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Chemical PC Code CAS No.

Allethrin 004001 584-79-2

Bioallethrin 004003 28057-48-9

S-Bioallethrin 004004 28434-00-6

D-Allethrin 004005 84030-86-4

Esbiothrin 004007 84030-86-4

Bifenthrin 128825 82657-04-3

Cyclethrin 004052 97-11-0

Cyfluthrin 128831 68359-37-5

Cyfluthrin, beta- 118831 68359-37-5

Cyhalothrin 128867 68085-85-8

Cyhalothin, lambda- 128897 91465-08-6

Cyhalothrin, gamma- 128807 76703-62-3

Cypermethrin 109702 52315-07-8

Cypermethrin, alpha- 209600 67375-30-8

Cypermethrin, beta- 118831 65731-84-2

Cypermethrin, zeta- 129064 52315-07-8

Cyphenothrin 129013 39515-40-7

Deltamethrin 097805 52918-63-5

Esfenvalerate 109303 66230-04-4

Fenvalerate 109302 51630-58-1

Fenfluthrin 109705 69409-94-5

Fenpropathrin 127901 39515-41-8

Imiprothrin 004006 72963-72-5

Metofluthrin 109709 240494-70-6

Permethrin 109701 52645-53-1

Prallethrin 128722 23031-36-9

Pyrethrins (not synthetic) 069001 8003-34-7

Resmethrin 097801 10453-86-8

Sumithrin (d-phenothrin) 069005 26002-80-2

Tau-fluvalinate 109302 102851-06-9

Tefluthrin 128912 79538-32-2

Tetramethrin 069003 7696-12-0

Tralomethrin 121501 66841-25-6
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Draft Science Policy Paper 

Proposed Common Mechanism Grouping for 

the 

Pyrethrins and Synthetic Pyrethroids 

May 19, 2009
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TABLE OF CONTENTS

1.0 Background ........................................................................................................... 5

2.0 Key Concepts ........................................................................................................ 6

3.0 Chemical Structure ............................................................................................... 7

3.1 Chirality of pyrethroids................................................................................. 9

3.2 Quantitative Structure Activity Relationships (QSAR)............................. 11

4.0 Toxicology of Pyrethroids.................................................................................. 12

4.1 Overview ...................................................................................................... 12

4.2 Voltage-Gated Sodium Channels (VGSC) ................................................. 13

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

4.2.2. Channel modulation by pyrethroids..................................................... 14

4.2.3. Relationship between pyrethroid-induced alterations in VGSC 

function & disrupted membrane excitability ....................................... 16

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

4.2.4.1. Voltage-clamp measurements......................................................... 16

4.2.4.2. Sodium channel cloning.................................................................. 17

4.2.5. Other molecular targets for pyrethroids.............................................. 18

4.3 In vivo profile............................................................................................... 23

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

4.3.2. Toxicity syndromes in rodents............................................................. 25

4.3.3. Information in humans .......................................................................... 30

4.4 Data in mixtures of pyrethroids ................................................................. 31

5.0 Proposed Common Mechanism Grouping under the FQPA ........................... 32

5.1 Weight of the Evidence............................................................................... 32

5.2 Summary...................................................................................................... 37

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

6.1. Pyrethroid Effects on Cloned VGSC Nav1.8.............................................. 38

6.2. Micro electrode arrays (MEA) Studies....................................................... 39

6.3. In Vivo Rodent Studies ............................................................................... 40

6.4. Proposed Subgrouping .............................................................................. 41

7.0 References........................................................................................................... 43

Attachment 1.0.Classification (as proposed by the Agency) and Structure of 

pyrethroids registered for use with the US EPA............................ 56
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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 Programs (OPP), a cumulative risk assessment begins with the identification of a group 

of chemicals referred to as a common mechanism group (CMG), that induce a common toxic 

effect by a common mechanism of toxicity. To date, OPP has conducted four cumulative risk 

assessments on the organophosphates (OPs), N-methyl carbamates (NMCs), chloracetanilides, 

and triazines. Pyrethroids are a class of synthetic insecticides which are structurally based on the 

pyrethrins, botanical insecticides extracted from Chrysanthemum cinerariaefolium (Soderlund et 

al. 2002). Pyrethroid exposure can occur from food, water, or non-occupational settings. Due, 

in large part, to changes in usage trends resulting from risk mitigation, residential use of OPs and 

NMCs has decreased. Consequently, potential exposure of the general public to pyrethroid 

pesticides has increased over the past decade. 

There is an extensive published literature concerning the toxicity of pyrethroids and potential 

modes of action of these compounds. In addition, in the last few years, EPA’s Office of Research 

and Development (ORD) and the Pyrethroid Working Group1 (PWG) have conducted 

independent, large-scale research efforts to evaluate aspects of pyrethroid exposure, 

pharmacokinetics (PK), mode/mechanism of action, and toxicity profiles. These research efforts 

have added to the scientific literature and also led to a meeting of The Federal Insecticide, 

Fungicide, and Rodenticide Act Science Advisory Panel (FIFRA SAP) in August, 2007, on a 

proposed approach to developing physiologically-based pharmacokinetic (PBPK) models for 

multiple pyrethroids (USEPA, 2007). The research by both EPA and the PWG was conducted, 

in large part, in anticipation of a future cumulative risk assessment on the pyrethroids. The 

purpose of this draft science policy document is to describe this scientific evidence which 

supports a proposal to designate the pyrethroids as a CMG under the FQPA. OPP is soliciting 

comments from the FIFRA SAP and the public on this document in June, 2009. 

This document follows OPP’s guidance on forming CMGs (2002) and also considers the 

concepts contained in the Mode of Action and Human Relevance Frameworks developed by 

EPA, WHO’s International Programme for Chemical Safety and the International Life Sciences 

Institute (USEPA, 1999; Sonich-Mullin et al. 2001; Meek et al. 2003; Seed et al. 2005; Boobis et 

al. 2006). Based on the current state of the science, OPP is proposing to designate the naturally 

occurring pyrethrins and synthetic pyrethroids form a CMG based on 1) shared structural 

characteristics; and 2) shared ability to interact with voltage-gated sodium channels (VGSC), 

resulting in disruption of membrane excitability in the nervous system, and ultimately 

neurotoxicity characterized by two different toxicity syndromes. As described in more detail 

below, OPP is further proposing to subgroup the pyrethroid CMG into two subgroups 

representing Type I and II pyrethroids. This subgrouping is consistent with historical 

categorization of pyrethroids and is supported by in vitro and in vivo studies from both older 

studies and new research. Pyrethroids considered for the CMG are listed in Appendix 1. 

 1 The Pyrethroid Working Group is a group of pesticide companies who own technical registrations for 

pyrethrins and some synthetic pyrethroids.
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The designation of Type I and Type II pyrethroids is based, in large part, upon 

differences in structure; the α-cyano moiety is absent in Type I pyrethroids and present in Type 

II. Type I members of the proposed CMG include allethrin, bifenthrin, permethrin, resmethrin, 

tefluthrin and tetramethrin, Type II members of the proposed CMG include cyfluthrin, 

cyhalothrin, cypermethrin, and deltamethrin. The absence/presence of the α-cyano group is 

correlated with length of time the sodium channel is inactivated (-CN=shorter; +CN=longer) 

which in turn corresponds with the two distinct toxicity syndromes (-CN=T syndrome; +CN=CS 

syndrome). The nature of these effects is discussed in more detail in section 4.0. Two 

pyrethroids, esfenvalerate and fenpropathrin, though structurally similar to Type II pyrethroids, 

are considered mixed Type I/II because they demonstrate properties of both groups during In 

vitro and in vivo evaluations. The Agency’s plans for future classification of the additional 

pyrethroids shown in Attachment 1 is described in Section 6.4 below. 

This document also describes some uncertainties considered including i) other endogenous 

channels (e.g., calcium and chloride) which have been proposed by some (Soderlund et al. 2002; 

Ray and Fry 2006; Clark and Symington 2007) as potential other target sites for pyrethroids; ii); 

sodium channel structural heterogeneity among tissue and across life stages with respect to 

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

pyrethroid-VGSC interaction. 

2.0 Key Concepts 

OPP’s has guidance documents for designating CMGs (USEPA 1999) and for developing 

cumulative risk assessments (USEPA 2002). These documents provide definitions for some key 

concepts and provide the guiding principles for the analysis contained in this proposal. OPP’s 

guidance documents and approaches to grouping chemicals for common mechanism and to 

developing cumulative risk assessments have been through extensive peer review with many 

reviews by the FIFRA SAP. Some key concepts included in the current analysis include:

• Mechanism of toxicity is defined as the major steps leading to a toxic effect following 

interaction of a pesticide with biological targets. All steps leading to an effect do not 

need to be specifically understood. Rather, it is the identification of the crucial events 

following chemical interaction that are required in order to describe a mechanism of 

toxicity. Generally, the more that is understood about the various steps in the pathway 

leading to an adverse effect, the more confident 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 receptor-related molecular response; in vivo it also leads to the molecular 

response and causes a number of intervening biological and morphological steps that 

result in an adverse effect. 

• A pesticide and another substance that are known to cause the same toxic effect in or at 

the same anatomical or physiological site or locus (e.g., same organ or tissue) are said to 

cause a common toxic effect. 

• A common mechanism of toxicity pertains to two or more pesticide chemicals or other 

substances that cause a common toxic effect to human health by the same, or essentially 
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the same, sequence of major biochemical events. Hence, the underlying basis of the 

toxicity is the 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 are toxic by a common mechanism from all pathways. Thus, 

cumulative risk assessments include multiple chemicals and multiple pathways (e.g., 

food, drinking water, consumer uses). 

• A common mechanism endpoint(s) is/are those common toxic effect(s) which are 

pertinent and sensitive endpoints associated with the common mechanism which will 

provide a scientifically sound basis for determining relative potency of chemicals in a 

cumulative risk assessment.

3.0 Chemical Structure 

The basic pyrethroid structure consists of an acid and alcohol moiety (Fig. 1). 

Pyrethroids, the synthetic derivatives of the pyrethrins, have evolved structurally over the past 

several decades. However, the basic components of pyrethrins, a chrysanthamic acid linked to 

an aromatic alcohol through an ester linkage have been conserved (Fig. 1). Structural 

modifications such as the addition of halogens to the chrysanthamic acid and aromatic alcohol 

moieties and the addition of the α-cyano group have increased photostability, insecticidal 

potency, and in some incidences, stereoisomerism of the pyrethroids. 
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 Pyrethrin I (Type I)

O

O

H

O

unsaturated side-chain cyclopropyl ring planar spacer methylene or equivalent bridge group

non-coplanar center of unsaturation

acid moiety alcohol moiety

central ester bond

* *

 Resmethrin (Type II) 

O

O

O

* *

 Cypermethrin (Type II)

 

O

O

Cl

Cl

N

O

 

* * *

* = chiral centers

Figure 1. Representative structures for pyrethrins (Pyrethrin I), Type I (resmethrin) and Type II 

(cypermethrin) pyrethroids.
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3.1 Chirality of pyrethroids

Pyrethroids can possess one to three chiral centers, resulting in two to eight enantiomers. 

For example, in the Type I pyrethroids, the presence of two chiral carbons, C1 and C2, in the in 

the cyclopropane ring of the chysthanthamic acid moiety produces 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 addition 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 example, the orientation of the C1 and C3 constituents in the cyclopropane ring 

of the chrysanthamic acid moiety determine the cis/trans structural confirmation, which can in 

turn impact potential toxicity and metabolism. Furthermore, the 1R configurations are 

insecticidal, whereas the enantiomeric 1S compounds, though physically identical, are without 

insecticidal activity (Elliot et al. 1974). Effects of pyrethroids on VGSC function have also been 

demonstrated to be stereo-isomer dependent (Lund and Narahashi 1982; Narahashi 1982). As 

will be discussed in Section 4.0, the insecticidal efficacy correlates with the ability of isomers to 

modify sodium channel kinetics. Furthermore, isomerism influences the metabolism of these 

compounds (See PK section 4.3.1 for more details). 

Ideally, there would be sufficient data to distinguish the pharmacodynamics (PD), PK, 

and environmental exposure of the active stereoisomer(s). For some, such as bioresmethrin, 

deltamethrin, and esfenvalerate, the registered products are enrichments of the more 

insecticidally active isomers. As such, for these, there is comparatively little uncertainty with 

respect to assessing the hazard and exposure. For permethrin, residue data and some limited 

toxicity information are reported as cis and trans. However, toxicity and exposure information 

available for the majority of pyrethroids are for the isomeric mixture. Specifically animal 

toxicity studies submitted for pesticide registration are performed with the isomeric mixture. In 

addition, residue data used to evaluate food, water, and residential exposure are typically 

reported as isomeric mixtures. Thus, although the Agency acknowledges the stereoisomerism as 

a consideration for pyrethroids, the ability of the Agency to quantitatively account for these in 

risk assessment is limited. Instead of assessing the active stereoisomer(s), the Agency typically 

assesses the risk to pyrethroids by evaluating the risk to the isomeric mixture. The resources 

required to collect such toxicity and exposure data on the isomers would be substantial. 

Although there is some uncertainty associated with evaluating the mixture instead of the active 

stereoisomer, the lack of such data does not prevent the Agency from developing risk 

assessments which are both health protective and scientifically sound2

. 

 2 With respect to the common mechanism grouping, pending the outcome of the SAP review and comments from 

the public, the Agency expects to identify the registered technical products (i.e., the isomeric mixture) as a potential 

member of the CMG, not the active stereoisomer(s).
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Figure 2. Stereochemistry of the pyrethroid acid moiety
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3.2 Quantitative Structure Activity Relationships (QSAR)

As stated above, one of the common features of pyrethroids is their common structural 

characteristics. With respect to similarity in chemical structure, OPP’s CMG guidance (2002)

states that: 

“Use of structural similarity as a starting point for grouping chemicals relies on the assumption 

that substances that are structurally analogous could contain a common toxophore3 (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, identification of a toxophore is important 

for evaluating a common PD interaction 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 the entire molecule at the site of action is required for 

interaction (Byberg et al. 1987; Soderlund et al. 2000). However, Ford et al. (2002) has 

performed a three-dimensional quantitative structure activity relationship (QSAR) analysis of 

pyrethroids. Conformational sampling of active pyrethroids using molecular dynamics and 

cluster analysis, taking into account the multitude of bond angles possible, revealed a common 

conformation in 39 of the 41 structures examined (Fig. 2). Type I and Type II pyrethroids both 

kill insects. Type I pyrethroids possess an effective ‘knockdown’ ability, debilitating insects 

within minutes. However, they may take days to kill, allowing the opportunity for recovery. 

Conversely, 

Type II pyrethroids lack the knockdown ability of Type I pyrethroids but are generally 

more potent at killing. The ‘knockdown’ and kill responses are also differentiated by their 

temperature dependence, negative for kill and positive for ‘knockdown’. Ford et al (2002)

related the putative toxophore to the killing potential, inherent to both Types I and II. The 

authors postulate that restriction about the ether linkage by an α-cyano group in Type II 

pyrethroids precludes the knockdown capacity associated with Type I pyrethroids. The 

unrestricted movement about the ether linkage requisite for ‘knockdown’ is not the most stable 

conformation, as reflected by its positive temperature dependence. 

 3 The CMG guidance defines the term “toxophore” with respect to toxic substances, as is akin to the term 

“pharmacophore” with respect to drug substances: the pharmacophore is that structural moiety of a drug 

substance or substances which imparts a desired pharmacological property. 
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Figure 3. Conformational structure of putative pyrethroid toxophore

As detailed in the following section, vertebrate VGSC structures are significantly more 

complex than insect channels. However, there is homology in structure and function between 

insect and mammalian sodium channels. Insect and mammalian VGSC are both composed of 

pore-forming α subunits with modifying proteins (mammalian β units and insect tipE proteins) 

and Type I and Type II pyrethroids cause similar modifications in channel kinetics in both 

species (Type I results in repetitive firing and Type II pyrethroids result in depolarizationdependent block of the action potential). It is therefore reasonable to assume the toxicophore 

identified in insects would be similar to that in mammals. As such, the Agency further assumes 

that a shared toxophore and chemical structure of pyrethroids provides part of the basis for 

grouping the pyrethroids as a CMG.

4.0 Toxicology of Pyrethroids

4.1 Overview

It is well established that pyrethroids modify the kinetics of the VGSCs in insect and 

mammalian neurons. The Agency has identified multiple lines of evidence which support the 

shared ability of the synthetic pyrethroids and pyrethrins to interact with the Na channels leading 

to depolarization of membranes in the nervous system, and ultimately neurotoxicity 

characterized by two different toxicity syndromes. This evidence is summarized below starting 

with the VGSC followed by mechanistic information from voltage clamp/patch clamp studies, 

use of neuronal networks (e.g., micro electrode arrays (MEAs)), in vivo toxicity studies, and PK. 

This paper does not provide an exhaustive review of this body of work but highlights and 

summarizes critical data relevant to common mechanism grouping findings. For extensive 

reviews, see Soderlund and Bloomquist (1989), Vijverberg and van den Berken (1990), 

Narahashi (1992), Clark (1995), Soderlund (1995), Bloomquist (1996), Narahashi (1996), Shafer 

et al (2005), Wolansky and Harrill (2008), Nemec(2006). 
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4.2 Voltage-Gated Sodium Channels (VGSC)

4.2.1. Protein structure of the VGSC

Briefly, the voltage-gated sodium channel α subunits consists of four homologous 

domains, each domain is comprised of six transmembrane helical protein segments forming the 

ion channel pore (Fig. 4). Expression of the α subunit alone is minimally sufficient to form a 

functioning channel. A voltage-sensing element located in the S4 transmembrane segment 

detects changes in membrane potential resulting in a conformation change of the channel leading 

to opening and rapid closing of the voltage dependent gating mechanism. In vertebrates, smaller 

auxiliary β subunits modulate α subunit gating, as well as insertion and distribution of the 

channel complex into the membrane (Isom et al. 1992; Isom et al. 1995; Isom 2001; Smith and 

Soderlund 2001). Insects have a single isoform of the α-subunit, expressed exclusively in the 

nervous system (Hong and Ganetzky 1994) and insects lack β subunits, per se. However, tipE 

protein has been suggested as a homologous, though structurally unrelated, protein to the 

vertebrate β subunit (Warmke et al. 1997). 

Figure 4: Schematic of the Transmembrane Voltage-Gated Sodium and Calcium Channel

In contrast to the simplicity of the insect sodium channel, mammalian VGSC are much 

more complex, and this complexity creates uncertainty in correlating effects on specific VGSC 

isoforms in vitro to in vivo symptomology caused by pyrethroid toxicity in mammals. There are 

at least 10 distinct genes that encode sodium channel α subunit proteins (Nav1.1-Nav1.9; Nav1.X) 

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

β4) (Yu et al. 2003) have been identified to date. The heterogeneity of the sodium channel can 

also be enhanced by the alternative mRNA splicing of some isoforms (Sarao et al. 1991; Schaller 

et al. 1992; Gustafson et al. 1993). Finally, the functional and pharmacological diversity of 

sodium channel expression varies by tissue and stage of development (reviewed in Meacham et 
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al (2008) and Mandel (1992)). The implications of this diversity on uncertainty related to actions 

of pyrethroids will be discussed further in the following sections.

4.2.2. Channel modulation by pyrethroids

The effects of pyrethroid exposure on neuronal tissue (both insects and mammals) have 

been extensively reviewed (Soderlund and Bloomquist 1989; Vijverberg and Van den Bercken 

1990; Narahashi 1992; Bloomquist 1993a; Clark 1995; Soderlund 1995; Bloomquist 1996; 

Narahashi 1996). 

Figure 5 depicts generalized pyrethroid effects on individual channels, whole-cell sodium 

currents, and action potentials (extracted from Shafer et al (2005)). Depolarization opens 

VGSCs (top left) allowing sodium to enter the cell. To limit sodium entry and depolarization 

length, VGSCs inactivate and must return to a “resting” state before reopening. Pyrethroids 

inhibit the function of two different “gates” that control sodium flux through VGSCs (top right), 

delaying inactivation (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 depolarization ends (bottom 

right, Sodium Current), resulting in a “tail” current (the notch at the end of example currents). If 

membrane voltage is examined, depolarization under normal circumstances generates a single 

action potential (bottom left). VGSCs modified by type I compounds (bottom right, Action 

Potential) depolarize the cell membrane above the threshold for action potential generation, 

resulting in a series of action potentials (repetitive firing). Type II compounds cause greater 

membrane depolarization, diminishing the sodium electrochemical gradient and subsequent 

action potential amplitude. Eventually, membrane potential becomes depolarized above the 

threshold for action potential generation (depolarization-dependent block) (Shafer et al. 2005). 

Modification of the Na channels in mammals has been shown to vary by Type I and II 

pyrethroids (Bottom right, Fig. 5). Natural pyrethrins and Type I synthetic pyrethroids are

typically characterized by slight prolongations for the sodium current tails (20 ms), resulting in

trains of action potentials accompanied with little or no effect on resting membrane potential. In 

contrast, pyrethroids containing the α-cyano moiety typically do not induce repetitive firing. 

Instead, Type II pyrethroids produce long-lasting sodium current tails (>>200ms) that ultimately

result in action potential block due to depolarization of resting membrane potential above the 

threshold for action potential generation. It has been estimated that as few as 1% of the sodium 

channels need be modified to render a cell hyperexcitable and induce repetitive firing (Song and 

Narahashi 1996; Motomura and Narahashi 2001).
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Figure 5. Effects of pyrethroids on Na channels, currents, and action potentials (From Shafer et al., 2005)
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4.2.3. Relationship between pyrethroid-induced alterations in VGSC 

function & disrupted membrane excitability 

The relationship between pyrethroid-induced alterations in VGSC function and disrupted 

membrane excitability (membrane depolarization and/or changes in firing of action potentials) is 

well established and has been demonstrated in a number of species in vitro. Using crayfish giant 

axon, Salgado and co-workers (1989) demonstrated temperature-dependent actions of fenvalerate 

on VGSC function as well as membrane excitability. In addition, voltage-dependent potassium 

currents were not altered by fenvalerate. Lund and Narahashi (1981) demonstrated that 

cyphenothrin disrupts sodium channel function, depolarized the membrane, and upon stimulation 

of the nerve, induces repetitive firing that is blocked by 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 

and depolarization and/or induce repetitive in peripheral nerves from (Vijverberg and Bercken 

1979), reviewed in Vijverberg and van den Berken (1982). Similar findings have also been 

reported in mammalian neurons in vitro, wherein pyrethroids modify VGSC function and 

produce changes in membrane excitability in dorsal 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. Following deltamethrin administration the typical period of 

supranormal nerve excitability of compound action potentials in rat tail nerve was increased from 

~30 msec to ~400 msec (Parkin and Le Quesne 1982). Similar changes in CAP were reported 

following administration of fenvalerate or allethrin (Nozaki et al. 1995). When dorsal root 

potentials were recorded from urethane anesthetized rats, cismethrin increased the amplitude of 

the potential by up to 142% (Smith 1980). Finally EEG measurements demonstrated that 

administration of cypermethrin produced bursts of epileptic activity following the first 

administration, and on subsequent days of administration, these were accompanied by shaking, 

myoclonus, and tonic-clonic seizures (Condés-Lara et al. 1999). Thus, data from in vitro studies 

indicate that pyrethroid modification of VGSC leads to changes in membrane excitability. In 

vivo, changes in neurophysiological measures occur that are consistent with alterations in action 

potentials, and correlate with behavioral changes.

4.2.4. Potential differences in tissue sensitivity to pyrethroids

4.2.4.1. Voltage-clamp measurements 

The earliest evidence of the ability of pyrethroids to disrupt sodium channel kinetics was 

achieved through voltage clamp techniques. Voltage clamp techniques allow measurement of 

voltage-gated membrane channel function by “clamping” the voltage difference across the 

membrane by electronic feedback (current injection), thus allowing controlled changes in 

membrane voltage. Changes in current injected to hold voltage at a constant level reflect changes 

in conductances across the membrane as a result of ion channel openings and closings. Thus, the 

membrane can be clamped to different voltages and resulting currents measured. Using solutions 

of different ionic composition as well as pharmacological compounds to block specific channels, 

it is possible to examine the behavior of different kinds of ion channels through which these 
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currents pass (Eckert et al. 1988). However, it should be pointed out that pharmacological 

antagonists to specific VGSC subunit combinations are not available; this precludes 

identification of the exact composition of VGSC subunits mediating pyrethroid sensitive currents 

in native mammalian neurons.

A number of voltage–clamp studies have demonstrated the selective ability of pyrethroids 

to perturb sodium channels based on chemical structure. Dosing with tetramethrin (Type I) 

caused the mean open time of Na channels to increase resulting in repetitive firing (Chinn and 

Narahashi 1986; Holloway et al. 1989). Deltamethrin and fenvalerate (Yu et al. 2003) increased 

mean open time 200 fold greater than noted for tetramethrin. These results are characteristic of 

the distinction commonly noted between Type I and Type II pyrethroids. 

Since individual neurons are known to contain multiple α and β subunits, it is not 

possible to determine the effect of the pyrethroids on individual channel subunit combinations 

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

sensitivity to pyrethroids, indicating channel isoform populations may differ with tissue type. 

Dorsal root ganglion (DRG) cells contain tetrodotoxin-sensitive (TTX-S) and tetrodotoxinresistant (TTX-R) sodium channels. TTX-R channels are susceptible to perturbation by both 

Type I and Type II pyrethroids while TTX-S channels are relatively less sensitive (Ginsburg and 

Narahashi 1993; Tatebayashi and Narahashi 1994; Song and Narahashi 1996; Narahashi et al. 

1998). These examples demonstrate the differential activity of Type I and Type II pyrethroids in 

multiple neuronal tissues. These differences in TTX-S and TTX-R VGSC sensitivity to 

pyrethroids are consistent with the sensitivity of the Nav1.8 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 specific manner. Further, there appear to be differences in pyrethroid 

effects on mammalian neurons that are dependent on the composition of the channel under study. 

Advances in molecular techniques have allowed researchers to express specific combinations of 

VGSC subunits in exogenous expression systems such as Chinese hamster ovary (CHO) cells 

and unfertilized Xenopus laevis oocytes. The isolation and expression of specific channel 

isoforms has allowed researchers to examine the direct impact of pyrethroids on these channels. 

For example, the expression of pore-forming α subunits Nav1.2 and Nav1.8 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, 

confirming their roles as modulators of pyrethroid activity on sodium channels (Smith and 

Soderlund 2001). When expressed in these systems, α subunits have demonstrated differential 

structure-dependent sensitivity to pyrethroids. Nav1.2 is sensitive to Type II pyrethroids, but 

insensitive to Type I pyrethroids (Smith and Soderlund 1998). Nav1.8, a principle carrier of 

TTX-R current (Smith and Soderlund 2001) in DRG neurons discussed in the preceding section, 

is sensitive to perturbation by both Type I and II pyrethroids resulting in characteristic 

modification of sodium currents (Choi and Soderlund 2006). These findings are supported by 

similar results in the voltage-clamp experiments previously reviewed. 
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Young rats (post-natal day 8) have been shown to be more sensitive to the acute toxicity 

of deltamethrin (Sheets et al. 1994) and cismethrin (Cantalamessa 1993) than adults but not to 

permethrin and cismethrin (Sheets 2000). PK differences between the immature and adult rats 

have been demonstrated (Cantalamessa 1993; Sheets et al. 1994; Anand et al. 2006). Meacham 

et al., (2008) explored the PD possibility of age-dependent pyrethroid toxicity using 

combinations of Nav 1.2 or Nav1.3 and β1 or β3 subunit expressed in oocytes. Nav1.3/β3 proved 

to be the most sensitive combination. Nav1.3/β3 is expressed embrionically while Nav1.2/β1

predominates in adults, supporting the hypothesis of developmentally-dependent PD differences.

Although these exogenous expression systems are useful in evaluating tissue and life 

stage differences in the Na channel populations (Trainer et al. 1997; Smith and Soderlund 1998, 

2001; Choi and Soderlund 2006; Meacham et al. 2008), such studies must be interpreted 

cautiously. High pyrethroid sensitivity of VGSC in neurons has been difficult to reproduce to 

date in channels expressed in oocytes or other systems, although effects of pyrethroids on all 

combinations of alpha and beta subunits have not been examined to date. Specifically, many of 

these In vitro studies have been conducted in non-mammalian systems, non-neuronal systems, 

and/or at temperatures lower than 37° C. 

Such differences may contribute to differences in sensitivity of the channel to pyrethroid 

modification, for example due to differences in post-translational modification, membrane 

composition or phosphorylation status. Even extrapolation from non-mammalian to mammalian 

exogenous expression systems is difficult. Studies examining pyrethroid effects on VGSC 

subunits expressed by transfection into mammalian cells often do not include co-expression of β 

subunits or concentration-response evaluations (Wang et al. 2001; Vickery et al. 2004; Dekker et 

al. 2005). Moreover, due to differences in techniques (electrophysiology vs. fluorescence 

measurements), studies with transfected mammalian cells are difficult to compare directly to 

studies utilizing oocytes. For example, Vickery et al (2004) showed that non-cyano pyrethroids 

(allethrin, permethrin or tetramethrin) were without effect on rat Nav1.2 expressed in Chinese 

hamster ovary cells, even when veratridine was present to activate VGSC. However, in the same 

study, deltamethrin and fenvalerate were also without effect individually and produced a small 

response in the presence of veratridine (Vickery et al. 2004).

From a risk assessment perspective, sodium channel cloning studies are interesting for 

purposes of evaluating species differences, potential population PD variability, and life stage 

differences. With respect to the proposal to form a CMG, this research highlights the importance 

of sodium channel interaction as a key event in pyrethroid toxicity. Specifically, although there 

is heterogeneity among the subunit combinations, the pathway of toxicity remains the same---

namely that sodium channel interaction as a critical and common key event in toxicity of 

pyrethroids. All the pyrethroids examined to date have the ability to modify VGSC kinetics in 

neurons. It is not necessary to incorporate this high level of specificity and/or variability with 

respect to the VGSC subunits into a risk assessment for human health and this level of detailed 

understanding is not necessary for forming a CMG. 

4.2.5. Other molecular targets for pyrethroids
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In the CMG guidance, OPP acknowledges that individual chemicals can exhibit multiple 

mechanisms of action and can have multiple molecular targets (USEPA, 1999). Specifically, the 

CMG guidance states: 

“Many substances can cause more than one toxic effect, depending upon level of exposure, and 

do so by different mechanisms of toxicity that take place at different sites of toxic action. 

However, a 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 proposed interaction with VGSCs as the common 

initial key event for determining the CMG. Pyrethroids, however, also have been reported to 

inhibit other types of ion channels, including chloride-permeable ion channels, voltage-gated 

calcium channels (Vijverberg and Bercken 1990; Narahashi 1996; Narahashi et al. 1998; Ray 

and Forshaw 2000; Ray 2001; Soderlund et al. 2002; Burr and Ray 2004; Shafer and Meyer 

2004) and ligand-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). While pyrethroid actions on VGSCs and 

neuronal excitability are well understood, very little is known about how effects on other target 

sites contribute to pyrethroid mammalian neurotoxicity (Ray 2001; Soderlund et al. 2002).
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A class of chloride-permeable channels has been proposed as potential targets for 

pyrethroid actions in the mammalian central nervous system (CNS) (Forshaw et al. 1993; Ray et 

al. 1997; Forshaw et al. 2000; Burr and Ray 2004). However, limitations of the supporting

studies justify cautious interpretation of the results with respect to the role of effects on this 

target in pyrethroid toxicity. A large conductance, chloride-permeable ion channel (denoted as 

‘maxi-chloride’) isolated using the ‘inside out patch’ electrophysiological technique is inhibited 

by various pyrethroid compounds. Pyrethroids inhibit these channels by reducing the probability 

that the channel will open. Information regarding concentration-response relationships is 

lacking, but several pyrethroids have been tested for their ability to modulate maxi-chloride 

channel opening In vitro. Deltamethrin, cypermethrin, cyfluthrin, fenpropathrin, and bioallethrin 

were effective between 10-5 to 10-6 M. Cismethrin, bifenthrin, cis-permethrin, cyfluthrin 

isomers, cyhalothrin, esfenvalerate, and tefluthrin had no significant effects a t 10-5 M. A 

distinct pattern for the Type I and II pyrethroids was not evident. 

The maxi-chloride channel has been detected only by utilizing the ‘inside-out patch’ 

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 

excitability. The electrophysiological parameters of the pyrethroid-sensitive maxi-chloride 

channel are homologous to those of the voltage-dependent anion channel (VDAC) family 

associated with the mitochondrial outer membrane (Dermietzel et al. 1994; Buettner et al. 2000). 

VDAC channels are non-specific pores that allow passage of anions and cations as well as small 

diameter molecules such as ATP and, possibly, cytochrome C across the mitochondrial outer 

membrane (Dermietzel et al. 1994; Sampson et al. 1997; Buettner et al. 2000). A plasma 

membrane associated form of the VDAC channel is normally present only in glial cells 

(Dermietzel et al. 1994; Buettner et al. 2000). It is still not known whether plasma membrane 

associated VDAC channels function in the regulation of membrane excitability or if they are 

expressed in neurons (Walz 2002). Available in vitro studies evaluating the possible interaction 

between pyrethroids and the chloride channels are limited in that the studies have been 

conducted utilizing only the N1E-115 neuroblastoma cell line and large-conductance chloride 

channels. Published data indicate N1E-115 cells are a glia X neuron hybrid that possesses 

characteristics of both cell types (Moolenaar and Spector 1977, 1978). Thus, these cells also 

contain glial characteristics and are not true neurons. Therefore, given the characteristics listed 

above, the maxi-chloride channels expressed in N1E- 115 cells are likely to be related to the 

VDAC family of channels. Reports that pyrethroids inhibit other volume sensitive anion 

channels (Culliford et al. 2004) support this conclusion.

In vivo studies in rats tested the ability of non-specific chloride channel agonists 

ivermectin and pentobarbitone to alleviate deltamethrin (2 mg/kg, iv)-induced neurotoxic signs. 

Some antagonism of behavioral measures resulting from deltamethrin exposure was noted with 

ivermectin and pentobarbitone. Neither compound was effective at reducing the in vivo signs of 

cismethrin neurotoxicity. There are also several limitations related to the in vivo data as well. 

Ivermectin is a competitive agonist of GABA-gated (GABAA) and glycine-gated chloride ion 

channel receptors present in the central and peripheral nervous systems (Abalis et al. 

1986)(Dawson et al. 2000)(Mascia et al. 1996; Huang and Casida 1997; Lu and Xu 2002; 

Hadipour-Jahromy and Daniels 2003)(Shan et al. 2001; Steinbach and Akk 2001). At the doses 

used for in vivo studies (Forshaw et al. 2000), the possibility that the in vivo signs of pyrethroid 
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neurotoxicity were alleviated by the actions of ivermectin on GABAA and glycine receptors, 

rather than on maxi-chloride channels, cannot be eliminated. Pentobarbitone is a sedative that 

also binds to, and activates, GABAA and glycine receptors (Hadipour-Jahromy and Daniels 

2003)(Mascia et al. 1996; Steinbach and Akk 2001; Lu and Xu 2002). At the doses used 

(Forshaw et al. 2000), the possibility cannot be eliminated that the decreased in vivo signs of 

pyrethroid neurotoxicity in the presence of pentobarbitone were the result of its action on 

GABAA and/or glycine receptors. The ability of ivermectin or pentobarbitone to competitively 

antagonize the pyrethroid dependent modulation of the maxi-chloride channel has not been 

tested. Given the lack of knowledge regarding the identity of the neuroblastoma maxi-chloride 

channel, and the questionable role of the VDAC channel, which is the most likely candidate for 

the ‘maxi-chloride’ channel, in regulation of membrane excitability, the results of the in vivo

studies must be interpreted with caution. The lack of specificity of ivermectin and pentobarbitone 

in these experiments precludes definitive conclusions regarding the target of pyrethroid action. In 

summary, the overall weight of scientific evidence for maxi-chloride channel effects as a basis 

for designating a CMG is not conclusive.

In addition to the voltage-gated sodium and chloride channels, In vitro evidence suggests 

that function of voltage-gated calcium channels (VGCCs) is altered by pyrethroids. VGCC α1

subunits are structurally similar to the α subunit of VGSCs, and like mammalian VGSCs, 

mammalian VGCCs are comprised of pore-forming α1 and auxiliary (β, λ, α2δ) subunits. 

Multiple isoforms of the VGCC α1 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 

release, Ca influx) approaches. Rather than provide an extensive review, (see Shafer and Meyer

(2004), several findings are highlighted here. Type II pyrethroids have been shown to increase 

Ca2+-dependent norepinehphrine 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 different isoforms of VGCC expressed in HEK293 cells, and to shift the kinetics of 

channel inactivation, but not activation (Hildebrand et al. 2004). More recently, 

electrophysiological studies with rat brain Cav2.2 expressed in Xenopus laevis ooctyes 

substantiated that deltamethrin modified Cav2.2 in vitro (Symington and Clark 2005). Using 

Ba2+ as a carrier for current through calcium channels, deltamethrin reduced peak current, caused 

a hyperpolarizing shift in potential activation and slowed activation and inactivation rates. The 

inhibition of peak current by deltamethrin appears inconsistent with 1) increased Ca2+ influx and 

enhanced neurotransmitter release reported using functional synaptosomal preparations 

(Symington and Clark 2005); 2) the lack of deltamethrin effects on spontaneous glutamate 

release at intact synapses in hippocampal neurons in vitro (Meyer and Shafer 2006); and 3) in 

vivo observations (Aldridge et al. 1978; Hossain et al. 2004) of increased neurotransmitter 

release following deltamethrin. Such differences suggest that other regulatory components of 

modulation Ca2+ influx and subsequent neurotransmitter release are necessary to elicit a 

neurophysiologic response that is consistent with in vivo symptoms. Overall, these and other 

uncertainties indicate that the potential role of direct effects of pyrethroids on VGCC in 

toxicological responses is presently not well enough understood to serve as the basis for a CMG. 
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Finally, there is evidence that pyrethroids may interfere with ligand-gated chloride 

channel currents. Briefly, when the inhibitory neurotransmitter γ-aminobutyric acid (GABAA) is 

released from the presynaptic nerve terminal, it binds to a postsynaptic receptor protein 

containing an intrinsic chloride ion channel (i.e. GABAA -activated chloride channel). When 

GABAA binds to its receptor, the chloride channel is opened and chloride ions flow into the cell 

resulting in an inhibitory post-synaptic potential which causes membrane hyperpolarization 

leading to a dampening affect on neuronal excitation, making it less likely that a post-synaptic 

action potential will occur (Costa 1998). GABAA antagonists, such as the chlorinated cyclodiene 

insecticides (e.g. toxaphene and lindane) and picrotoxinin (Lawrence et al. 1985), have an 

excitatory effect by blocking GABAA -mediated chloride current, resulting in hyperexcitability 

convulsions, and death (Bloomquist 1993b). Receptor agonists like diazepam (central) and RO5-

4864 (peripheral) increase the affinity of GABAA for its receptor causing an increased frequency 

of chloride channel opening and increased inhibition. Deltamethrin and fenvalerate, Type II 

pyrethroids, have shown the ability to weakly antagonize GABAA 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). Conversely, 

deltamethrin had no effect on the amplitude of GABAA -induced chloride current in cultured 

newborn DRG cells at a concentration that produced modifications of VGSC current (Ogata et 

al. 1988). In an in vivo study, Crofton and Reiter (1987) evaluated the combinatorial effects of 

cismethrin (Type I) and deltamethrin (Type II) on neurobehavioral function (i.e., acoustic startle 

response (ASR) and motor activity) in conjunction with controls for GABAA receptor activated 

chloride current, picrotoxin, muscimol, or chlordiazepoxide. Only deltamethrin and picrotoxin 

showed a dose-additive effect indicating an interaction at the picrotoxin binding site of the 

GABAA 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 granule cells. These authors concluded that pyrethroid effects on GABAA receptors 

were unlikely to account for pyrethroid induced changes in hippocampal synaptic transmission 

(Gilbert et al. 1989; Joy et al. 1990) or dorsal root ganglion potentials (Smith 1980). While in 

vitro ligand-mediated chloride channel studies indicate that GABAA 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 GABAA receptors is insufficient.

In summary, chloride channels have demonstrated susceptibility to some 

pyrethroids, however, interpretation of the in vitro results with respect to the purpose of 

the CRA groupings are confounded by the potential with VDAC channels and choice of 

cell line; in vivo results are compromised due to the potential antagonism of ivermectin 

and pentobarbitone on voltage-gated chloride channels and GABAA and glycine 

receptors. Voltage-gated calcium channels are potential sites of action of pyrethroids;

however the inconsistency of data from different types of preparations and compounds 

limits the utility of using this information for the purposes of determining a mechanism of 

action. Type II Pyrethroids have also demonstrated the ability to disrupt the ligandmediated chloride current by antagonizing GABAA receptors In vitro and a potential 

interaction at the picrotoxin binding site of the GABAA receptor-ionophore complex in 

vitro. While these pathways may be susceptible to perturbation by pyrethroids In vitro, 
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evidence from other studies indicate that GABAA receptors are less sensitive to 

pyrethroids than VGSC, and not all in vivo pyrethroid effects are clearly not mediated by 

actions on GABAA receptors. Furthermore, the limited number of chemicals examined 

does not provide a sufficiently broad representation of the pyrethroid class of pesticides

very well. Therefore, while these mechanisms may be involved in the overall 

neurotoxicity inherent with pyrethroids, there is not enough evidence supporting their 

inclusion into the finding of a common mechanism.

4.3 In vivo profile

The previous sections focus on effects on pyrethroids on the VGSC and membrane 

excitability. The following text summarizes data from in vivo studies, primarily in rodents. 

4.3.1. Pharmacokinetics 

Pyrethroid pharmacokinetics in rats have been described for a limited number of 

compounds. However, as a class, pyrethroids are lipophilic compounds with relatively short 

mammalian in vivo half-lives, being absorbed and eliminated relatively quickly. Focusing on 

plasma (as a measure of bioavailability) and brain (as a primary target tissue) compartments, 

half-life estimates are described herein for deltamethrin, lambda-cyhalothrin, and permethrin. 

Kim et al. (2008) administered deltamethrin IV (2 mg/kg) and orally (0.4, 2, and 10 mg/kg). 

Following a 2 mg/kg dose, the oral and IV plasma half-lives were similar, 15.8 and 13.3 hours, 

respectively. Following 2 and 10 mg/kg oral dose, brain half-lives were 12.5 and 19.5 hours, 

respectively, similar to plasma. Anadón et al. (1996) estimated a slightly longer plasma half-life, 

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

estimated half-lives for several sections of the brain ranged from 18 to 33 hours, similar to Kim 

et al. (2008) estimates. Estimates for lambda-cyhalothrin half-lives 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 were achieved under 

4 hours (deltamethrin <2; lambda-cyhalothrin 2.9; and permethrin 3.5 hours) and maximum brain 

concentrations under 8 hours (deltamethrin 1-7.3; and lambda-cyhalothrin 2.2-3.1)were generally 

achieved between under 2 hours. Finally, the oral bioavailability of deltamethrin in the Kim et 

al. (2008) and Anadón (1996) studies were similar at 18 and 14.4 %, respectively. Oral 

absorption rates for lambda-cyhalothrin (67.4%) and permethrin (60.7%) were greater.

Pyrethroids do not require metabolic activation to result in neurotoxicity. Indeed, 

metabolic 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 half-lives of pyrethroids vary considerably by 

compound (Table 1, (Godin et al. 2007; Scollon et al. 2009). Generally, the pyrethrins and firstgeneration pyrethroids tested (e.g. allethrin and resmethrin) have considerably shorter half-lives 

than the Type II pyrethroids (e.g. deltamethrin and β-cypermethrin), often differing by orders of 

magnitude (Wolansky and Harrill 2008). Furthermore, the relative clearance rates are structure 

dependent. Cis-isomers are subject to oxidation through numerous liver enzymes in mammals 
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(Anand et al. 2006; Godin et al. 2007; Scollon et al. 2009) while the trans-isomers are 

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

Oxidative clearance is a relatively slow mechanism compared to hydrolysis by carboxylesterases. 

Therefore, the half-lives of trans-isomers are generally shorter by 2 to 10 fold than their ciscounterparts. It has been postulated that since the cis-isomers are more persistent in vivo, they 

are inherently more toxic from a pharmacokinetic perspective. 

 

Table 1. Intrinsic clearance rates of selected pyrethroids from rat hepatic macrodomes

Pyrethroid Intrinsic clearance 

(ml/min/kg bw ± SD)

Allethrin; S-Bioallethrin 1218 ± 106

Bifenthrina 224 ± 20

Cyfluthrina 260 ± 27

Cypermethrina 104 ± 63

Deltamethrinb 89±23.7

Esfenvalerateb 108.2± 2.2

Permethrina 529 ± 19(cis);

438 ± 46 (trans) 

Resmethrina 1515 ± 367 (trans)

a

(Scollon et al. 2009);

b

(Godin et al. 2007)

Pyrethroids share many physical chemical and PK properties as a class (i.e., molecular 

weight, liphophilicity, common metabolic pathways, and disposition). The Agency is taking 

advantage of these similarities by using a “family modeling approach”. Conceptually, “family 

modeling” begins with developing a hypothesis based model for a single representative 

chemical. In this approach, a single model structure may be applied to all pyrethroids with 

similarities in the number of compartments and the physiological processes that determine the 

absorption, disposition, metabolism and elimination are described in those compartments. For 

individual pyrethroids, the model incorporates chemical specific parameters, such as, hepatic 

clearance rates, partition coefficients, coefficients of permeability. The initial models are 

developed and evaluated in rodents. Through PBPK modeling evaluations in the rodent, 

constants that are largely invariant across pyrethroids tested are identified, calibrated and 

retained in the model structure. Likewise, parameters that are determined to be compounddependent through relevant in vivo and in vitro studies are corrected accordingly. For example, 

while the enzymes involved in pyrethroid metabolism are the same across chemicals, the 

metabolic rates are not. Thus the mathematical description of metabolism remains the same 

across chemicals, but the rate constants are chemical specific. Once the ability to properly scale 

and incorporate the derived constants in the PBPK model in rodents is evaluated, the models can 

be scaled to humans by incorporating the appropriate species-specific physiological, chemical 

and biochemical parameters. Deltamethrin is used by EPA as the representative chemical as the 

pharmacokinetics and metabolism of this pyrethroid are well characterized and isomeric 

composition is not an issue (Godin et al. 2007). Based upon this version, PBPK models for 

several other pyrethroids identified by their exposure and hazard potential are being considered 

for development. 
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4.3.2. Toxicity syndromes in rodents

Pyrethroids have historically been classified according to the acute intoxication 

syndromes they exhibit in vivo. Initial studies examined “high-dose” pyrethroid exposures, 

doses resulting in near-death or death (Table 2). 

Verscholyle and Aldridge (1980) investigated toxicity following rat IV tail injection of 36 

pyrethroids. Signs of intoxication generally fell into one of two categories; the T-syndrome 

characterized by aggressive sparring, increased sensitivity to external stimuli and fine whole 

body tremors, and the CS-syndrome characterized by initial pawing and burrowing, salivation, 

and course whole body tremors (choreoathetosis). Eighteen pyrethroids were classified as Ttype, 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 not make any absolute 

conclusions regarding the relationship between cyan- and non-cyano-containing pyrethroids, 

however, linking the association between structure and function was highly significant. 

Lawrence and Casida (Lawrence and Casida 1982) administered 29 pyrethroids to mice 

via intracerebral (ICV) injections of 29 pyrethroids. The pyrethroids were classified as Type I or 

Type II, equivalent to the T- and CS- syndromes described by Verscholyle and Aldridge 

(Verschoyle and Aldridge 1980). The ICV injections were used to bypass primary metabolism 

and correlate intoxication with pyrethroid concentration in neuronal tissue directly. Study 

findings indicate α-cyano containing pyrethroids (Type II) produced choreoathetosis, 

convulsions and salivation, whereas compounds lacking the α-cyano group (Type I) produced 

tremors and convulsions, supporting the division of pyrethroids based on structure and 

intoxication syndromes. Furthermore, Gammon et al. (Gammon et al. 1981) classified 

pyrethroids as either Type I or Type II based on their effects on cercal sensory nerve recordings 

in vivo and In vitro and on 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 restlessness, lack of coordination, hyperactivity, prostration, and paralysis in 

vivo. Type II pyrethroids did not induce repetitive firing in the cercal sensory nerve. The 

prolonged discharges in the CNS were longer and of lower frequency than the Type I 

pyrethroids. In vivo symptomology also distinguished the Type II pyrethroids. Cockroaches 

became ataxic and lack of coordination with periods of convulsions and intense hyperactivity, 

sometimes associated with salivation. Periods of sustained contractions with the extension of 

metathoracic legs were particularly noticeable. Fenpropathrin and an oxime O-αcyanophenoxybenzyl ether were classified as Type I based on their action on the cercal sensory 

nerve but resulted in symptoms associated with Type II pyrethroids. It is therefore clear from 

these early studies that pyrethroids toxicity can be separated into two subgroups, Type I and 

Type II, based on structure and in vivo symptomology.
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Table 2. Behavioral characterization of pyrethroid toxicity of pyrethroids included in the Proposed 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 II

Cypermethrin CS II II

Deltamethrin CS II II

As recently reviewed (Wolansky and Harrill 2008), more contemporary studies were 

designed to detect behavioral changes at a broader range of doses 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, 

sematosensory response, social/sexual interactions and behavior to handling, and the functional 

observational battery (FOB). Although the number of pyrethroids with available data for 

coordination, neuromuscular response, social/sexual interactions, and cognitive response 

(Wolansky and Harrill 2008) is limited, these studies support a similar finding, that pyrethroids 

can be separated into two major groups based on neurological signs. Table 3 below provides an 

overview of behavioral changes observed in rodent studies (extracted from Wolansky and Harrill 

2008).

Motor activity is an apical measurement of nervous system function commonly measured 

in acute and sub-chronic neurotoxicity guideline studies (OPPTS Guidelines 870.6200, 

870.6200b). Motor activity measured as changes in ambulatory movement of rodents is currently 

the endpoint with the most extensive dose-response data for a large number of both Type I and II 

pyrethroids. Pyrethroids have been repeatedly shown dose-dependent decreases in motor activity 

in rodents following acute exposures. Past work (Crofton and Reiter 1984; Crofton and Reiter 

1987; Crofton and Reiter 1988; Gilbert et al. 1990; McDaniel and Moser 1993; Crofton et al. 

1995) has demonstrated decreased motor activity for eleven pyrethroids using the same test 

method (i.e., figure-eight mazes). In addition, decreases in motor activity have been 

demonstrated in other testing devices after acute or short-term exposure, including permethrin 

(Hoy et al. 2000), fenvalerate (Spinosa et al. 1999), cyhalothrin (Righi and Palermo-Neto 2003), 

cyhalothrin (Hornychova et al. 1995; Ratnasooriya et al. 2002), and cypermethrin (Hornychova 

et al. 1995). The acute motor depressant effect has been also observed in mice after oral exposure 

to fenvalerate (Mandhane and Chopde 1997) and deltamethrin (Chanh et al. 1984). A limited 

number of studies have observed increases or no change in motor activity (Mitchell et al. 1988; 

Husain and Husain 1996; Dayal 2003). These reports of increased activity are difficult to

interpret due to the use of commercial formulations rather than a technical product. Commercial 

formulations routinely contain very low concentrations of pyrethroids (e.g., 2-5%) and contain 

solvents and other ‘inerts’. Overall, the available studies on the effects of pyrethroids on motor 

activity suggest a common effect, i.e., decreased activity, on this apical neurobehavioral 

endpoint. 
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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 effects observed in studies of rodents following acute 

exposure to Type I and Type II pyrethroids. Different stages of the poisoning syndromes are 

noted at the far-left of the table. Note that the clinical landmarks produced under different routes 

of exposure are similar despite differences in body size and species. 1

Gammon et al. (1982), 

2

Lawrence & Casida (1982), 3Ray (1982), 4Verschoyle & Aldridge (1980), 5Wright et al., 1988, 

6

Souyri & Hoellinger (1983), 7Cremer (1983), 8Soderlund et al (2002), 9McDaniel & Moser 

(1993). †

Deltamethrin and other cyano compounds induces little or no salivation with icv dosing 

(Gray & Soderlund, 1985).
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Data from studies on auditory startle 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 proposed reflex path is short, consisting of auditory nerve, posteroventral 

cochlear nucleus, the nucleus reticularis pontis caudalis, and motor neurons in the spinal cord 

(Davis et al. 1982), and is sensitive to a variety of drugs and toxicants making the reflex a useful 

animal model of sensorimotor reactivity (Lee et al. 1996). Auditory startle data have 

demonstrated divergent patterns related to pyrethroid structure (Crofton and Reiter 1984; Crofton 

and Reiter 1988). Possible groupings include Type I compounds producing an increase in startle 

amplitude (e.g., permethrin, bifenthrin, cismethrin); Type II compounds that produce a decrease 

in startle amplitude (e.g., deltamethrin, cypermethrin, cyfluthrin); and compounds that produce 

alterations in the auditory startle reflex parameters not predicted by the absence or presence of 

the α-cyano group (fenvalerate) (Wolansky and Harrill 2008). With respect to the proposed 

common mechanism grouping, the auditory startle studies support the conclusion that Type I and 

Type II pyrethroids exhibit different in vivo toxicity outcomes. 

The FOB consists of a series of behavioral measurements from standardized tests such as 

posture, convulsions, tremors, salivation, mobility, rearing, and an array of sensory observations 

- touch, startle, and pain to name a few. In the case of pyrethroids, the FOB is designed to 

evaluate potential neurotoxicity by examining behaviors such as open field, home cage, sensory 

observations, physiological, and neuromuscular observations. McDaniel and Moser (1993)

described the neurobehavioral syndromes produced by high acute doses of permethrin (Type I) 

and cypermethrin (Type II) in rats using a FOB. Permethrin produced whole body tremors, 

ataxia, greater resistance to removal from home cage, and increases in arousal, handling 

reactivity, and the response to touch or experimenter approach. Cypermethrin caused salivation 

and urination, splayed limbs, choreoathetosis, increased foot splay, resistance to removal from 

home cage, decreased arousal, and decreased response to touch or tail pinch. There were also a 

number of neuromuscular effects that were affected by both chemicals including grip strength, 

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 clinical signs from high dose 

acute toxicity studies (Verschoyle and Aldridge 1980; Lawrence and Casida 1982), demonstrate 

that that methods for observation of clinical signs (e.g., FOB) can be used to systematically 

evaluate and potentially quantify changes in behaviors in rats exposed to pyrethroids. Moreover, 

McDaniel and Moser (1993) demonstrated that the FOB can be tailored to evaluate 

neurobehavioral syndromes specific to pyrethroids and can thus be used as a tool to compare the 

toxicity profile of different pyrethroids. 
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OPP has received acute neurotoxicity data on more than 15 registered pesticides, most of 

these include FOB observation in rats post-dosing, typically 2 to 8 hours after exposure. As part 

of the on-going work to re-evaluate the pyrethroids as part of registration review 

(http://www.epa.gov/oppsrrd1/registration_review/), OPP has performed a preliminary review of 

the available regulatory acute neurotoxicity studies. Typically, neurotoxic signs are observed at 

or near peak time of effect in these studies. As a result, these studies are often the most sensitive 

study in the available database and are therefore are often used by OPP to derive points of 

departure4 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 consistency between i) laboratory techniques and equipment; ii) the quality, duration and 

temporal spacing of observations; iii) lack of specificity in reporting (e.g., fine vs. whole body 

tremors; iv) route of administration; v) dosing vehicle and volume; vi) isomer concentration; vii) 

formulation; viii) 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; E.G. Williamson 1989; Lepeshkin et al. 1992; 

Villarini et al. 1995; Axelrad et al. 2002).

The PWG conducted a special FOB study designed to characterize effects classically 

associated with T and CS pyrethroid toxicity for 12 test chemicals (1 pyrethrin, 5 Type I and 6 

Type II pyrethroids) (Nemec 2006). The FOB consisted of 56 observations covering home cage, 

sensory, handling, physiological, open field, and neuromuscular parameters. The special FOB 

study offers two principle advantages lacking amongst the literature studies and registrant 

submitted data; 1) specific neurological behaviors that address the issue of common toxic effect 

and 2) consistency among testing and observational parameters. Each chemical was tested was 

at 2 or 3 doses, minimally and moderately effective doses were used to establish a spectrum of 

neurotoxicological effects. 

 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.
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In the evaluation of pyrethroid toxicity profiles, the PWG used principal component analysis 

and dissimilarity indices to evaluate the clustering of four independent data sets around common 

dimensions (Breckenridge and Holden 2007). These datasets included: 

1. 56 behavioral and physiological parameters from the special FOB study (Nemec 

2006); 

2. 8 electrophysiological parameters from patch clamp experiments conducted on five 

different sodium channels constitutively expressed in Xenopus oocyte (Choi and 

Soderlund 2006; Soderlund 2006); 

3. Indices of efficacy (maximum response), potency (log EC50) and binding (slope of the 

logistic dose-response function) 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 for voltage-gated chloride channels in neuronal membranes 

from mouse neuroblastoma cells (Burr and Ray 2004).

This analysis identified four primary patterns of variation in the FOB data. Two of these 

patterns described behaviors associated with CS behaviors (lower temperature, excessive 

salivation, impaired mobility) and two described behaviors associated with the T syndrome 

(elevated temperature, tremors, myoclonus). Non-cyano pyrethroids tended to have a high 

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

the CS factors. Pyrethroids with the strongest CS and T responses were deltamethrin/ λcyhalothrin and resmethrin/bifenthrin, respectively. Fenpropathrin and pyrethrin had weak 

responses for either the C or TS characteristics. The consistency of the special FOB study with 

those of Verschoyle and Aldridge (1980) and Lawrence and Casida (1982) is remarkable. 

Notably groupings of Type I and Type II pyrethroids based on symptoms of in vivo toxicity are 

similar in studies conducted four decades apart.

Based upon the FOB, sodium channel, calcium channel, and chloride channel studies, the 

PWG proposed that the pyrethroids separate into Type I and Type II subgroups based on the 

absence or presence of the α-cyano group. 

4.3.3. Information in humans 

Pyrethroids are generally thought be less hazardous than OPs and NMCs and generally 

human incidences with synthetic pyrethroids and pyrethrins are low (Power and Sudakin 2007). 

Recent studies have, however, shown an increase in reported incidences following exposure to 

pyrethroids (Power and Sudakin 2007; Walters et al. 2009). The clinical manifestations of

pyrethroid exposure have been recently reviewed by Bradberry et al, (2005). Dermal exposure 

can result in transient paraesthesias with many pyrethroids. Inhalation exposure can result 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). 
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There are few epidemiology studies reporting effects of pyrethroids. Permethrin has been 

evaluated the Agricultural Health Study (AHS; Alavanja et al. (2003), Rusiecki, et al (2009)).

Kamel et al (2005, 2007) have evaluated statistical associations between self-reported pesticide 

use with neurological symptoms with cumulative use of insecticides classified by chemical type 

among licensed pesticide applicators enrolled in the AHS from 1993 to 1997 (n =18, 782). The 

investigators found increased risk associated with use of OPs among applicators who had never 

experienced doctor-diagnosed pesticide poisoning or high exposure events (OR: 1.6_1.7 for 

highest category of use) and a small increase in risk associated with use of carbamates, but there 

was little effect of permethrin.

4.4 Data in mixtures of pyrethroids

EPA’s default assumption when evaluating multi-chemical risk is to assume dose 

additivity. This assumption is thought to be particularly appropriate when evaluating chemicals 

which share a common mechanism of action. Chemicals which share a mechanism of toxicity 

will share a common molecular target. As such mixture studies which support the dose 

additivity hypothesis also indirectly support the common mechanism findings since dose 

additivity suggests that the chemicals of different potencies act as dilutions of each other. 

To date, there has been limited research conducted on the potential effects of exposure to 

pyrethroid mixtures. Two in vitro studies have shown that initial exposure of sodium channels to 

fenvalerate (Song and Narahashi 1996) and deltamethrin (Tabarean and Narahashi 1998; 

Motomura and Narahashi 2001) caused use-dependent VGSC modification, typical of α-cyano 

containing pyrethroids. Subsequent exposure to tetramethrin (Type I) resulted in the slow tail 

decay being replaced by a more rapidly decaying current, characteristic of pyrethroids lacking 

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

al. (1996) concluded that tetramethrin may displace fenvalerate or interact allosterically from 

sodium channel protein binding sites in an ex vivo rat dorsal root ganglion preparation. While 

these reports suggest that pyrethroids do not act in an effect-additive manner, there are a number 

of possible confounds in making definitive conclusions from these reports. First, the 

electrophysiological work (Ray and Fry 2006) used dose levels that exceeded known LD50

dosages. In addition, all of these previous reports lacked either by study design or statistical 

approach, the ability to test the hypothesis of additivity. The use of rigorous statistical models is 

critical for testing hypotheses of effect- or dose-addition, and determining whether antagonism or 

synergism exists (Feron and Groten 2002; Hertzberg and Teuschler 2002; Gennings 2004; 

LeBlanc and Olmstead 2004; Teuschler 2007). Overall, these limited in vitro data suggest there 

may be complicated outcomes from in vitro mixtures at the molecular or cellular level.

In an in vivo assessment of the dose-addition of pyrethroids , Wolansky and colleagues, 

tested a mixture of low doses of both Type I and II pyrethroids (Wolansky and Harrill 2008; 

Wolansky et al. 2009). With a mixture composed of individual chemical at doses 30% lower that 

the threshold for neurobehavioral effects, that the effects of this mixture pyrethroids on motor 

activity was accurately predicted by a single chemical required dose additivity model (Wolanksy 

et al. 2009). While the data of Wolansky et al 2009 demonstrate dose-addition of low dose 

effects for both Type I and Type II pyrethroids, these data need to be interpreted with caution. 

Motor activity is an apical measure of the disruption of nervous system function (Reiter and 
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McPhail 1979), that is decreased by a wide variety of pesticides. Thus the specificity for a 

causative mode-of-action based linking of effects on VGSCs to apical measures of neural 

function like motor activity are extremely difficult to demonstrate. Because a mechanistic 

understanding is not available for pyrethroid induced decreases in motor activity, these data on a 

non-specific endpoint have limited utility in supporting a dose-additivity assumption for a CMG 

as established per the requirements of the FQPA. 

5.0 Proposed Common Mechanism Grouping under the FQPA 

5.1 Weight of the Evidence 

The above sections provide a survey of key studies which 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, disrupting neuronal firing patterns and ultimately resulting in the signs of pyrethroid 

toxicity. Specifically, evidence of a common toxicity pathway and common toxic effects for 

pyrethroids can be found from studies evaluating effects on 1) the sodium channel from both 

mammalian and non-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 modification of VGSC, alterations in membrane 

excitability, and clinical signs and symptoms are completely understood, particularly in 

mammals. Data in non-mammalian species provide support for the proposed mechanism where 

mammalian data are lacking. Moreover, there is a strong correlation between the pyrethroid 

structure, kinetic modulation of the sodium channel, and phenotypical clinical syndrome in 

rodents. Pyrethroids lacking an α-cyano moiety are found to have a delayed deactivation of 

sodium channels resulting in extended tail currents of approximately 20 ms and are associated 

with clinical effects defined as the T syndrome. On the other hand, pyrethroids containing an αcyano moiety delay deactivation of sodium channels resulting in prolonged tail currents of 

approximately 500 ms to minutes and are associated with clinical effects defined as the CS 

syndrome. 
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Based on this review OPP is proposing that the naturally occurring pyrethrins and 

synthetic pyrethroids form a CMG based on 1) shared structural characteristics; and 2) shared 

ability to interact with voltage-gated sodium channels (VGSC), resulting in disruption of 

membrane excitability in the nervous system, and ultimately neurotoxicity characterized by two 

different toxicity syndromes. The proposed pathway to toxicity is illustrated in the Figure 6. 

The presence of pyrethroids at the target tissue causes a disruption in the VGSC, leading to an 

alteration of action potentials, and ultimately resolving as clinical neurotoxicity characteristic of 

pyrethroid poisoning.

Figure 6. Proposed common toxic pathway of the pyrethroids

Although there is an extensive body of literature on pyrethroids mechanism of toxicity 

and toxicity syndromes, the Agency acknowledges that some science issues remain. 

Target 

Tissue Dose

VGSC 

Alterations

In Vivo 

Clinical 

Signs

Altered 

Neuronal

Excitability

All pyrethroids 

modify the kinetics of 

VGSC activation and 

inactivation in 

mammalian 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 toxicity
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a) Other endogenous channels (e.g., calcium, chloride, GABA) which have been proposed by 

some (Soderlund et al. 2002; Ray and Fry 2006; Clark and Symington 2007) as potential other 

target sites for some pyrethroid.

A key task in forming a CMG is to distinguish those molecular target(s) and pathways of 

toxicity which are common among the group members and those which are not. OPP’s CMG 

guidance notes that some chemicals may exhibit multiple mechanisms of toxicity but that OPP 

will group substances that cause multiple toxic effects by a common mechanism “provided at 

least one of the toxic effects is common among the substances.” 

Calcium, chloride, and GABAA channels have all shown the potential to interact with 

pyrethroids (Section 4.2.5). However, as described in more detail above, the data are not 

sufficiently robust to support a common mechanism grouping for these. In contrast, each 

member of the proposed CMG is believed to interact with the VGSC. The Agency is not aware 

of any data demonstrating that any pyrethroids lack an interaction with VGSCs. In other words, 

all pyrethroids tested in mammalian neurons have shown interaction with VGSCs. As such, 

interaction with the VGSCs provides the common molecular target for pyrethroids. The Agency 

does acknowledge that interaction between pyrethroids and these other channels may mediate the 

toxicity of specific pyrethroids but this does not preclude the formation of the CMG. 

b) Na channel structural heterogeneity among tissue and across life stages with respect to 

alpha- and beta-subunits. 

Another important issue when evaluating mechanistic information for a CMG finding is 

the concept that multiple tissues and/or sites can exhibit the same key events leading to toxicity 

but have different dose-response relationships or different tissue sensitivities. This issue is 

applies to VGSC structural heterogeneity. As described in Section 4.2, mammalian sodium 

channels are comprised of α and β subunits that exist in multiple isoforms, giving rise to tissue, 

regional and life 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 comprising 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 human embryonic kidney (HEK) cells. 

With respect to the current proposal to designate the pyrethroids as a CMG, data from this 

approach has provided evidence to support modification of sodium channel function by 

pyrethroids as a key event in neurotoxicity (Vais et al. 2001). Data from these in vitro systems 

have also shown differences in pyrethroid sensitivity of various sodium channel isoforms that 

may influence differences in pyrethroid sensitivity based on tissue, region (Choi and Soderlund 

2006), life stage (Meacham et al. 2008) or species, including differences between rodents and 

humans (Vais et al. 2000; Tan and Soderlund 2009).

The pyrethroid sensitivity of all possible 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, mammalian neurons is confounded by at least two significant uncertainties; 1) exogenous 

expression of VGSC subunits for these studies requires the use of non-mammalian and/or non-
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neuronal cell types, and differences in post-translational modification of subunits, their assembly 

and insertion into the membrane, or modification by other cellular processes (e.g. 

phosphorylation, glycosylation, etc) may contribute to differences in sensitivity of the channels 

to pyrethroid modification, and 2) the subunit composition of VGSC when recording sodium 

currents in mammalian neurons is unknown and difficult to determine due to the expression of 

multiple VGSC subunits within a neuron. Thus, it is currently not possible to associate 

pyrethroid effects with specific combinations of VGSC subunits. Despite this, data from this line 

of scientific investigation support the hypothesis that effects on VGSC are an initial and common 

key event in 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, for purposes of defining the CMG, the “major steps leading to a 

toxic effect” need to be considered and that “All steps leading to an effect do not need to be 

specifically understood (1999).” Thus, although differences in the sensitivity of different VGSC 

subunit combinations may influence region, tissue or life stage differences in sensitivity, or may 

influence 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. 

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

In the cumulative risk assessments for the OPs and NMCs, the common mechanism 

groupings were based on the ability of these pesticides to inhibit the enzyme acetylcholinesterase 

(AChE)5 leading to accumulation of acetylcholine and ultimately to neurotoxicity. As such, 

AChE inhibition provides the first key event in the pathway to neurotoxicity for these pesticides. 

AChE inhibition (for example, blood AChE inhibition) is in an easily measurable biomarker in 

multiple species, tissues, and life stages. Thus, AChE inhibition provides a scientifically robust 

and health protective endpoint for use as the common mechanism endpoint in the cumulative risk 

assessment. Such a readily quantifiable in vivo biomarker for pyrethroid-modified VGSCs is 

not available for the pyrethroids. 

 5 OPs inhibit AChE via phosphorylation; NMCs inhibit AChE via carbamaylation. These different chemical 

reactions result in different patterns of toxicity with respect to time to peak effect and time to recovery.
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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 associated with pyrethroid exposure in vivo, particularly at high doses. 

In newer studies performed by ORD and the PWG, the same pyrethroids were tested in three 

testing systems at three different levels of biological organization: channel, cells, and whole 

animal. The older literature and newer studies support common findings, namely that 

pyrethroids share the ability to interact with VGSC leading to disruptions of membrane 

excitability, and that pyrethroids share the ability to cause neurobehavioral sequele in rodents. 

The relationship between pyrethroid-induced alterations in VGSC function and disrupted 

membrane excitability (membrane depolarization and/or changes in firing of action potentials) is 

well established and has been demonstrated in a number of species in vitro. Across multiple 

invertebrate and vertebrate species, including mammals, pyrethroid-induced modification of 

VGSC function results in alterations in neuronal membrane excitability that are readily measured 

using neurophysiological 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 cannot provide a quantitative link between alterations in 

sodium channels in vivo, and changes in the function of the nervous system as evidenced by 

neurobehavioral effects in mammals. A quantitative link between the molecular target and 

downstream adverse outcomes is hampered by the lack of an easily accessible in vivo/in situ/ex 

vivo measure of (biomarker for) pyrethroid modified VGSC and a lack of a complete 

understanding of how subsequent changes in sodium channel function result in specific 

neurological outcomes. Despite this, the scientific evidence correlating pyrethroid-induced 

changes in VGSC function with neurotoxicity is 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. 
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5.2 Summary 

In summary, although some uncertainties exist, OPP believes that there is a convincing 

amount of data from multiple lines of evidence and from studies spanning several decades which 

support a common mechanism grouping for the naturally occurring pyrethrins and synthetic 

pyrethroids under the FQPA. This 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 sodium channel kinetics, leading to alterations in membrane 

excitability and firing potentials.

• Neurotoxic Effects - Pyrethroid toxicity is manifested through neurological 

syndromes described as either T (fine tremors), CS (choreoathetosis and salivation), 

or some combination thereof, depending on the structure. Open literature supports a 

correlation between the modification in sodium channel kinetics and the resulting 

syndrome.

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

As described in more detail above there are many in vitro and in vivo studies which 

suggest that pyrethroid toxicity in manifested by two profiles. The following text will not repeat 

the above review. Instead, the following focuses on several studies conducted by the PWG or 

EPA’s ORD (Table 4). The following studies (Choi and Soderlund 2006; Nemec 2006; 

Wolansky et al. 2006; Shafer et al. 2008; Losa et al. 2009) are unique in that they were 

conducted on the same pyrethroids with the purpose of evaluating the common toxic effects of 

pyrethroids. The studies represent three different levels of biological complexity (sodium 

channel, nerve cell, whole animal) and provide a remarkably similar separation of the 11 

chemicals. 
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Figure 7. Differences in Type I and Type II pyrethroids which support forming two sub-groups 

within the Proposed CMG 

6.1. Pyrethroid Effects on Cloned VGSC Nav1.8

As part of the studies sponsored by the PWG, Choi and Soderlund (2006) examined the 

interactions of 11 pyrethroid compounds on function of the Nav1.8 VGSC expressed in Xenopus

oocytes. The Nav1.8 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) rates as 

well as induction of tail currents upon repolarization of the membrane. Based on the alterations 

in the activation kinetics, as well as the time constants for tail current decay (tau1,tau2; Table 6), 

the 11 pyrethroid compounds could be clearly separated into 2 groups. The first group is 

comprised of the Type I compounds that produced modest increases in activation rate (tauact <12) 

and tail current decay described by a single exponential, while the second group is comprised of 

the Type II compounds, which produced large increases in the channel activation rate (tauact > 

15) and tail current decay described by two exponential (one fast and one slow) components. 

Even considering the uncertainties detailed above regarding exogenous expression systems such 

as oocytes, these results demonstrate at the most basic level, two distinct groups of compounds 

based on actions at the level of the VGSC.

Type II

Sodium 

Current

Less potent, 

generally, in 

mammalian neural 

networks in vitro 

(e.g., MEAs) 

T 

syndrome 

in vivo

Action

Potential

Sodium 

Current

Action

Potential

Type I

More potent,

generally, in 

mammalian neural 

networks in vitro 

(e.g., MEAs) 

CS syndrome 

in vivo
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6.2. Micro electrode arrays (MEA) Studies

Newer technologies have allowed researchers to examine how pyrethroids affect 

mammalian neural networks in vitro. Primary neuronal cell cultures can be grown on 

microelectrode arrays (MEAs), Figure 8, allowing the measurement of spontaneous 

electrophysiological activity. While voltage-clamp techniques allow measurement of channel 

function in an individual neuron, MEAs measure function of networks of neuronal cells, and are 

thus a “summed” measure of effects of compounds on all ion channel constituents (e.g. Na, Ca) 

in the cells and network. Thus, multiple VGSC as well as VGCC isoforms contribute to the 

overall responses measured with MEAs. While this precludes direct examination of individual 

isoform interaction with pyrethroids, pharmacological approaches may be used to gain inferences 

regarding the relative importance of different ion channel types to the pyrethroid response. 

Glutamate is a major excitatory neurotransmitter in the mammalian CNS and effects of 

pyrethroids on glutamatergic networks have been characterized using MEA techniques. Effects 

of pyrethroids on glutamatergic networks using spontaneously active cortical (Losa et al. 2009)

and hippocampal (Shafer et al. 2008) cultures have recently been examined. In hippocampal 

cultures, pharmacological approaches indicated that pyrethroid-induced changes could be 

minimally explained via effects on Na channels. These data are consistent with voltage-clamp 

measurements of permethrin and deltamethrin effects on miniature excitatory post-synaptic 

currents (mEPSCs) in hippocampal neurons (Meyer and Shafer 2006). Thus, these data support 

the modulation of VGSC as a key event in pyrethroid induced changes in neuronal excitability.

Figure 8. Photograph of neurons on a MEA. There are 59 electrodes on each MEA “chip” and each 

electrode can record firing rates and patterns of any neurons in “contact” with it.

Losa and colleagues determined IC50 values for inhibition of spontaneous activity in 

glutamate networks for 11 pyrethroids (Losa et al. 2009) using primary cultures of cortical 

neurons. The values ranged from 25 to 1685 nM (Table 4); Type I and II pyrethroids resulted in 
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higher and lower IC50 values, respectively. Esfenvalerate and fenpropathrin resulted in 

intermediate values.

Table 4. IC50 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

In an effort to determine the relative potency of pyrethroids, Wolansky et al (2006) 

determined acute dose-response functions for 11 pyrethroids in rats using motor activity. 

Activity was measured for 1 hour in figure 8 mazes during the time of peak effect. It is difficult 

establish a common toxic effect using motor activity because the specificity for a causative 

mode-of-action based linking of effects on VGSCs to apical measures of neural function like 

motor activity are extremely difficult to demonstrate. However, the determined ED30s, that is the 

effective dose which causes a 30% decrease in motor activity, generally demonstrate a separation 

of Type I and Type II pyrethroids based on potency with some exceptions. 

The PWG conducted a special FOB study designed to characterize effects classically 

associated with T and CS pyrethroid toxicity for 12 test chemicals (1 pyrethrin, 5 Type I and 6 

Type II pyrethroids)(Nemec 2006). The FOB consisted of 56 observations covering home cage, 

sensory, handling, physiological, open field, and neuromuscular parameters. Four primary 

patterns of variation were observed in the FOB data. Two of these patterns described behaviors 

associated with CS behaviors (lower temperature, excessive salivation, impaired mobility) and 

two described behaviors associated with the T syndrome (elevated temperature, tremors, 

myoclonus). Non-cyano pyrethroids tended to have a high loading on the T factors whereas 

cyano-containing pyrethroids tended to have high loading on the CS factors. Pyrethroids with 

the strongest CS and T responses were deltamethrin/ λ-cyhalothrin and resmethrin/bifenthrin, 

respectively. Fenpropathrin and pyrethrin had weak responses for either the C or TS 

characteristics. 
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Table 5. Behavioral characterization of pyrethroid toxicity by PWG 

Pyrethroid WIL*

Allethrin; S-Bioallethrin T

Bifenthrin T

Cyfluthrin CS

Cyhalothrin, lambda- CS

Cypermethrin CS

Deltamethrin CS

Esfenvalerate MIXED

Fenpropathrin T

Permethrin T

Resmethrin T

Tefluthrin T

6.4. Proposed Subgrouping

Table 6 below combines the information described in Sections 6.1-6.3 and thus 

summarizes data at the channel level (Nav 1.8 kinetics), the tissue level (MEA IC50s) and the 

behavioral level (WIL FOB data). 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, with two chemicals, esfenvalerate and fenpropathrin, representing 

mixed Type I/II. 

OPP is proposing to subgroup the pyrethrins and pyrethroids into Type I (lacking alphacyano moiety) and Type II (bearing alpha-cyano moiety) based upon differences in structure, 

channel gating activity, and neurobehavioral effects. This conclusion is based on the following 

characteristics:

• Structure – based on the absence or presence of an alpha-cyano moiety

• Sodium channel kinetics – Type I pyrethroids cause slight prolongations of the 

sodium current tails (20 ms), often resulting in long trains of action potentials. Type 

II pyrethroids significantly prolong sodium current tails (200ms to minutes) typically 

resulting in increased resting membrane potential and can induce action potential 

block. 

• Toxicity syndromes – Type I and Type II pyrethroids generally elicit C and TS 

syndromes, respectively. Exceptions, including esfenvalerate and fenpropathrin, 

elicit symptoms that are a combination of the T and CS syndromes.
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Table 6. Summary of key data supporting the proposed subgrouping into Type I and Type II 

Pyrethroid

Nav 1.8 Kineticsa MEA IC50s

d

WILe

Activationb tau1c tau2c

Allethrin; S-Bioallethrin 3.9 1.9 NA 1525 T

Bifenthrin 3.9 4 NA 439 T

Permethrin 2.5 2.3 NA 719 T

Resmethrin 3.3 2.6 NA 1685 T

Tefluthrin 3.8 4.3 NA 499 T

Esfenvalerate 10.7 13.4 NA 809 MIXED

Fenpropathrin 10.8 12.4 NA 1518 T

Cyfluthrin 23.6 7.1 95.9 305 CS

Cyhalothrin, lambda- 22.3 9.4 84.7 25 CS

Cypermethrin 12.6 6.9 55.8 181 CS

Deltamethrin 15.7 5.3 52.1 175 CS

a

Gating kinetics of the rat Nav 1.8 sodium channel isoform expressed in Xenopus laevis oocytes (Choi and 

Soderlund, 2006)

b

time constants (ms)for the activation of pyrethroid-modified channels c

time constants (ms) for the fast (tau1) and slow (tau2) components of tail current delay

d

MEA IC50 values exhibited by XX tissue (Shafer SOT poster??) e

Neurotoxic syndrome expressed by rats following high doses of pyrethroids as defined by FOB observations 

(WIL Study)

Attachment 1 provides the structures of the preliminary list of pyrethrins and synthetic 

pyrethroids being considered in the CMG. Attachment 1 also provides assignments of potential 

sub-grouping (i.e., Type I or II) to 11 pyrethroids and the pyrethrins. These assignments were 

made based on the information in Table 6 and the results of the special FOB for pyrethrins. As 

of May, 2009, five additional pyrethroids (cyphenothrin, imiprothrin, prallethrin, tetramethrin, 

and d-phenothrin) were being tested in a special FOB study similar to the study sponsored by the 

PWG. OPP will assign the sub-grouping for these five following receipt and review of the ongoing studies. For the remaining pyrethroids, OPP will first evaluate existing data from In vitro

and/or in vivo studies for effects consistent with Type I or II effects. In the absence specific data 

to inform a particular sub-group, OPP plans to use structure (i.e., presence/absence of an α-cyano 

group) as the determining factor for assigning individual pyrethroids to a sub-group.
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Attachment 1.0. Classification (as proposed by the Agency) and Structure of pyrethroids 

registered for use with the US EPA.

Name Type I/II Structure

Allethrin (s-Bioallethrin, dallethrin) I

Bifenthrin I

Cyfluthrin (β-cyfluthrin) II

Cyhalothrin (γ/λcyhalothrin) II

Cypermethrin (α/β/θ/ζcypermethrin) II

Deltamethrin II

Fenpropathrin Mixed I/II

Fenvalerate (esfenvalerate) Mixed I/II
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Name Type I/II Structure

Permethrin I

Resmethrin (bioresmethrin, 

cismethrin) I

Tefluthrin I

Tetramethrin

Pending Agency 

Decision; Special FOB

Study On-going

Cyphenothrin

Pending Agency 

Decision; Special FOB 

Study On-going

Fluvalinate (τ-fluvalinate) Pending Agency Decision

Imiprothrin

Pending Agency 

Decision; Special FOB 

Study On-going

Metofluthrin Pending Agency Decision
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Name Type I/II Structure

Phenothrin (d-phenothrin, 

sumithrin)

Pending Agency 

Decision; Special FOB 

Study On-going

Prallethrin

Pending Agency 

Decision; Special FOB 

Study On-going

Tralomethrin Pending Agency Decision

Pyrethrins I

Cinerin I Pending Agency Decision

Cinerin II Pending Agency Decision

Jasmolin I Pending Agency Decision

Jasmolin II Pending Agency Decision