington and Clark 2005); 2) the lack of deltamethrin effects on spontaneous glutamate release at in tact synapses in hippocampal neurons in vitro (Meyer and Shafer 2006); and 3) in vivo observations (Aldridge et al. 1978; Hossain et al. 2004) of increased neurotransmitter release fo llowing deltamethrin. Such differences suggest that other regulator y components of modulation Ca 2+ influx and subsequent neurotransmitter release are necessary to elicit a neurophysiologic response that isPage 20 of 58 consistent with in vivo symptoms. Moreover, using primary cultures of mouse neocortical neurons, Cao et al. (2011b) have rec ently shown that pyrethroids stimulate calcium enty into the neurons subsequent to thier actions on VGSCs. Overall, these uncertainties and findings indicate that the potential role of di rect effects of pyrethroids on VGCC in toxicological responses is presently not well enough understood to serve
as the basis for a CMG.

Finally, there is evidence that pyrethro ids may interfere with ligand-gated chloride channel currents. Briefly, when the inhibitory neurotransmitter γ -aminobutyric acid (GABA A ) is released from the presynaptic nerv e terminal, it binds to a postsynaptic receptor protein containing an intrin sic chloride ion channel (i.e. GABA A -activated chloride channel). When GABA A binds to its receptor, the chloride channel is opened
and chloride ions flow into the cell resulti ng in an inhibitory post-synaptic potential which causes membrane hyperpolarization lead ing to a dampening affect on neuronal excitation, making it less likely that a pos t-synaptic action potential will occur (Costa 1998). GABA A antagonists, such as the chlori nated 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 (Bloom quist 1993b). Receptor agonist s like diazepam (central) and RO5-4864 (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, hav e shown the ability to weakly antagonize
GABA A binding 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). Conver sely, deltamethrin 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 combinator ial effects of cismethrin (Type I) and deltamethrin (Type II) on neur obehavioral function (i.e., acoustic startle response (ASR) and motor activity) in conjunction with controls for GABA A receptor activated chloride current, picrotoxin, muscimol, or chlordiazepoxide. Only deltamethrin and picrotoxin showed a dose-additive effect i ndicating an interaction at the picrotoxin binding site of the GABA A receptor-ionophore complex. Conversely, other in vivo rodent studies using anesthetized rats, failed to show any impact of GABA antagonists on GABA-mediated inhibition of hippocampal gr anule cells. These author s concluded that pyrethroid effects on GABA A receptors were unlikely to account for pyrethroid induced changes in hippocampal synaptic transmission (G ilbert et al. 1989; Joy et al. 1990) or dorsal root ganglion potentia ls (Smith 1980). While in 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
conclusions 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 dem onstrated susceptibility to some pyrethroids, however, interpretation of the in vitro results with respect to the purpose ofPage 21 of 58 the CRA groupings are confounded by the pot ential with VDAC channels and choice of cell line; in vivo results are compromised due to t he potential antagonism of ivermectin and pentobarbitone on voltage-gated chloride channels and GABA A and glycine receptors. Voltage-gated calciu m channels are potential sites of action of pyrethroids; however the inconsistency of data from di fferent types of preparations and compounds limits the utility of using this information for the purposes of determining a mechanism of
action and there is direct evidence that VGCC activity is secondary to VGSC activation. Type II Pyrethroids have also demonstrated the ability to disrupt the ligand-mediated chloride current by antagonizing GABA A receptors in vitro and a potential interaction at the picrotoxin bindi ng site of the GABA A receptor-ionophore complex in vitro . While these pathways may be susceptible to perturbation by pyrethroids in vitro , evidence
from 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 nu mber of chemicals examined does not provide a sufficiently broad repres entation 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 focused on the e ffects of pyrethroids on the VGSC and membrane excitability. The follo wing 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, pyrethro ids are lipophilic compounds with relatively
short mammalian in vivo half-lives, being absorbed and elim inated relatively quickly. Focusing on plasma (as a measure of bioav ailability) and brain (as a primary target tissue) compartments, half-life estimates are described herein for deltamethrin, lambdacyhalothrin, and permethrin. Kim et al. (2008) administered deltameth rin 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-lives were 12.5 and 19.5 hours, respectively, similar to plasma. Anadón et al. (1996) estimat ed 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 halflives for several sections of the brain ranged from 18 to 33 hours, si milar to Kim et al. (2008) estimates. Estimates for lambda-cyhal othrin half-lives following 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 concentrations were achieved under 4 hours (deltamethrin <2; lambda-cyhalothrin 2.9; and permethrin 3. 5 hours) and maximum brain concentrationsPage 22 of 58 under 8 hours (deltamethrin 1-7.3; and lambda-cy halothrin 2.2-3.1). Finally, the oral bioavailability of deltamethrin in the Kim et al. (2008) and Anadón (1996) studies were similar at 18 and 14.4 %, respectively. Or al absorption rates fo r lambda-cyhalothrin (67.4%) and permethrin (60.7%) were greater.

Pyrethroids do not require metabolic activa tion to result in neurotoxicity. Indeed,
metabolic detoxification 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 detoxification and clearance. In vitro, mammalian clearance halflives of pyrethroids vary considerably by compound (Table 1). Generally, the pyrethrins and first-generation pyrethroids tested (e.g. a llethrin and resmethrin) have considerably shorter half-lives than the Type II pyrethroids (e.g. deltamethrin and β -cypermethrin), often differing by orders of magnitude (W olansky and Harrill 2008). Furthermore, the relative clearance rates are structure dependen t. Cis-isomers are subject to oxidation through numerous liver enzymes in mammals (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 car boxylesterases. Ther efore, 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 ci s-isomers are more persistent in vivo , they are inherently more toxic from a pharmacokinetic perspective.

Table 1. Intrinsic clearance rates of select ed pyrethroids from rat hepatic microsomes

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)

4.3.2. Toxicity syndromes in rodents

Pyrethroids have historically been classifi ed according to the acute intoxication syndromes they exhibit in vivo . Initial studies examined “high-dose” pyrethroid exposures, doses resulting in ne ar-death or death (Table 2).Page 23 of 58 Verscholyle and Aldridge (1980) investigated toxicity following rat IV tail injection of 36 pyrethroids. Signs of intoxication gen erally fell into one of two categories; the Tsyndrome characterized by aggressive sparring, increased sensitivity to external stimuli and fine whole body tremors, and the CS-sy ndrome characterized by initial pawing and burrowing, salivation, and course whole body tremors (choreoat hetosis). 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 were associated with the CS symptoms while those lacking the α -cyano generally resulted in T symptoms. The authors did no t make any absolute conclusions regarding the relationship between cy an- and non-cyano-containing pyre throids, however, linking
the association between structure and function was highly significant.

Lawrence and Casida (1982) administe red 29 pyrethroids to mice via intracerebral (ICV) injections. The pyreth roids were classified as Type I or Type II, equivalent to the T- and CS- syndromes de scribed by Verscholyle and Aldridge (1980). The ICV injections were used to bypass pr imary metabolism and correlate intoxication with pyrethroid concentration in neuronal ti ssue directly. Study findings indicate α

cyano containing pyrethroids (Type II) pr oduced choreoathetosis, convulsions and salivation, whereas compounds lacking the α -cyano group (Type I) produced tremors and convulsions, supporting the division of pyrethroids based on structure and intoxication syndromes. Furt hermore, Gammon et al. (1981) classified pyrethroids as either Type I or Type II based on their effects on in vivo and in vitro cercal sensory nerve recordings and on symptomology produced in dosed cockroaches. Type I pyrethroids produced repetitive firing in the cercal sensory nerve in vitro and trains of cercal sensory spikes accompanied by restle ssness, lack of coordination, hyperactivity, prostration, and paralysis in vivo . Type II pyrethroids did not induce repetitive firing in the cercal sensory nerve. The prolonged di scharges in the CNS were longer and of lower frequency than the Type I pyrethroids. In vivo symptomology also distinguished the Type II pyrethroids. Cockroaches bec ame ataxic and lack of coordination with periods of convulsions and intense hyperactivi ty, sometimes associated with salivation.
Periods of sustained contractions with the extension of metathoracic legs were particularly noticeable. F enpropathrin and an oxime O- α -cyanophenoxybenzyl ether were classified as Type I based on their acti on on the cercal sensory nerve but resulted in symptoms associated with Type II pyrethroid s. It is therefore cl ear from these early studies that pyrethroids toxicity can be sepa rated into two subgroups, Type I and Type II, based on structure and in vivo symptomology.

Table 2. Behavioral characterization of pyrethroid toxicity of pyrethroids included in the CMG from peer-reviewed literature

Pyrethroid Verschoyle 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 IIPage 24 of 58 Cypermethrin CS II II

Deltamethrin CS II II

As recently reviewed (Wolansky and Ha rrill 2008), more contemporary studies were designed to detect behavioral changes at a broader range of doses including 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, se matosensory response, social/sexual interactions and behavior to handling, and the functional observational battery (FOB). Although the number of pyreth roids 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 provides an overview of behavioral changes observ ed 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 neurotoxic ity guideline studies (OPPTS Guidelines 870.6200, 870.6200b). Motor activity measured as changes in ambulatory movement of rodents is currently the endpoint with the most extensive dose-response data for a large number of both Type I and II pyrethroids. Pyrethroids have repeatedly shown dosedependent decreases in motor activity in r odents 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; Croft on et al. 1995) has demonstrated decreased motor activity for eleven pyrethroids using the same test method (i.e., figure-eight mazes). In addition, decreas es in motor activity hav e been demonstrated in other testing devices after acute or short-term exposure, including pe rmethrin (Hoy et al. 2000), fenvalerate (Spinosa et al. 1999), cy halothrin (Righi and Palermo-Neto 2003), cyhalothrin (Hornychova et al. 1995; Ra tnasooriya et al. 2002), and cypermethrin (Hornychova et al. 1995). The acute motor depressant effect has also been observed in mice after oral exposure to fenvalerate (Mandhane an d Chopde 1997) and deltamethrin (Chanh et al. 1984). A limited nu mber of studies have obser ved increases or no change in motor activity (Mitchell et al. 1988; Hu sain and Husain 1996; Dayal et al. 2003). These reports of increased activity are difficu lt 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 ac tivity, on this apical neurobehavioral endpoint.Page 25 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) Hyperexcitability 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 effect s observed in studies of rodents following acute exposure to Type I and Type II pyreth roids. Different stages of the poisoning syndromes are noted at the far-l eft of the table. Note t hat the clinical landmarks produced under different routes of exposure are similar despit e 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 (1983), 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).Page 26 of 58 Data from studies on auditory star tle and FOB support the conclusion that pyrethroids 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 reflex pat h 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) . This response mechanism is sensitive to a variety of drugs and toxicants making the refl ex a useful animal model of sensorimotor reactivity (Lee et al. 1996). Auditory star tle 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., delta methrin, cypermethrin, cyfluthrin); and compounds that produce alterations in t he 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 common mechanism grouping, the auditory startle studies support the conclusion that Type I and Type II pyrethroid s exhibit different in
vivo toxicity outcomes.

The FOB consists of a series of b ehavioral measurement s from standardized tests such as posture, convulsions, tremors, salivation, mobility, r earing, and an array of sensory observations - touch, startle, and pain to name a few. In the case of pyrethroids, the FOB is designed to eval uate potential neurotoxic ity by examining behaviors such as open field, home cage, sensory observations, physiological, and
neuromuscular observations. McDani el and Moser (1993) described the neurobehavioral syndromes produced by high ac ute doses of permethrin (Type I) and cypermethrin (Type II) in rats using a FOB. Permethrin produced w hole body tremors, ataxia, greater resistance to removal fr om home cage, and increases in arousal, handling reactivity, and the response to touc h or experimenter approach. Cypermethrin caused salivation and urination, splayed limb s, choreoathetosis, increased foot splay, resistance to removal from home cage, dec reased arousal, and decreased response to touch or tail pinch. There were also a number of neuromuscula r effects that were affected by both chemicals including gr ip strength, neuromuscular tone, and gait changes. This work along with other data from FOB studies (Soderlund et al. 2002; Wolansky et al. 2007), and observations of clinic al 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 systematically evaluate and potentially quantify changes in behaviors in rats exposed to pyrethroids. Moreover, McDaniel and Moser (1993) demons trated that the FOB can be tailored to evaluate neurobehavioral syndromes specific to pyrethroids and can thus be used as a tool to compare the toxicity profile of different pyrethroids.Page 27 of 58 OPP has received acute neurotoxicity fo r several pyrethroids, including 17 guideline studies and several additional non-guide line studies. Most of these studies included 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 for registration review 3 , OPP has performed a preliminary review of the available regulatory acute neurotoxicity studies. Typically, neurotoxic signs are observ ed at or near peak time of effect in these studies. As a result, these studies are oft en the most sensitive st udy in the available
database and are therefore ar e 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 for 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 cons istency between i) laboratory techniques and equipment; ii) the quality, dur ation 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 concentra tion; vii) formulation; viii) and 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; Williamson et al. 1989; Lepeshkin et al. 1992; Villarini et al. 1995; Axelrad et al. 2002).

Special FOB studies designed to characterize effects cl assically associated with T and CS pyrethroid toxicity for 17 test chemicals (Nemec 2006; Herberth 2010). The
FOB consisted of 56 observations cove ring home cage, sensory, handling, physiological, open field, and neuromuscular param eters. The special FOB study offers two principle advantages lacki ng amongst the literatur e studies and regi strant submitted data; 1) specific neurological behaviors that address the issue of common toxic effect and 2) consistency among testing and observati onal parameters. Each chemical was tested with 2 or 3 doses. Minimally and m oderately effective doses were used to establish a spectrum of neurot oxicological effects.

3 http://www.epa.gov/oppsrrd1/registration_review/
4 Point of departure (PoD) — the value from a dose -response curve 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.Page 28 of 58 In an evaluation of pyrethroid toxicity pr ofiles for 12 pyrethroids, principal component analysis and dissimilarity indices were used to evaluate the clustering of four independent data sets around common dim ensions (Breckenridge and Holden 2007).
These datasets included:

1. 56 behavioral and physiological param eters from the sp ecial FOB study (Nemec 2006); 2. 8 electrophysiological parameters fr om patch clamp experiments conducted on five different sodium channels 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 func tion) were calculated for 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 fo r voltage-gated chloride channels in neuronal membranes from mouse neuroblastoma cells (Burr and Ray 2004).

This analysis identified four primary pattern s of variation in t he FOB data. Two of these patterns described behaviors associated with CS behaviors (l ower temperature, excessive salivation, impaired mobility ) and two described behaviors associated with the T syndrome (elevated tem perature, tremors, myoclonus). Non-cyano pyrethroids tended to have a high loading on the T fact ors whereas cyano-containing pyrethroids tended to have high loading on the CS factors. Pyrethroids with the strongest CS and T responses were deltamethrin/ λ -cyhalothrin and resmethrin/b ifenthrin, respectively. Fenpropathrin and pyrethrin had weak responses for either the C or TS characteristics. The consistency of the special FOB study wit h those of Verschoyle and Aldridge (1980) and Lawrence and Casida (1982) is remarkable . Notably, grouping s of Type I and Type II pyrethroids based on symptoms of in vivo toxicity are similar in studies conducted four decades apart.

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 pyrethroid s (Power and Sudakin 2007; Walters et al. 2009). The clinical manifestations of pyre throid exposure have been recently reviewed by Bradberry et al. (2005). De rmal exposure can result in transient paraesthesias with many pyrethroids. Inhalation exposure can re sult in upper airway irritation. Ingestion can result in systemic intoxication that includes nausea, vomiting, abdominal pain, increased secretions, and in rare, severe cases, coma and seizures (Power and Sudakin 2007).

4.4 Data in mixtures of pyrethroidsPage 29 of 58 EPA’s default assumption when evaluating mu lti-chemical risk is to assume dose additivity. This assumption is thought to be particularly appropriate when evaluating
chemicals which share a common mechanism of action. Chemicals which share a mechanism of toxicity will share a common mo lecular target. As such mixture studies which support the dose additivity hypothes is also indirectly support the common mechanism findings since dose additivity su ggests that the chemicals of different potencies act as dilutions of each other.

There has been limited research conducted on the potential effects of exposure to pyrethroid mixtures. Two in vitro studies have shown that init ial exposure of sodium channels to fenvalerate (Song and Narahas hi 1996) and deltamethrin (Tabarean and Narahashi 1998; Motomura and Narahas hi 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 modifica tions were observed. Song et al. (1996) concluded that tetramethrin may displace fenvalerate or in teract allosterically from sodium channel protein binding sites in an ex vivo rat dorsal root ganglion preparation. While these reports suggest that pyrethroids do not ac t in an effect-additive manner, there are a number of possible confounds in making definitive conclusions from these reports. First, the electrophysiological work (Ray and Fr y 2006) used dose levels that exceeded
known LD 50 dosages. In addition, these previous reports lacked either by study design or statistical approach, the abilit y to test the hypothesis of additivity. The use of rigorous
statistical models is critical for testi ng hypotheses of effect- or dose-addition and determining whether antagonism or syner gism exists (Feron and Groten 2002; Hertzberg and Teuschler 2002; Gennings 2004; LeBlanc and Olmstead 2004; Teuschler 2007). Overall, these limited data suggest there may be complicated outcomes from in
vitro mixtures at the molecu lar or cellular level.

More recently, Cao et al. (2011a) designed an in vitro study to specifically address potential additivity of pyrethroids. Pyrethroid-induced increases in sodium influx were determined in prim ary cultures of cerebrocorti cal neurons using the sodiumsensitive dye sodium-binding benzofur an isophthalate (SBFI) . Concentrationdependent responses were determined for 11 pyrethroids, including Type I and Type II pyrethroids. Depsite differences in indivi dual compound potencies, the mixture of all 11 pyrethroids were additive when an appropr iate statistical model was used.

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; Wolanksy et al. 2009). With a mixture composed of individual
chemical at doses 30% lower that the threshold for neurobehavioral effects, motor activity was accurately predicted by a singl e-chemical dose additivity model (Wolanksy et al. 2009). While the data of Wolansky et al. (2009) de monstrate dose-addition of low dose effects for both Type I and Type II pyrethroids, these data need to be interpreted with caution. Motor activity is an apical measure of the disrupt ion of nervous systemPage 30 of 58 function (Reiter and McPhail 1979) that is de creased by a wide variety of pesticides. Thus the specificity for a causative mode-of -action based linking of effects on VGSCs to apical measures of neural function like mo tor activity are extremely difficult to
demonstrate. Because a mechanistic underst anding is not available for pyrethroid induced decreases in motor activity, thes e data on a non-specific endpoint have limited utility in supporting a dose-additivity assump tion for a CMG as established per the requirements of the FQPA.

5.0 Common Mechanism Grouping under the FQPA

5.1 Weight of the Evidence

The above sections provide a survey of key studies which evaluate 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, disrupti ng neuronal firing patterns and ultimately resulting in the signs of pyrethroid toxicity . Specifically, evidence of a common toxicity pathway and common toxic effe cts for pyrethroids can be f ound from studies evaluating effects on 1) the sodium channel from both mammalian and non-mammalian in 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 modifi cation of VGSC, alterations in membrane excitability, and clinical signs and symptom s are completely understood, particularly in mammals. Data in non-mammalian species provide su pport for the mechanism where mammalian data are lacking. Moreover, there is a strong correlation between the pyrethroid structure, kinetic modulati on 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 current s of approximately 20
ms and are associated with clinical effects defined as the T syndrome. On the other hand, pyrethroids containing an α -cyano moiety delay deactiv ation 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.Page 31 of 58 Based on this review, OPP has determined that the naturally occurring pyrethrins and synthetic pyrethroids form a CMG based on 1) shared structural characteristics; and 2) shared ability to interact with volt age-gated sodium channels (VGSC), resulting in disruption of membrane excitability in the nervous system, ultimately leading to neurotoxicity characterized by two different to xicity syndromes. The 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 alte ration of action potentials, and ultimately resolving as clinical neurotoxicity c haracteristic of pyrethroid poisoning.

Figure 6. Common toxic pathway of the pyrethroids

Although there is an extensiv e 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

neurons Changes in VGSC kinetics produce
alterations in neuronal excitability. It has been
estimated that
modification of <1% of
VGSC alters neuronal excitability Changes in neuronal excitability underlie the clinical signs of pyrethroid toxicityPage 32 of 58 a) Na channel structural heterogeneity among tissue and across life stages with respect to alpha- and beta-subunits.

Another important issue when evaluatin g 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 sensitivities. This issue is applies to VG SC 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 stage heterogeneity in sodium channel expression (Plummer and Meisler 1999; Goldin 2001). Mammalian neurons typically express multiple isoforms of both α and β subunits, making it difficult to determine the composition of subunits compri sing sodium channel currents in native neurons. Specific combinations of VGSC subunits can be expressed exogenously in systems such as unfertilized frog oocytes or non-neuronal huma n embryonic kidney (HEK) cells. With respect to the current designation of t he 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 in vitro systems have also shown differences in pyrethroid sensitivity of various sodium channel isoforms that may influence di fferences in pyrethroid sensitivity based on tissue, region (Choi and Soderlund 2006) , life stage (Meacham et al. 2008) or species, including differences between r odents and humans (Vais et al. 2000; Tan and Soderlund 2009).

The pyrethroid sensitivity of all po ssible combinations of mammalian VGSC subunits has 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, mamma lian neurons is confounded by at least two significant uncertainties; 1) exogenous expre ssion of VGSC subunits for these studies requires the use of non-mammalian and/or non- neuronal cell types, and differences in post-translational modification of subunits, their assembly and insertion into the membrane, or modification by other ce llular processes (e.g. phosphorylation, glycosylation, etc) may contri bute to differences in sensit ivity of the channels to pyrethroid modification, and 2) the subuni t composition of VGSC when recording
sodium currents in mammalian neurons is unknown and difficult to determine due to the expression of multiple VGSC su bunits 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 invest igation support the hypothesis that effects on
VGSC are an initial and common key event in pyrethroid 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 understanding is not necessary for purposes of designating a CMG. Instead, fo r purposes of defining the CMG, the “major steps leading to a toxi c 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 mayPage 33 of 58 influence region, tissue or life stage differences in sensitivity, or may influence the types of toxic signs, the common key event in all cases, and the basis for the common mechanism grouping, is disruption of VGSC function.

b) Uncertainty associated with a lack of an in vivo biomarker for pyrethroid-VGSC
interaction.

In the cumulative risk assessment s for the OPs and NMCs, the common mechanism groupings were based on the abilit y 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 inhibition provides a scientifically robust and health protective endpoint for use as the common mechani sm endpoint in the cumulative risk assessment. Such a readily quantifiable in vivo biomarker for pyrethroid-modified
VGSCs is not available fo r the pyrethroids.

There is a large body of evidence on the in vitro effects of pyrethroids on sodium channels. Similarly there are multiple studies performed in rodents evaluating the neurobehavioral changes associat ed 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 di fferent levels of biological organization: channel, cells, and whole animal. The older literature and newer studies support common findings, namely that pyrethroids s hare the ability to interact with VGSC leading to disruptions of me mbrane excitability, and that py rethroids share the ability to
cause neurobehavioral s equele in rodents.

The relationship between pyrethroid-induc ed alterations in VGSC function and disrupted membrane excitabilit y (membrane depolarization and/or changes in firing of action potentials) is well established and has been demonstrated in a number of species
in vitro . Across multiple invertebrate and vert ebrate species, including mammals, pyrethroid-induced modification of VGSC func tion results in alterations in neuronal membrane excitability that ar e readily measured using n europhysiological 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 c annot provide a quantitative link between
alterations in sodium channels in vivo , and changes in the function of the nervous system as evidenced by neurobehavioral effe cts 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

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.Page 34 of 58 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 substantial. 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.Page 35 of 58 5.2 Summary

In summary, although some uncertainties exist, there is a convincing amount of data from multiple lines of evidence and fr om studies spanning several decades which support a common mechanism grouping for t he naturally occurring pyrethrins and synthetic pyrethroids under the FQPA. Th is conclusion 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 sodi um channel kinetics, leading to
alterations in membrane exci tability and firing potentials.

 Neurotoxic Effects - Pyrethroid toxicity is manifested through neurological syndromes described as either T (fi ne tremors), CS (choreoathetosis and salivation), or some combination ther eof, depending on the structure. Open literature supports a correlation between the modification in sodium channel kinetics and the resulting syndrome.

6.0 Potential to Sub-Group the Pyre throids by Type I & Type II

As described in more det ail above there are many in vitro and in vivo studies which suggest that pyrethroid toxicity is m anifested by two profiles. The following text will not repeat the above revi ew. Instead, the following focuses on several studies conducted by the PWG or EPA’s ORD (Tabl e 6). 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 levels of biological complexity (s odium channel, nerve cell, whole animal) and provide a remarkably similar sepa ration of the 11 chemicals.Page 36 of 58Figure 7. Differences in Type I and Type II pyrethroids which support forming two subgroups within the CMG

6.1. Pyrethroid 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 pyreth roid 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 channel and produced characteristic changes in channel function, including alterations in activation (channel opening) and inactivation (channel closing) ra tes as well as induction of tail currents upon repolarization of the membrane. Based on t he alterations in the activation kinetics,
as well as the time constant s for tail current decay (tau 1 ,tau 2 ; Table 6), the 11 pyrethroid compounds could be clearly separated into 2 gr oups. 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 exponentia l. The second group is comprised of the Type II compounds that produced large increas es in the channel activation rate (tau act > 15) and tail current decay described by two exponential (one fast and one slow) components. Even considering the uncertain ties detailed above regarding exogenous expression systems such as oocytes, these resu lts demonstrate at the most basic level, two distinct groups of co mpounds 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 vivoPage 37 of 58 6.2. Micro electrode arrays (MEA) Studies

Newer technologies have allowed researc hers to examine how pyrethroids affect
mammalian neural networks in vitro . Primary neuronal cell cu ltures can be grown on microelectrode arrays (MEAs), Figure 8, allowing the measur ement of spontaneous electrophysiological activity. While volt age-clamp techniques al low measurement of
channel function in an indi vidual neuron, MEAs measure function of networks of neuronal cells, and are thus a “summed” meas ure of effects of compounds on all ion channel constituents (e.g. Na, Ca) in the ce lls and network. Thus, multiple VGSC as
well as VGCC isoforms contribute to the ov erall responses measured with MEAs. While this precludes direct examination of indivi dual 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 neurotransmi tter in the mammalian CNS and effects of pyrethroids on glutamatergic networks have been characteriz ed using MEA techniques. Effects of pyrethroids on glutamatergic networks using s pontaneously active cortical (Losa et al. 2009) and hippocampal (Shafer et al. 2008) cult ures have recently been examined. In hippocampal cultures, pharmacological approac hes indicated that pyrethroid-induced changes could be minimally explained via e ffects on Na channels. These data are consistent with voltage-clam p measurements of permethri n and deltamethrin effects on miniature excitatory post-synaptic curr ents (mEPSCs) in hippocampal neurons (Meyer and Shafer 2006). Thus, these data support t he 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 pyre throids (Losa et al. 2009) using primaryPage 38 of 58 cultures of cortical neurons . The values ranged from 25 to 1685 nM (Table 4); Type I and II pyrethroids resulted in higher and lower IC 50 values, respectively. Esfenvalerate and fenpropathrin resulted in intermediate values.

Table 4. IC 50 values for glutamate network inhibition

Pyrethroid Type-based on

structure MEA IC50s

Cyhalothrin, 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 Vivo Rodent Studies

As discussed in section 6.0, special FOB study designed to characterize effects classically associated with T and CS pyrethro id toxicity for 17 test chemicals (1 pyrethrin, 5 Type I and 6 Type II pyrethroid s)(Nemec 2006). The FOB consisted of 56 observations covering home cage, sensor y, handling, physiological, open field, and neuromuscular parameters. F our primary patterns of vari ation were observed in the
FOB data. Two of these patterns described behaviors associated with CS behaviors (lower temperature, excessive salivati on, impaired mobility) and two described behaviors associated with the T syndrome (eleva ted temperature, trem ors, myoclonus). Non-cyano pyrethroids tended to have a high loading on the T factors whereas cyanocontaining pyrethroids tended to have high loadi ng on the CS factors. Pyrethroids with the strongest CS and T responses were deltamethrin/ λ -cyhalothrin and resmethrin/bifenthrin, respectively. F enpropathrin and pyrethrin had weak responses
for either the C or TS characteristics.

Table 5. Behavioral characterization of pyrethroid toxicity by PWG

Pyrethroid WIL*
Allethrin; S-Bioallethrin T

Bifenthrin T Cyfluthrin CSPage 39 of 58 Cyhalothrin, lambda- CS Cypermethrin CS

Deltamethrin CS

Esfenvalerate MIXED Fenpropathrin T

Permethrin T

Resmethrin T

Tefluthrin T

6.4. Potential to Subgroup

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. Examination of the table indicates a clear pattern that the 11 pyrethroids separate into 2 groups based on structure, ion channel gating activity, and neurobehavioral effects, wit h two chemicals, esfenv alerate and fenpropathrin, representing mixed Type I/II.

In the event that refinements to the CRA are needed, there is the potential to subgroup the pyrethrins and pyrethroids 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. Th is conclusion is based on the following
characteristics:

 Structure – based on the absence or pres ence of an alpha-cyano moiety

 Sodium channel kinetics – Type I pyrethroids cause slight prolongations of the sodium current tails (20 ms), oft en resulting in long trains of action potentials. Type II pyrethroids significantly prolong sodium current tails (200ms to minutes) typically result ing in increased resting membrane potential and can induce acti on potential block.

 Toxicity syndromes – Type I and Type II pyrethroids generally elicit C and TS syndromes, respectively. Excepti ons, including esfenvalerate and fenpropathrin, elicit sympt oms that are a combinat ion of the T and CS syndromes.Page 40 of 58 Table 6. Summary of key data supporting the 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; SBioallethrin 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 activati on 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 primary neuronal cultures (Scollon et al. 2010)

e Neurotoxic syndrome expressed by rats following high doses of pyrethroids as defined by FOB observations (Weiner et al. 2009))

Attachment 1 provides the structures of the preliminary list of pyrethrins and synthetic pyrethroids included in the CMG. Attachment 1 also provides assignments (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 resu lts of the special FOB for pyrethrins. Five additional pyrethroids (cyphenothrin, im iprothrin, prallethrin, tetramethrin, and dphenothrin) were tested in a special FOB study similar to the study sponsored by the PWG (Herberth 2010) and have been categorized as Type I or Type II. For all remaining pyrethroids, OPP fi rst evaluated existing data from in vitro and/or in vivo studies for effects consistent with Type I or II effects. In the abs ence specific data to inform a particular sub-group, OPP used st ructure (i.e., presence/absence of an α

cyano group) as the determining factor fo r assigning individual pyrethroids to a subgroup.Page 41 of 58 7.0 References

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EPA’s classification and structure of pyre throids 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

Cyphenothrin II

Deltamethrin IIPage 56 of 58 Name Type I/II Structure

Fenpropathrin Mixed I/II

Fenvalerate (esfenvalerate) Mixed I/II

Flucythrinate II

Flumethrin II

Fluvalinate ( τ -fluvalinate) II

Imiprothrin I

Metofluthrin I

Permethrin IPage 57 of 58 Name Type I/II Structure

Prallethrin I

Resmethrin (bioresmethrin,

cismethrin) I

Phenothrin (dphenothrin, sumithrin) I

Tefluthrin I

Tetramethrin I

Tralomethrin II

Pyrethrins I

Cinerin I IPage 58 of 58 Name Type I/II Structure

Cinerin II I

Jasmolin I I

Jasmolin II I