UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: August 28, 2013 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: Common Mechanism Grouping for the Pyrethrins and Synthetic Pyrethroids 

PC Code: See Below 

Decision No.: 455436 

Petition No.: None 

.Risk Assessment Type: None 

TXR No.: 0056776 , 

MRID No.: None 

DP Barcode: 394576 

Registration No.: None 

Regulatory Action: None 

Case No.: None 

CAS No.: See Below 

40 CFR: None -· 

FROM: Edward Scallon, Ph.D., Toxicologist 

Anna Lowit, Ph.D., Senior Scientist 

Health Effects Division (7509P) 

ti ; ._j -{,~ ,· £-.:5 ci--~~_c._/, " 

~"'/ 2:7! 

THROUGH: 

TO: Dana Friedman, Team Leader 

Risk Management and Implementation Branch 2 

Pesticide Re-Evaluation Division (7508P) 

•,_ 

The Office of Pesticide Programs has determined that the pyrethrins and the synthetic 

pyrethroids cause toxicity through the same mode of action, interaction with the voltage-gated 

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

common mechanism group was presented and supported by a FIFRA Scientific Advisory Panel 

(June 2009). Following the meeting and the consideration of public comments, the draft science 

paper was duly posted in the Pyrethrins/Pyrethroid Cumulative Risk Assessment docket 

(http://www.regulations.gov;EPA-HQ-OPP-2011-0746) on November 9, 2011. Since the 

November 9 posting of the draft science document, the Agency has updated the draft version. 

This memo is accompanied by the final version of Science Policy Paper: Common Mechanism 

Grouping for the Pyrethrins and Synthetic Pyrethroids. The attached document is fmal. 

Page 1 of2 
Page 2 of 2 

Chemical PC Code CAS No. 

Allethrin 004001 584-79-2

Bioallethrin 004003 28057-48-9 

S-Bioallethrin 004004 28434-00-6 

D-Allethrin 004005 84030-86-4 

Esbiothrin 004007 84030-86-4 

Bifenthrin 128825 82657-04-3

Cyclethrin 004052 97-11-0 

Cyfluthrin 128831 68359-37-5

Cyfluthrin, beta- 118831 68359-37-5

Cyhalothrin 128867 68085-85-8

Cyhalothin, lambda- 128897 91465-08-6

Cyhalothrin, gamma- 128807 76703-62-3

Cypermethrin 109702 52315-07-8

Cypermethrin, alpha- 209600 67375-30-8

Cypermethrin, beta- 118831 65731-84-2

Cypermethrin, zeta- 129064 52315-07-8

Cyphenothrin 129013 39515-40-7

Deltamethrin 097805 52918-63-5

Esfenvalerate 109303 66230-04-4

Fenvalerate 109302 51630-58-1 

Fenfluthrin 109705 69409-94-5 

Fenpropathrin 127901 39515-41-8

Flumethrin 036007 69770-45-2 

Fluvalinate 109302 69409-94-5 

Imiprothrin 004006 72963-72-5

Metofluthrin 109709 240494-70-6

Permethrin 109701 52645-53-1

Prallethrin 128722 23031-36-9

Pyrethrins (not synthetic) 069001 8003-34-7

Resmethrin 097801 10453-86-8

Sumithrin (phenothrin) 069005 26002-80-2

Tau-fluvalinate 109302 102851-06-9

Tefluthrin 128912 79538-32-2

Tetramethrin 069003 7696-12-0

Tralomethrin 121501 66841-25-6
Science Policy Paper 

 

Common Mechanism Grouping for the 

Pyrethrins and Synthetic Pyrethroids 

August 28, 2013 
Page 2 of 58 

TABLE OF CONTENTS 

1.0 Background ........................................................................................................... 3

2.0 Key Concepts ........................................................................................................ 4

3.0 Chemical Structure ............................................................................................... 5

3.1 Chirality of pyrethroids ................................................................................. 7

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

4.0 Toxicology of Pyrethroids .................................................................................. 10

4.1 Overview ...................................................................................................... 10

4.2 Voltage-Gated Sodium Channels (VGSC) ................................................. 11

4.2.1. Protein structure of the VGSC .............................................................. 11

4.2.2. Channel modulation by pyrethroids..................................................... 12

4.2.3. Relationship between pyrethroid-induced alterations in VGSC 

function & disrupted membrane excitability ....................................... 14

4.2.4. Potential differences in tissue sensitivity to pyrethroids................... 14

4.2.4.1. Voltage-clamp measurements ......................................................... 14

4.2.4.2. Sodium channel cloning .................................................................. 15

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

4.3 In vivo profile ............................................................................................... 21

4.3.1. Pharmacokinetics .................................................................................. 21

4.3.2. Toxicity syndromes in rodents ............................................................. 22

4.3.3. Information in humans .......................................................................... 28

4.4 Data in mixtures of pyrethroids ................................................................. 28

5.0 Common Mechanism Grouping under the FQPA ............................................. 30

5.1 Weight of the Evidence ............................................................................... 30

5.2 Summary ...................................................................................................... 35

6.0 Potential to Sub-Group the Pyrethroids by Type I & Type II ........................... 35

6.1. Pyrethroid Effects on Cloned VGSC Nav1.8 .............................................. 36

6.2. Micro electrode arrays (MEA) Studies ....................................................... 37

6.3. In Vivo Rodent Studies ............................................................................... 38

6.4. Potential to Subgroup ................................................................................. 39

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

Attachment 1.0............................................................................................................ 55
Page 3 of 58 

1.0 Background 

The Food Quality Protection Act (USEPA,1996) requires EPA to take into account 

“available evidence concerning the cumulative effects on infants and children of such 

residues and other substances that have a common mechanism of toxicity.” As 

described in detail in the cumulative risk assessment (CRA) guidance (USEPA, 2002), 

developed by the Office of Pesticide 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 cumulative risk assessments on the 

organophosphates (OPs), N-methyl carbamates (NMCs), chloracetanilides, triazines, 

and most recently, pyrethroids (Oct. 4, 2011). Pyrethroids are a class of synthetic 

insecticides which are structurally based on the pyrethrins, botanical insecticides 

extracted from Chrysanthemum cinerariaefolium (Soderlund et al. 2002). Pyrethroid 

exposure can occur from food, water, or non-occupational settings. Due, in large part, 

to changes in usage trends resulting from 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 body of published literature concerning the toxicity of 

pyrethroids and potential modes of action of these compounds. In addition, over the 

last decade, EPA’s Office of Research and Development (ORD) and the Pyrethroid 

Working 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 science policy document is to describe this scientific 

evidence which supports the designation of pyrethroids as a CMG under the FQPA. 

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 has determined that the naturally occurring pyrethrins and synthetic 

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

ability to interact with 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, the pyrethroid 

CMG may be divided into two subgroups representing Type I and Type II pyrethroids, 

however, all pyrethroids were considered in a single group for the CRA. This 

 

1

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

pyrethrins and some synthetic pyrethroids. 
Page 4 of 58 

subgrouping is consistent with historical categorization of pyrethroids and is supported 

by in vitro and in vivo information from both older studies and new research. 

Pyrethroids considered for the CMG are listed in Appendix 1. 

The designation of Type I and Type II pyrethroids is based, in large part, upon 

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

in Type II. Type I members of the CMG include allethrin, bifenthrin, imiprothrin, 

metofluthrin, permethrin, resmethrin, phenothrin, tefluthrin, tetramethrin, sumithrin, 

prallethrin, and pyrethrins; Type II members include cyfluthrin, cyhalothrin, 

cypermethrin, cyphenothrin, deltamethrin, flucythrinate, flumethrin, and fluvalinate. 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 illicit effects intermediary between Type I and Type II 

pyrethroids during in vitro and in vivo evaluations. Additional discussion on the 

classification of pyrethroids can be found in Section 6.4. 

This document also describes some uncertainties considered including i) other 

endogenous channels (e.g., calcium and chloride) which have been proposed as 

potential 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 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. 

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 are: 

 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 receptorrelated molecular response; in vivo it also leads to the molecular response and 
Page 5 of 58 

causes a number of intervening biological and morphological steps that result in 

an adverse effect. 

 A pesticide and another substance that are known to cause the same toxic effect 

in or at the same anatomical or physiological site or 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 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 

and will provide a scientifically sound basis for determining relative potency of 

chemicals in a cumulative risk assessment. 

3.0 Chemical Structure 

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

Pyrethroids, the synthetic derivatives of 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 i) the addition of halogens to the 

chrysanthamic acid and aromatic alcohol moieties and ii) the addition of the α-cyano 

group, have increased photostability, insecticidal potency, and in some incidences, 

stereoisomerism of the pyrethroids. 
Page 6 of 58 

 Pyrethrin I (Type I) 

O

O

H

O

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

non-coplanar center of unsaturation

acid moiety alcohol moiety

central ester bond

* *

 Resmethrin (Type I) 

O

O

O

* *

 Cypermethrin (Type II) 

 

O

O

Cl

Cl

N

O * * *

* = chiral centers 

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

(cypermethrin) pyrethroids. 
Page 7 of 58 

3.1 Chirality of pyrethroids 

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

enantiomers. For example, in the Type I 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). Furthermore, 

interaction with the (Lund and Narahashi 1982; Narahashi 1982), ability to modify 

sodium channel kinetics (Section 4.0), and metabolism (PK section 4.3.1) are also 

isomer dependent. 

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 sound. 
Page 8 of 58 

Figure 2. Stereochemistry of the pyrethroid acid moiety 
Page 9 of 58 

3.2 Quantitative Structure Activity Relationships (QSAR) 

As stated above, one of the 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 

toxophore2

 (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. 3). Type I and Type II pyrethroids both kill insects. Type I 

pyrethroids possess an effective ‘knockdown’ ability, debilitating insects within minutes. 

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

Type II pyrethroids lack the knockdown 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. 

 

 

2

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

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

substance or substances which imparts a desired pharmacological property. 
Page 10 of 58 

Figure 3. Conformational structure of putative pyrethroid toxophore

As detailed in the following section, 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). 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 depolarization-dependent block of the action potential). 

It is therefore reasonable to assume the toxicophore identified in insects would be 

similar to that in mammals. As such, the Agency further assumes 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, ultimately 

leading to 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)), and in vivo toxicity studies. This paper does not 

provide an exhaustive review of this body of work but highlights and summarizes critical 

data relevant to common mechanism grouping findings. For extensive reviews, see 

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

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

al (2005), Wolansky and Harrill (2008), Nemec (2006). 

 
Page 11 of 58 

4.2 Voltage-Gated Sodium Channels (VGSC) 

4.2.1. Protein structure of the VGSC 

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

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

forming the ion channel pore (Fig. 4). Expression of the α subunit alone is minimally 

sufficient to form a functioning channel. A voltage-sensing element located in the S4 

transmembrane segment detects changes in membrane potential resulting in a 

conformation change of the channel leading to opening and rapid closing of the voltage 

dependent gating mechanism. In vertebrates, smaller auxiliary β subunits modulate α

subunit gating, as well as insertion and distribution of the channel complex into the 

membrane (Isom et al. 1992; Isom et al. 1995; Isom 2001; Smith and Soderlund 2001). 

Insects have a single isoform of the α-subunit, expressed exclusively in the nervous 

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

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

vertebrate β subunit (Warmke et al. 1997). 

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

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

much more complex, and this complexity 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 
Page 12 of 58 

channel expression varies by tissue and stage of development (reviewed in Meacham 

et al. (2008) and Mandel (1992)). The implications of this diversity on uncertainty 

related to actions of pyrethroids will be discussed further in 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 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).
Page 13 of 58 

Figure 5. Effects of pyrethroids on Na channels, currents, and action potentials (From Shafer et al., 2005) 
Page 14 of 58 

4.2.3. Relationship between 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 causing depolarization and/or induce repetitive in peripheral nerves 

(Vijverberg and Bercken 1979, 1982). Similar findings have also been reported in 

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

changes in membrane excitability in 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 
Page 15 of 58 

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 

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 tetrodotoxin-resistant (TTX-R) sodium channels. 

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

while TTX-S channels are relatively less sensitive (Ginsburg and Narahashi 1993; 

Tatebayashi and Narahashi 1994; Song and Narahashi 1996; Narahashi et al. 1998). 

These examples demonstrate the 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
Page 16 of 58 

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. 

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 agedependent 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 artificial settings may alter post-translational modification, 

membrane composition, or phosphorylation status, leading to differences in channel 

sensitivity to pyrethroid modification. 

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 noncyano 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 CMG, this research highlights 
Page 17 of 58 

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 

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 determined interaction with VGSCs is 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) or are secondary to perturbation of the VGSC (Cao et 

al. 2011b). 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).

 
Page 18 of 58 

A class of chloride-permeable channels has been proposed as potential targets 

for pyrethroid actions in the mammalian central nervous system (CNS) (Forshaw et al. 

1993; Ray et al. 1997; Forshaw et al. 2000; Burr and Ray 2004). However, limitations of 

the supporting studies justify cautious interpretation of the results with respect to the 

role of effects on this target in pyrethroid toxicity. A large conductance, chloridepermeable 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 at 10-5 M. A 

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

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

patch’ electrophysiological technique (Walz 2002), thus, it is difficult to associate 

pyrethroid induced changes in maxi chloride-channel function with any other alterations 

in cellular function or excitability. The electrophysiological parameters of the pyrethroidsensitive 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 largeconductance 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-induced neurotoxicity. Some 

antagonism of behavioral measures following deltamethrin exposure was noted. 

Neither compound was effective at reducing the in vivo signs of cismethrin neurotoxicity. 

There are also several limitations related to the in vivo data as well. Ivermectin is a 

competitive agonist of GABA-gated (GABAA) and glycine-gated chloride ion channel 

receptors present in the central and peripheral nervous systems (Abalis et al. 1986; 
Page 19 of 58 

Mascia et al. 1996; Huang and Casida 1997; Dawson et al. 2000; Shan et al. 2001; 

Steinbach and Akk 2001; Lu and Xu 2002; Hadipour-Jahromy and Daniels 2003). At 

the doses used for in vivo studies (Forshaw et al. 2000), the possibility that the in vivo 

signs of pyrethroid neurotoxicity were 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 (Mascia et al. 1996; Steinbach and Akk 2001; Lu and Xu 2002; HadipourJahromy and Daniels 2003). 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 
Page 20 of 58 

consistent with in vivo symptoms. Moreover, using primary cultures of mouse 

neocortical neurons, Cao et al. (2011b) have recently shown that pyrethroids stimulate 

calcium enty into the neurons subsequent to thier actions on VGSCs. Overall, these 

uncertainties and findings indicate that the potential role of direct effects of pyrethroids 

on VGCC in toxicological responses is presently not well enough understood to serve 

as the basis for a CMG. 

Finally, there is evidence that 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 
Page 21 of 58 

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 and there is direct evidence that VGCC activity is secondary to VGSC activation. 

Type II Pyrethroids have also demonstrated the ability to disrupt the ligand-mediated 

chloride current by antagonizing 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, 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 focused on the effects of 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, lambdacyhalothrin, 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 halflives 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 concentrations were achieved under 4 hours (deltamethrin <2; 

lambda-cyhalothrin 2.9; and permethrin 3.5 hours) and maximum brain concentrations 
Page 22 of 58 

under 8 hours (deltamethrin 1-7.3; and lambda-cyhalothrin 2.2-3.1). Finally, the oral 

bioavailability of deltamethrin in the Kim et al. (2008) and Anadón (1996) studies were 

similar at 18 and 14.4 %, respectively. 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 halflives of pyrethroids vary considerably by compound (Table 1). Generally, the pyrethrins 

and first-generation 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 (Anand et al. 2006; Godin et al. 2007; 

Scollon et al. 2009) while the trans-isomers are metabolized by the liver enzymes as 

well as hepatic carboxylesterases (Ross et al. 2006). Oxidative clearance is a relatively 

slow mechanism compared to hydrolysis by carboxylesterases. Therefore, the half-lives 

of trans-isomers are generally shorter by 2 to 10 fold than their cis- counterparts. It has 

been postulated that since the cis-isomers are more persistent in vivo, they are 

inherently more toxic from a pharmacokinetic perspective. 

 

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

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) 

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). 
Page 23 of 58 

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 Tsyndrome 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 T-type, 12 as CS-type and 2 compounds resulted in 

intermediate symptoms. It was also noted that pyrethroids containing the α-cyano 

group were associated with the CS symptoms while those lacking the α-cyano generally 

resulted in T symptoms. The authors did 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 (1982) administered 29 pyrethroids to mice via 

intracerebral (ICV) injections. The pyrethroids were classified as Type I or Type II, 

equivalent to the T- and CS- syndromes described by Verscholyle 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 αcyano 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. (1981) classified pyrethroids as 

either Type I or Type II based on their effects on in vivo and in vitro cercal sensory 

nerve recordings and on symptomology produced in dosed cockroaches. Type I 

pyrethroids produced repetitive firing in the cercal sensory nerve in vitro and trains of 

cercal sensory spikes accompanied by 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-α-cyanophenoxybenzyl ether 

were classified as Type I based on their action on the cercal sensory nerve but resulted 

in symptoms associated with Type II pyrethroids. It is therefore clear from these early 

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

II, based on structure and in vivo symptomology. 

Table 2. Behavioral characterization of pyrethroid toxicity of pyrethroids included in the CMG 

from peer-reviewed literature 

Pyrethroid Verschoyle and 

Aldridge (1980) 

Lawrence and 

Casida (1982) 

Gammon et al. 

(1981) 

Allethrin; S-Bioallethrin T I I 

Permethrin T I I 

Resmethrin T I I 

Fenpropathrin CS/T I/II I/II 

Esfenvalerate CS II II 
Page 24 of 58 

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 repeatedly shown dosedependent 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 also been 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 et al. 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. 

 
Page 25 of 58 

Table 3. Comparison of the signs of pyrethroid poisoning across rodent species and different 

routes of exposure (Extracted from Wolansky and Harrill, 2008)

Mice Rat 

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

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

Mild 

poisoning 

signs 

Hyperactivity 

Circling (mostly ipsilateral 

direction) 

Hyperexcitability 

Aggressive 

sparring 

Restlessness 

Chewing; nosing; 

pawing; burrowing 

Episodes of restlessness 

Chewing; nosing; pawing burrowing; 

head bobbing; whole body shakes 

↑ Sensitivity Salivation ↑ Responsivity Salivation 

 ↑ Responsivity Salivation 

↑↓ responsivity 

Aggressive sparring 

Moderate to 

severe 

poisoning 

signs 

Whole body 

tremors 

Tremors 

followed 

by CA 

Fine tremors 

progressing to 

whole body 

Impaired 

movements 

followed by CA 

(tail, limbs) 

Fine tremors 

progressing to 

whole body 

Tremor followed 

by CA 

Prostration Sinuous 

writhing 

Un-coordinated 

twitches 

Hind limb extensor 

rigidity; rolling gait 

Spontaneous 

muscular 

contractions 

Motor 

incoordination; 

↑ hindlimb 

extensor tone 

Hyperthermia Normothermia 

or hypothermia Hyperthermia Normothermia or 

hypothermia 

Nearly 

lethal 

syndrome 

Clonic 

seizures 

Tonic 

seizures 

Rigor 

occasionally 

just before 

death 

Loss of 

Consciousness; 

Tonic seizures 

Clonic seizures 

Loss of 

consciousness; 

severe writhing 

spasms; tonic 

seizures 

Death, at 10-60 min Death, at 0.5-2 h Death, at 2-8 h 

Table 3 is a compendium of clinical 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), 2Lawrence & Casida (1982), 3Ray (1982), 

4

Verschoyle & Aldridge (1980), 5Wright et al., 1988, 6Souyri & Hoellinger (1983), 

7

Cremer (1983), 8Soderlund et al (2002), 9McDaniel & Moser (1993). †Deltamethrin and 

other cyano compounds induces little or no salivation with icv dosing (Gray & 

Soderlund, 1985). 

 
Page 26 of 58 

 Data from studies on auditory 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 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). This response mechanism 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 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. 

 
Page 27 of 58 

OPP has received acute neurotoxicity for several pyrethroids, including 17 

guideline studies and several additional non-guideline studies. Most of these studies 

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

part of the on-going work to re-evaluate the pyrethroids for registration review3, 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; Williamson et al. 1989; 

Lepeshkin et al. 1992; Villarini et al. 1995; Axelrad et al. 2002). 

Special FOB studies designed to characterize effects classically associated with 

T and CS pyrethroid toxicity for 17 test chemicals (Nemec 2006; Herberth 2010). 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 with 2 or 3 doses. Minimally and moderately effective doses were used to 

establish a spectrum of neurotoxicological effects. 

 

 

3

 http://www.epa.gov/oppsrrd1/registration_review/ 

4 Point of departure (PoD) — the value from a dose-response curve that is at the low end of the 

observable data and that is the toxic dose that serves as the ‘starting point’ in extrapolating a risk to the 

human population.
Page 28 of 58 

In an evaluation of pyrethroid toxicity profiles for 12 pyrethroids, principal component 

analysis and dissimilarity indices were used 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/ λ-cyhalothrin and resmethrin/bifenthrin, respectively. 

Fenpropathrin and pyrethrin had weak responses for either the C or TS characteristics. 

The consistency of the special FOB study with 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. 

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). 

4.4 Data in mixtures of pyrethroids 
Page 29 of 58 

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. 

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, 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 data suggest there may be complicated outcomes from in 

vitro mixtures at the molecular or cellular level. 

More recently, Cao et al. (2011a) designed an in vitro study to specifically 

address potential additivity of pyrethroids. Pyrethroid-induced increases in sodium 

influx were determined in primary cultures of cerebrocortical neurons using the sodiumsensitive dye sodium-binding benzofuran isophthalate (SBFI). Concentrationdependent responses were determined for 11 pyrethroids, including Type I and Type II 

pyrethroids. Depsite differences in individual compound potencies, the mixture of all 11 

pyrethroids were additive when an appropriate statistical model was used. 

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

colleagues, tested a mixture of low doses of both Type I and II pyrethroids (Wolansky 

and Harrill 2008; Wolanksy et al. 2009). With a mixture composed of individual 

chemical at doses 30% lower that the threshold for neurobehavioral effects, motor 

activity was accurately predicted by a single-chemical 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 
Page 30 of 58 

function (Reiter and 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 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 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 α-cyano moiety delay deactivation of sodium channels 

resulting in prolonged tail currents of approximately 500 ms to minutes and are 

associated with clinical effects defined as the CS syndrome. 

 
Page 31 of 58 

Based on this review, OPP has determined that the naturally occurring pyrethrins 

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

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

in disruption of membrane excitability in the nervous system, ultimately leading to 

neurotoxicity characterized by two different toxicity syndromes. The pathway to toxicity 

is illustrated in the Figure 6. The presence of pyrethroids at the target tissue causes a 

disruption in the VGSC, leading to an alteration of action potentials, and ultimately 

resolving as clinical neurotoxicity characteristic of pyrethroid poisoning. 

Figure 6. 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 
Page 32 of 58 

a) Na channel structural heterogeneity among tissue and across life stages with 

respect to alpha- and beta-subunits. 

Another important issue when 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 designation of 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-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 
Page 33 of 58 

influence region, tissue or life stage differences in sensitivity, or may influence the types 

of toxic signs, the common key event in all cases, and the basis for the common 

mechanism grouping, is disruption of VGSC function. 

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

interaction. 

In the cumulative risk 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. 

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 

 

5

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

reactions result in different patterns of toxicity with respect to time to peak effect and time to recovery. 
Page 34 of 58 

modified VGSC and a lack of a complete understanding of how subsequent changes in 

sodium channel function result in specific neurological outcomes. Despite this, the 

scientific evidence correlating pyrethroid-induced changes in VGSC function with 

neurotoxicity is substantial. Given the availability of extensive studies on the 

mechanisms and toxic effects of pyrethroids, the lack of an in vivo biomarker does not 

preclude the common mechanism grouping. 

 
Page 35 of 58 

5.2 Summary 

In summary, although some uncertainties exist, there is a convincing amount of 

data from multiple lines of evidence and 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 Potential 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 is 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 6). The following studies (Choi and 

Soderlund 2006; Nemec 2006; Wolansky et al. 2006; Shafer et al. 2008; Losa et al. 

2009) are unique in that they were conducted on the same pyrethroids with the purpose 

of evaluating the common toxic effects of pyrethroids. The studies represent three 

different levels of biological complexity (sodium channel, nerve cell, whole animal) and 

provide a remarkably similar separation of the 11 chemicals. 
Page 36 of 58 

Figure 7. Differences in Type I and Type II pyrethroids which support forming two subgroups within the 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. The second group is comprised of the 

Type II compounds that 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
Page 37 of 58 

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 
Page 38 of 58 

cultures of cortical neurons. The values ranged from 25 to 1685 nM (Table 4); Type I 

and II pyrethroids resulted in higher and lower 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 

As discussed in section 6.0, special FOB study designed to characterize effects 

classically associated with T and CS pyrethroid toxicity for 17 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 cyanocontaining 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. 

Table 5. Behavioral characterization of pyrethroid toxicity by PWG 

Pyrethroid WIL* 

Allethrin; S-Bioallethrin T 

Bifenthrin T 

Cyfluthrin CS 
Page 39 of 58 

Cyhalothrin, lambda- CS 

Cypermethrin CS 

Deltamethrin CS 

Esfenvalerate MIXED

Fenpropathrin T 

Permethrin T 

Resmethrin T 

Tefluthrin T 

6.4. Potential to Subgroup 

Table 6 below combines the information described in Sections 6.1-6.3 and thus 

summarizes data at the channel level (Nav 1.8 kinetics), the tissue level (MEA IC50s) 

and the behavioral level. Examination of the table indicates a clear pattern that the 11 

pyrethroids separate into 2 groups based on structure, ion channel gating activity, and 

neurobehavioral effects, with two chemicals, esfenvalerate and fenpropathrin, 

representing mixed Type I/II. 

 

In the event that refinements to the CRA are needed, there is the potential to 

subgroup the pyrethrins and pyrethroids into Type I (lacking alpha-cyano moiety) and 

Type II (bearing alpha-cyano moiety) based upon differences in structure, channel 

gating activity, and neurobehavioral effects. 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. 
Page 40 of 58 

Table 6. Summary of key data supporting the subgrouping into Type I and Type II 

Pyrethroid 

Nav 1.8 Kineticsa MEA IC50sd

WILe

Activationb tau1c tau2c

Allethrin; SBioallethrin 3.9 1.9 NA 1525 T 

Bifenthrin 3.9 4 NA 439 T 

Permethrin 2.5 2.3 NA 719 T 

Resmethrin 3.3 2.6 NA 1685 T 

Tefluthrin 3.8 4.3 NA 499 T 

Esfenvalerate 10.7 13.4 NA 809 MIXED 

Fenpropathrin 10.8 12.4 NA 1518 T 

Cyfluthrin 23.6 7.1 95.9 305 CS 

Cyhalothrin, lambda- 22.3 9.4 84.7 25 CS 

Cypermethrin 12.6 6.9 55.8 181 CS 

Deltamethrin 15.7 5.3 52.1 175 CS 

a

Gating kinetics of the rat 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 primary neuronal cultures (Scollon et al. 2010) 

e

Neurotoxic syndrome expressed by rats following high doses of pyrethroids as defined by FOB 

observations (Weiner et al. 2009)) 

Attachment 1 provides the structures of the preliminary list of pyrethrins and 

synthetic pyrethroids included in the CMG. Attachment 1 also provides assignments 

(i.e., Type I or II) to 11 pyrethroids and the pyrethrins. These assignments were made 

based on the information in Table 6 and the results of the special FOB for pyrethrins. 

Five additional pyrethroids (cyphenothrin, imiprothrin, prallethrin, tetramethrin, and dphenothrin) were tested in a special FOB study similar to the study sponsored by the 

PWG (Herberth 2010) and have been categorized as Type I or Type II. For all 

remaining pyrethroids, OPP first evaluated 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 used structure (i.e., presence/absence of an αcyano group) as the determining factor for assigning individual pyrethroids to a 

subgroup. 
Page 41 of 58 

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Attachment 1.0. 

EPA’s classification and structure of pyrethroids registered for use with the US EPA. 

Name Type I/II Structure 

Allethrin (s-Bioallethrin, 

d-allethrin) I 

Bifenthrin I

Cyfluthrin (β-cyfluthrin) II

Cyhalothrin (γ/λcyhalothrin) II

Cypermethrin (α/β/θ/ζcypermethrin) II

Cyphenothrin II 

Deltamethrin II
Page 56 of 58 

Name Type I/II Structure 

Fenpropathrin Mixed I/II

Fenvalerate 

(esfenvalerate) Mixed I/II

Flucythrinate II 

Flumethrin II 

Fluvalinate (τ-fluvalinate) II 

Imiprothrin I 

Metofluthrin I 

Permethrin I
Page 57 of 58 

Name Type I/II Structure 

Prallethrin I 

Resmethrin 

(bioresmethrin, 

cismethrin) 

I

Phenothrin (dphenothrin, sumithrin) I 

Tefluthrin I

Tetramethrin I

Tralomethrin II

Pyrethrins I

Cinerin I I
Page 58 of 58 

Name Type I/II Structure 

Cinerin II I

Jasmolin I I

Jasmolin II I