Toxicology 171 (2002) 3–59 www.elsevier.com/locate/toxicol

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David M. Soderlund a,*, John M. Clark b, Larry P. Sheets c, Linda S. Mullin d, Vincent J. Piccirillo e, Dana Sargent f, James T. Stevens g, Myra L. Weiner h

a Department of Entomology, New York State Agricultural Experiment Station, Cornell Uni6ersity, Gene6a, NY 14456, USA b Department of Entomology, Uni6ersity of Massachusetts, Amherst, MA 01003, USA c Bayer Corporation, 17745 South Metcalf A6enue, Stilwell, KS 66085, USA d DuPont Crop Protection, Stine-Haskell Research Center, P.O. Box 30, Newark, DE 19714, USA e VJP Consulting, Inc., 22636 Glenn Dri6e, Suite 304, Sterling, VA 20164, USA f A6entis CropScience, 2 T.W. Alexander Dri6e, P.O. Box 12014, Research Triangle Park, NC 27709, USA g Syngenta Crop Protection, Inc., 410 Swing Road, Greensboro, NC 27409, USA h FMC Corporation, P.O. Box 8, Princeton, NJ 08543, USA Received 25 September 2001; accepted 10 October 2001

Abstract

The Food Quality Protection Act (FQPA) of 1996 requires the United States Environmental Protection Agency to consider the cumulative effects of exposure to pesticides having a ‘common mechanism of toxicity.’ This paper reviews the information available on the acute neurotoxicity and mechanisms of toxic action of pyrethroid insecticides in mammals from the perspective of the ‘common mechanism’ statute of the FQPA. The principal effects of pyrethroids as a class are various signs of excitatory neurotoxicity. Historically, pyrethroids were grouped into two subclasses (Types I and II) based on chemical structure and the production of either the T (tremor) or CS (choreoathetosis with salivation) intoxication syndrome following intravenous or intracerebral administration to rodents. Although this classification system is widely employed, it has several shortcomings for the identification of common toxic effects. In particular, it does not reflect the diversity of intoxication signs found following oral administration of various pyrethroids. Pyrethroids act in vitro on a variety of putative biochemical and physiological target sites, four of which merit consideration as sites of toxic action. Voltage-sensitive sodium channels, the sites of insecticidal action, are also important target sites in mammals. Unlike insects, mammals have multiple sodium channel isoforms that vary in their biophysical and pharmacological properties, including their differential sensitivity to pyrethroids. Pyrethroids also act on some isoforms of voltage-sensitive calcium and chloride channels, and these effects may contribute to the toxicity of some compounds. Effects on peripheral-type benzodiazepine receptors are unlikely to be a principal cause of pyrethroid intoxication but may contribute to or enhance convulsions caused by actions at other target sites. In contrast, other putative target sites that have been identified in vitro do not appear to play a major role in pyrethroid intoxication. The diverse toxic actions and pharmacological effects of pyrethroids suggest that simple additivity

* Corresponding author. Tel.: +1-315-787-2364; fax: +1-315-787-2326. E-mail address: [email protected] (D.M. Soderlund).

0300-483X/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0300-483X(01)00569-8 4 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 models based on combined actions at a single target are not appropriate to assess the risks of cumulative exposure to multiple pyrethroids. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: FQPA; Pyrethroids; Toxicity; Sodium channel; Calcium channel; Chloride channel; Common mechanism

1. Introduction detail and only as they relate to the principal emphasis of this review. This document is in- The Food Quality Protection Act (FQPA) of tended to serve as a contemporary review of the 1996 dramatically altered the regulation of pesti- state of the science in the field of pyrethroid cides in the United States. This Act, which neurotoxicology and to inform and aid the regula- amended both the Federal Insecticide, Fungicide tory review of pyrethroids under the common and Rodenticide Act and the Federal Food, Drug mechanism statute of the FQPA. and Cosmetics Act, requires the United States Environmental Protection Agency (EPA) to con- sider a number of factors relative to the toxicol- 2. Structures and insecticidal properties of ogy of pesticides, including the cumulative effects pyrethroids of exposure to pesticides having a ‘common mech- anism of toxicity’. The methods for identifying 2.1. What is a pyrethroid? pesticides having a common mechanism of toxic- ity, and the ramifications of such a determination, The term ‘pyrethroid’ is commonly used to are only now beginning to be clarified. designate a synthetic insecticide that is derived Pyrethroids are a class of synthetic insecticides structurally from the natural pyrethrins, the six that have been designed and optimized based on insecticidal constituents of pyrethrum extract the structures of the pyrethrins, the six insecticidal (Fig. 1). Decades of research and development by constituents of the natural insecticide pyrethrum the agrochemical industry and by government and (Elliott, 1995). Since the 1970s the pyrethroids academic research laboratories have resulted in a have been widely used to control insect pests in wide range of pyrethroid structures and a multi- agriculture and public health. By the mid-1990s, tude of uses in agricultural, veterinary, medical pyrethroid use had grown to represent 23% of the and household pest control. The development of U.S. dollar value of the world insecticide market, synthetic pyrethroid insecticides has involved an ranking second only to organophosphorus com- iterative process of structural modification and pounds among insecticide classes (Casida and biological evaluation, so that the array of present- Quistad, 1998). day commercial and experimental insecticides This paper reviews the data available from the identified as ‘pyrethroids’ includes compounds published literature and from unpublished studies that are several conceptual steps removed from by pyrethroid manufacturers on the toxicity and the pyrethrins. In this review, we focus specifically mechanisms of toxic action of the pyrethroids in on pyrethrum (Fig. 1) and the following synthetic mammals. Pyrethroids as a class are acute neuro- pyrethroid insecticides that are registered for use toxicants, although some individual compounds in the United States and many other parts have been associated with the production of toxic of the world, either as fully racemic mixtures, effects on other organ systems in long-term feed- mixtures of specified isomers, or single resolved ing studies. In the context of the implementation isomers (Fig. 2): allethrin (both bioallethrin and of the FQPA and the need to discern common S-bioallethrin), bifenthrin, cyfluthrin (including mechanisms of toxicity, this review therefore fo- beta-cyfluthrin), cyhalothrin (including lambda- cuses on the neurotoxic actions of pyrethroids in cyhalothrin), cypermethrin (including zeta- mammals. Other topics (e.g. insecticidal activity; cypermethrin), deltamethrin, fenpropathrin, fen- toxicokinetics and metabolism) are covered in less valerate (including esfenvalerate), permethrin, D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 5

Fig. 1. Structures of the six natural pyrethrins. resmethrin, tefluthrin, tetramethrin, and covered by the sequential replacement of struc- tralomethrin. tural elements of the pyrethrins with novel structural moieties that were selected to conserve 2.1.1. De6elopment of synthetic pyrethroids the molecular shape and physical properties of the The discovery and development of synthetic template structure. Because the pyrethrins are es- pyrethroid insecticides has been the subject of ters of a cyclopropanecarboxylic acid and a cy- numerous reviews. The summary that follows is clopentenolone alcohol (Fig. 1), synthetic based primarily on a recent comprehensive review modifications typically held one of these major by Elliott (1995) and on earlier sources cited domains of the molecule constant while introduc- therein. ing new structural features in the other. Although The principal drawback of pyrethrum as an the early stages of this process made reference to insecticide is its instability in light and air, which the natural pyrethrins as templates, subsequent limits its effectiveness in crop protection and stages employed newly discovered synthetic other insect control contexts in which residual pyrethroids with desirable insecticidal activity, activity is essential. The development of synthetic stability, and other properties as the templates for pyrethroids is the result of efforts to modify the the further design of new compounds. structure of the natural pyrethrins (Fig. 1) in Both the historical development of pyrethroids order to increase photostability while retaining as an insecticide class and the structural diversity the potent and rapid insecticidal activity and rela- of pyrethroids are illustrated by the compounds tively low acute mammalian toxicity of currently registered for use in the United States pyrethrum. Most synthetic pyrethroids were dis- (Fig. 2). Allethrin, one of the earliest of synthetic 6 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 Fig. 2. Structures of synthetic pyrethroids registered for use in the United States. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 7 pyrethroids still in current use, represents one The structural diversity of synthetic pyrethroids initial synthetic approach, the replacement of the was further enhanced by the discovery that the pentadienyl side chain of pyrethrin I with a sim- 2,2-dimethylcyclopropanecarboxylic acid moiety pler, synthetically more accessible moiety having of the pyrethrins and most synthetic compounds similar steric and electronic properties. Other could be replaced by an a-isopropylphenylacetic early synthetic analogues were designed by replac- acid moiety. This new series of compounds led to ing the center of unsaturation represented by the the discovery of the commercial insecticide fen- alkenyl side chain of the alcohol moieties of valerate (Fig. 2). More radical changes based on pyrethrins and allethrin by an aromatic sub- this series included replacement of the central stituent. The next significant step in pyrethroid ester bond with structures (e.g. etofenprox; see development involved the replacement of the cy- Fig. 3) that retained the overall configuration of clopentenolone ring of the pyrethrin and allethrin the molecule. These series yielded candidate com- alcohols with an alternative unsaturated hetero- mercial insecticides with pyrethroid-like insectici- cyclic moiety, resulting in resmethrin (Fig. 2). dal activity, but examples of these compounds are This compound not only exhibited increased pho- not registered for use in the United States. tostability but also was substantially more potent as an insecticide and lower in acute mammalian 2.1.2. Structure–acti6ity relationships for toxicity than pyrethrin I. The combination of insecticidal acti6ity these desirable properties in a single molecule It is difficult to identify a structural relationship provided a strong impetus to search for new between compounds as diverse as the non-ester compounds with greater activity and photostabil- pyrethroid etofenprox and pyrethrin I without ity. Examples of other commercially important knowledge of the structures of the entire pyrethroids that are cyclopropanecarboxylate es- pyrethroid series. Nevertheless, pyrethroids as di- ters of modified alcohol moieties include te- verse structurally as these two compounds con- tramethrin, bifenthrin, and tefluthrin (Fig. 2). form to a single, operationally defined Permethrin (Fig. 2) proved to be the first syn- structure–activity relationship for insecticidal ac- thetic pyrethroid with sufficient photostability for tivity that is based on the physical properties, agricultural use. When compared to resmethrin, shape and three-dimensional configuration of the this compound contains structural replacements entire molecule (Elliott et al., 1974a). Fig. 3 iden- in both the alcohol moiety (3-phenoxybenzyl for tifies the principal elements of the pyrethroid 5-benzyl-3-furylmethyl) and the acid moiety (chlo- structure–activity relationship and illustrates rines for methyl groups) that confers enhanced these elements with four structurally divergent photostability without loss of insecticidal activity. pyrethroid insecticides. It is evident from this Inclusion of a a-cyano substituent in the 3-phe- analysis that there is no specific substructure, noxybenzyl alcohol moiety, as in deltamethrin reactive entity, or molecular moiety that can be (Fig. 2), produced compounds with much greater identified as the toxophore conferring pyrethroid- insecticidal potency than permethrin but with sim- like insecticidal activity. Instead, such activity ap- ilar photostability. Synthetic pyrethroids related parently results from the appropriate fitofthe in structure to permethrin and deltamethrin, entire molecule at the site of action, and a wide which constitute the largest chemical subfamily of variety of chemical structures have been shown to pyrethroids in current use, include cypermethrin, satisfy the requirements for fit at that site based cyfluthrin, cyhalothrin, fenpropathrin and on insecticidal activity data. tralomethrin (Fig. 2). These compounds exhibit The significance of overall molecular shape in structural features that confer an expanded range the action of pyrethroids is most clearly evident in of insecticidal activity, enhanced overall insectici- the stringent stereospecificity of insecticidal ac- dal potency, modified photostability, or other de- tion. The presence of two chiral centers at carbon- sirable properties when compared to pyrethrum 1 and carbon-3 of the chrysanthemic acid moiety or earlier synthetic pyrethroids. of pyrethrin I produces two pairs of 8 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

Fig. 3. Essential features of pyrethroid structure–activity relationships illustrated by pyrethrin I and three synthetic pyrethroids. diastereomers, which are designated trans and cis ety is structurally congruent with 1R cyclo- based on the orientation of the C-1 and C-3 propanecarboxylates and gives insecticidal esters, substituents in relation to the plane of the cyclo- whereas the corresponding 2R esters are inactive propane ring (Fig. 4). The acid moieties of the (Fig. 4). Stereospecific determinants of insecticidal natural pyrethrins are exclusively in the 1R,trans activity are implicit even in achiral acid moieties, configuration. When esters were prepared from such as 2,2,3,3-tetramethylcyclopropanecarboxylic the four resolved chrysanthemic acid isomers, acid, the acid moiety of the insecticide fen- those with the R configuration at cyclopropane propathrin. Removal of one methyl group from C-1 were insecticidal, whereas the enantiomeric this symmetrical acid creates a chiral cyclo- 1S compounds, though physically identical, were propanecarboxylate that obeys the same struc- without insecticidal activity (Elliott et al., 1974a). ture–activity rules as those elaborated for This profound stereospecificity extends to com- chrysanthemic acid isomers (Elliott et al., 1974a). pounds such as fenvalerate, in which the 2S Stereoisomerism is a less common feature of configuration of the non-cyclopropane acid moi- pyrethroid alcohol moieties. Nevertheless, when a D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 9 chiral center is present in the alcohol moiety at Confirmation that voltage-sensitive sodium the carbon bearing the hydroxyl group (e.g. al- channels are the principal sites of insecticidal ac- lethrin and deltamethrin; see Fig. 2), only one tion of pyrethroids has emerged from studies of epimer has high insecticidal activity even when the molecular genetics of mechanisms of esterified to an acid moiety that contains the pyrethroid resistance that involve reduced nerve appropriate stereochemical configuration for high sensitivity (Soderlund, 1997; Soderlund and Knip- insecticidal activity. ple, 1999). In the housefly, the kdr (‘knockdown resistance’) and super-kdr (enhanced knockdown 2.2. Mechanism of insecticidal action resistance) traits confer resistance to all pyrethroids by reducing the sensitivity of the fly Pyrethroids are known to alter the normal nervous system to these compounds. Genetic link- function of insect nerves by modifying the kinetics age analyses mapped these traits close to the of voltage-sensitive sodium channels, which medi- principal voltage-sensitive sodium channel gene of ate the transient increase in the sodium permeabil- the housefly (designated Vssc1). Moreover, kdr- ity of the nerve membrane that underlies the nerve like resistance in other insect species has been action potential (Soderlund and Bloomquist, mapped to sodium channel genes that are or- 1989; Bloomquist, 1993a). The action of pyrethroids on insect and other invertebrate thologous to Vssc1. DNA sequence analyses of sodium channels, and the correlation of these Vssc1 coding sequences from susceptible and re- effects with insecticidal activity, has been reviewed sistant housefly strains identified a single mutation extensively (Sattelle and Yamamoto, 1988; Soder- common to all resistant strains and a second lund and Bloomquist, 1989; Narahashi, 1992; mutation found only in the highly-resistant super- Bloomquist, 1993a; Soderlund, 1995; Narahashi, kdr strains. Insertion of these mutations into a 1996). A more detailed summary of the actions of susceptible Vssc1 cDNA, followed by functional pyrethroids on sodium channels will be presented analysis of the susceptible and specifically mu- in a subsequent section of this review. tated sodium channels, showed that these muta- tions were sufficient to account for the resistance caused by the kdr and super-kdr traits. Therefore, the sodium channels encoded by the Vssc1 gene of the housefly and by orthologous genes of other insect species must be the principal sites of insecti- cidal action of pyrethroids. An unanticipated finding from these molecular genetic studies was the insensitivity to pyrethroids of sodium channels containing the two mutations associated with the super-kdr resistance trait (Lee et al., 1999; Lee and Soderlund, 2001). It remains to be determined whether sodium channels from super-kdr insects, when assayed in their native neuronal environment, are equally insensitive to pyrethroids. If the insensitivity of expressed chan- nels is confirmed in assays with native neuronal preparations, then the modest but clearly demon- strable toxicity of pyrethroids to super-kdr insects may involve one or more target sites other than

Fig. 4. Stereochemical determinants of insecticidal activity in the voltage-sensitive sodium channel encoded by pyrethroid acid moieties. the Vssc1 gene. 10 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

3. Mammalian toxicity 3.1.2. Structure–toxicity relationships Determination of the acute toxicity of the re- 3.1. Acute toxicity solved optical isomers of several pyrethroids fol- lowing intravenous administration to rats 3.1.1. Acute toxicity to mammals (Verschoyle and Aldridge, 1980) and intracerebral Prior to 1970, there was little available informa- administration to mice (Lawrence and Casida, tion on the acute toxicity to mammals of the 1982) permitted the elucidation of structure–ac- natural pyrethrins or the limited group of syn- tivity relationships for acute mammalian toxicity. thetic pyrethroids known at that time. The first The principal features of these relationships, sum- systematic study of pyrethroid toxicity (Ver- marized below, are drawn from a more detailed schoyle and Barnes, 1972) compared the acute analysis published elsewhere (Gray and Soder- oral and intravenous toxicities to rats of lund, 1985). pyrethrum, pyrethrin I, pyrethrin II, bioallethrin, The neurotoxicity of pyrethroids to mammals isomers and isomer mixtures of resmethrin, and a depends on the stereochemical configuration at structural relative of resmethrin. This study docu- cyclopropane C-1 or the homologous position in mented the modest oral toxicity of the tested compounds lacking the cyclopropanecarboxylate compounds, the significant intravenous toxicity of moiety (see Fig. 4). Only esters of 1R cyclo- the pyrethrins and bioallethrin, and the profound propanecarboxylates and isosteric 2S isomers of differences in the toxicity of resmethrin isomers non-cyclopropane acids are neurotoxic, whereas by both routes of administration. the corresponding 1S cyclopropanecarboxylates The discovery during the 1970s of numerous and their sterically equivalent 2R acyclic analogs new pyrethroids with the potential for widespread are without measurable toxicity even when admin- use in agriculture stimulated more extensive stud- istered at high doses directly to the CNS (Gray ies of pyrethroid toxicity both in the context of and Soderlund, 1985). This relationship parallels academic research and in support of the registra- the stereospecificity of insecticidal action of tion of insecticide products. As a result, there now pyrethroids (see Section 2.1.2). exists a large and diverse set of data on the acute In mammals, the absolute configuration at cy- toxicity of pyrethroids to mammals by routes of clopropane C-3 of cyclopropanecarboxylate esters administration relevant to human exposure (e.g. of primary alcohols (e.g. resmethrin, permethrin) oral) as well as routes (e.g. intravenous, intraperi- also strongly influences toxicity (Table 2). Typi- toneal, intracerebral) not directly relevant to hu- cally, pyrethroids from this group of compounds man exposure but instead chosen for the precise having the 1R,cis configuration (e.g. [1R,cis]-per- experimental manipulation of dose and the direct methrin and NRDC 157, Table 2) are both insec- delivery of toxicant to the presumed target organ. ticidal and toxic to mammals, whereas the Table 1 summarizes the oral toxicity to rats of corresponding pyrethroids having the 1R,trans several pyrethroids that are currently registered configuration (e.g. [1R,trans]-permethrin, Table for use in the United States. With few exceptions, 2), though similar in insecticidal potency, lack these compounds have acute oral LD50 values measurable acute toxicity to mammals. Initially, following administration in vegetable oils lying the low toxicity of these 1R,trans compounds to between 50 and 500 mg/kg and are therefore mammals was ascribed to their rapid hydrolytic considered to be moderately toxic (EPA Category detoxication by liver esterases (see Section 4.3), II). As illustrated in Table 1, the acute oral toxic- but intracerebral dosing experiments (Lawrence ity of any pyrethroid varies widely depending on and Casida, 1982) demonstrated that these com- the administration vehicle used. In contrast to the pounds had very low intrinsic toxicities even when moderate oral toxicity of most pyrethroids, the the impact of biotransformation was removed. pyrethroids as a class exhibit very low levels of This dependence of the acute toxicity of cyclo- systemic toxicity following dermal exposure propanecarboxylate esters of primary alcohols on (Clark, 1995). stereochemical configuration at C-3 is generally D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 11

Table 1 Acute oral toxicities of pyrethroids to rats

a Compound Strain Vehicle LD50 (mg/kg) Reference

Male Female

Bioallethrin S Corn oil 395 410 (Furnax and Audegond, 1985a) S-Bioallethrin SCorn oil 370 320 (Furnax and Audegond, 1985b) Bifenthrin S Corn oil70.1 53.8 (Freeman, 1982) Lambda-cyhalothrin ACorn oil 79 56 (Southwood, 1985a) Aqueous299 433 (Allen and Leah, 1990) suspensionb Cyfluthrin WAcetone+peanut oil 155 160 (Heimann, 1987) Cypermethrin S Corn oil 297 372 (Freeman, 1987) Undiluted 7654 7180 (Rand, 1983) Zeta-cypermethrin S Corn oil 134 86 (Freeman, 1989) Deltamethrin S87Corn oil 95 (Varsho, 1996) Aqueous\5000 \5000 (Myer, 1989) suspensionc Fenpropathrin SCorn oil 70.6 66.7 (Hiromori et al., 1983) Fenvalerate S Corn oil 370d 370d (Bilsback et al., 1984) Esfenvalerate S87Corn oil 87e e (Bilsback et al., 1984) Permethrin W Corn oil 1200e 1200e (Killeen, 1975) Undiluted 8900e 8900e (Killeen, 1975) Pyrethrum S Corn oil 710 320 (Gabriel, 1992) Resmethrin SCorn oil 1695 1640 (Glomot, 1979) Tefluthrin A Corn oil 21.8 34.6 (Southwood, 1985b) Tralomethrin SSesame oil 99 157 (Audegond et al., 1981a) Aqueous\1000 and \1000 and (Audegond et al., 1981b) suspensionf B5000 B5000

a Strain: A, Alderley Park; S, Sprague–Dawley; W, Wistar. b Containing 0.5% hydroxypropylmethylcellulose+0.1% polysorbate 80. c Containing 1% methylcellulose. d Exact LD50 not calculated; mortality less at higher doses. e Combined value for males and females. f Containing 0.25% carboxymethylcellulose+0.2% polysorbate 80. applicable to this class, but two significant excep- mammalian toxicity, this effect is highly tions are known: [1R,cis]-phenothrin lacks mea- stereospecific: the a-R epimers of compounds that surable toxicity to mammals (see Table 2), retain the appropriate configurations for high tox- whereas [1R,trans]-ethanomethrin1 (a close struc- icity in the acid moiety have no demonstrable tural relative of bioresmethrin) is a potent central toxicity when injected directly into the brain (e.g. neurotoxicant in mammals (Lawrence and Casida, NRDC 156B, Table 2). The a-cyano substituent 1982). also indirectly alters structure–toxicity relation- In mammals, as in insects, the presence of an ships in the acid moiety. The most dramatic ef- a-cyano substituent in S configuration in the 3- fects are seen with the 1R,trans cyclopropane- phenoxybenzyl alcohol moiety also greatly en- carboxylates of 3-phenoxybenzyl alcohol (e.g. hances acute neurotoxicity. In the case of [1R,trans]-permethrin, Table 2), which exhibit ex- tremely low toxicity to mammals; addition 1 5-Benzyl-3-furylmethyl-3-cyclopentylidenemethyl-2,2-di- of an a-cyano substituent in the S configuration methylcyclopropanecarboxylate, also designated RU 11679. to these esters produces compounds (e.g. 12 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

Table 2 Effects of pyrethroid structure and stereochemistry on intracerebral acute toxicity to mice

m a Name or designation X IsomerR LD50 ( g/g brain)

\ Phenothrin CH3 trans H 8600 \ Phenothrin CH3 cis H 4300 Permethrin C1 trans H \860 Permethrin C1cis H 11 NRDC 157 Br cis H 5.4b

Cyphenothrin CH3 trans S-CN 12 Cyphenothrin CH3 cis S-CN 3.9 Cypermethrin C1 trans S-CN 1.6 Cypermethrin C1 cis S-CN 0.6 De tamethrin Br cis S-CN 0.5, 1.2 NRDC 156B Br cis R-CN \860b

a Data from Lawrence and Casida (1982) except were noted. b Data from Ghiasuddin and Soderlund (1985), recalculated on the basis of mg/g brain weight.

[1R,trans,aS]-cypermethrin, Table 2) with signifi- 3.2. Signs of pyrethroid intoxication cant neurotoxicity to rodents. These results demonstrate that, whereas Two studies published in the early 1970s iden- pyrethroid structure–toxicity relationships in tified two distinct syndromes associated with the mammals are generally congruent with structure– acute toxicity of pyrethroids to rats. Verschoyle activity relationships in insects, there are never- and Barnes (1972) provided the first systematic theless significant differences. These differences, as description of the signs of pyrethroid intoxication well as the individual compounds noted above in rats following oral and intravenous dosing. that are exceptions to the general structure–activ- These authors noted the same syndrome of intox- ity rules, underscore the risks of generalizing ication for pyrethrins, bioallethrin, resmethrin, mammalian structure–toxicity relationships be- and NRDC 1082 (an experimental pyrethroid yond compounds for which direct comparative structurally related to resmethrin) by either route data exist. of administration. This syndrome included hyper- Available structure–toxicity data show that sensitivity and aggression followed by stimulus-in- acute toxicity of pyrethroids to mammals depends duced bouts of general tremor, convulsive on the shape and three-dimensional configuration twitching, coma, and death. The principal differ- of the entire molecule. The absence of any ence observed between oral and intravenous dos- single structural feature or reactive moiety that is ing was the speed of onset of intoxication. required to produce pyrethroid-like toxicity im- plies that there is no common toxophore that mediates the acute toxicity of pyrethroids to 2 5-Benzyl-3-furylmethyl-2,2,3,3-tetramethylcyclopropane- mammals. carboxylate. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 13

The publication of the discovery of deltamethrin which was designated the CS (choreoathetosis (Elliott et al., 1974b), the first pyrethroid contain- with salivation) syndrome, whereas four produced ing the a-cyano-3-phenoxybenzyl moiety, was ac- the T syndrome of intoxication. One a-cyano-3- companied by a brief report describing the acute phenoxybenzyl ester and one compound in which toxicity of deltamethrin to rats (Barnes and Ver- the a-cyano group was replaced by an a-ethynyl schoyle, 1974). This report noted that the signs of group were found to produce elements of both deltamethrin intoxication following either oral or syndromes (tremor with salivation). This classifi- intravenous administration, which involved saliva- cation of the signs of pyrethroid intoxication into tion without lacrimation followed by jerking leg two principal syndromes was confirmed in studies movements and progressive writhing convulsions of the intracerebral toxicity of 29 pyrethroids to (choreoathetosis), were distinctly different than mice (Lawrence and Casida, 1982). The major those reported by these authors for other difference in the findings of these two studies was pyrethroids (Verschoyle and Barnes, 1972). Inter- that fenpropathrin, classified as a T syndrome estingly, the signs of deltamethrin intoxication in compound in the first study, was classified as freely-moving rats following direct infusion into exhibiting both syndromes of intoxication in the three different regions of the central nervous sys- second study. tem (lateral ventricle, cisterna magna, or sub- An alternative nomenclature (Types I and II) arachnoid space) produced signs of intoxication has been proposed for subgroups of pyrethroids specific to each site of administration that were based not only on the syndromes of intoxication not observed following intravenous administration produced in mammals (Lawrence and Casida, 1982) but also on their chemical structures, their (Brodie, 1985). Whereas region-specific infusion signs of poisoning in insects and their actions on also produced some of the motor signs typical of insect nerve preparations (Gammon et al., 1981). deltamethrin intoxication by other routes, admin- The Type I/II nomenclature has been widely istration of deltamethrin by these routes failed to adopted in the literature and is often used in a produce either choreoathetosis or salivation. manner parallel to the T/CS nomenclature, so that These results suggest that multiple target tissues or Type I compounds are generally considered to regions of the central nervous system are involved produce the T syndrome of intoxication and Type in producing the signs of toxicity observed follow- II compounds are considered to produce the CS ing intravenous administration and suggest that syndrome (Lawrence and Casida, 1982). However, the signs associated with deltamethrin intoxication we have chosen to employ the T/CS nomenclature may be route-dependent. in this review because it reflects the historic clas- A landmark study (Verschoyle and Aldridge, sification of pyrethroid action in terms most 1980) described both the acute toxicity and the clearly related to the mammalian toxicology of signs of intoxication of 36 pyrethroids following these compounds. intravenous administration, thereby establishing a Although these studies provide an important taxonomy of pyrethroid intoxication in mammals conceptual framework for interpreting the toxic that persists to the present. Of the 18 esters of actions of pyrethroids in mammals, they are not various primary alcohols examined in this study, comprehensive from the perspective of compounds 15 compounds produced signs of intoxication cor- currently registered for use. Of the 14 registered responding to those first described for pyrethrins pyrethroid structures specifically considered and pyrethroids (Verschoyle and Barnes, 1972), in this review, pyrethrins and eight synthetic which was designated the T (tremor) syndrome, pyrethroids (allethrin, cypermethrin, deltamethrin, whereas the remaining three compounds did not fenvalerate, fenpropathrin, permethrin, res- produce any detectable signs of intoxication at the methrin, and tetramethrin) were included in at highest doses tested. Of the 17 esters of a-cyano-3- least one of the original comparative studies and phenoxybenzyl alcohol examined, 12 produced therefore could be considered to be classified in signs of intoxication like those first described for terms of intoxication syndrome within the T/CS deltamethrin (Barnes and Verschoyle, 1974), classification scheme. However, five currently 14 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 registered compounds (bifenthrin, cyfluthrin, cy- that are currently registered for use in the U.S. As halothrin, tefluthrin, and tralomethrin) were not a screen for neurotoxicity, these studies rely on included in either study. Therefore, their tentative neurobehavioral tests that include clinical obser- inclusion in the T/CS classification scheme must vations, a procedurally standardized functional be based on studies done in other laboratories by observational battery (FOB), measurements of methods other than those used in the two studies motor activity in an automated device, and termi- that generated the classification of syndromes. nal microscopic examination of neural tissues. In Given that the primary classification study (Ver- acute studies, the FOB and motor activity tests schoyle and Aldridge, 1980) did not describe a are performed at least four times: prior to treat- simple relationship between pyrethroid structure ment, at the time of peak effects (when signs of and intoxication syndrome and also identified intoxication are most pronounced, determined for some compounds with signs of intoxication that each compound in a preliminary range-finding did not fall neatly into either the T or CS class, study) following the administration of a single any assumptions about intoxication syndrome dose, and again 7 and 14 days following treat- produced by compounds other than those con- ment. In subchronic studies, animals are also tained in the two canonical classification studies tested on four occasions: prior to the initiation of must be made with great caution. Moreover, both treatment and again during weeks 4, 8 and 13 of studies on which the classification of symptoms is sustained treatment. Animals are also evaluated based employed routes of administration (intra- for clinical signs of intoxication by cage-side ob- venous or intracerebral) that are not relevant to servations that are performed at least once daily routes of human exposure. Although the initial for acute studies and once weekly for the sub- descriptions of the T and CS syndromes suggested chronic studies. that the signs of intoxication were independent of The regulatory neurotoxicity studies summa- the route of administration (Verschoyle and rized here were performed in several different Barnes, 1972; Verschoyle and Aldridge, 1980), it is laboratories, using similar, but not identical, pro- not clear from the available data that this general- cedures. The FOB used in each of these studies is ization extends to all pyrethroids or to all routes based on the procedure described by Moser of administration. Without a corresponding data (1989), in which the observer was blind with set on the signs of intoxication following oral respect to the animal’s dose group assignment to administration under comparable experimental ensure an unbiased assessment. The FOB begins conditions, any assumptions about the universal- with cage-side observations that are conducted ity of the T/CS classification scheme for all routes prior to disturbing the animal and then continues of exposure must also be made with caution. with a detailed physical examination. Next, the animal is placed into an open field to facilitate 3.3. Beha6ioral assessments of pyrethroid observations including gait, activity level, posture, neurotoxicity and any unusual behaviors. At the end of this period, the reaction to various stimuli is evalu- 3.3.1. Regulatory neurotoxicity studies: ated, along with assessments of pupil size, pupil background and methodology response, and aerial righting. The FOB typically Acute and subchronic adult neurotoxicity concludes with measurements of grip strength, screening studies, conducted in accordance with landing foot splay, and body weight. Laboratories the regulatory guidelines3 provided by the U.S. are required to establish the adequacy of observer EPA, represent a potential source of additional training using reference compounds as positive information about the actions of the pyrethroids controls for the production of well-defined effects. It is also necessary to control inter-observer varia- 3 U.S. Environmental Protection Agency, Office of Pesti- tion if there is more than one observer for a given cides and Toxic Substances, Health Effects Test Guideline study. Thus, the standardized procedures em- 870.6200, Neurotoxicity Screening Battery (August 1998). ployed in these tests offer the potential for signifi- D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 15 cant comparability across studies and provide following intravenous administration (Verschoyle added confidence in the accuracy and consistency and Aldridge, 1980) with those recorded in the of the observations when compared to more rou- FOB employed in regulatory neurotoxicity stud- tine cage-side observations. ies. For example, the observation of ‘abnormal The experimental design for the acute neurotox- hindlimb locomotion’ associated with the CS syn- icity study is the most comparable of all studies drome (Verschoyle and Aldridge, 1980) is too performed under regulatory guidelines to the pub- vague to be correlated with specific responses lished studies that established the classical syn- recorded from the FOB. However, because others dromes of pyrethroid intoxication (Verschoyle have described hindlimb impairment with and Aldridge, 1980; Lawrence and Casida, 1982). deltamethrin as increased hindlimb extensor tone In acute neurotoxicity studies, the animals are (Ray, 1982b) or splayed hindlimbs (Gray and tested using the FOB at the time of peak effects, Rickard, 1982), evidence of hindlimb impairment when clinical signs of toxicity are most apparent. in regulatory neurotoxicity studies that involves The highest dose tested is one that produces clear similar changes in posture (splayed hindlimbs or evidence of toxicity, i.e. approximately the highest hunched posture) and gait (exaggerated hindlimb dose that animals are expected to survive. One flexion or hindlimbs splayed or dragging) may be important difference between such studies and the considered to be consistent with the CS syndrome. previous studies that established the taxonomy of On the other hand, distinctions between the T and pyrethroid intoxication is the route of administra- CS syndromes based on an increased response to tion. For all of the examples summarized here, the stimuli (increased sensitivity to stimuli with the T pyrethroid was administered orally, by gavage, syndrome and increased startle response with the rather than by the intravenous (Verschoyle and CS syndrome), and tremors (fine or coarse) (Ver- Aldridge, 1980) or intracerebral (Lawrence and schoyle and Aldridge, 1980) are insufficiently clear Casida, 1982) route. to permit their correlation with observations The study design for the subchronic neurotoxic- recorded in the FOB employed in regulatory ity study is less comparable to the published studies. studies that defined pyrethroid intoxication syn- dromes (Verschoyle and Aldridge, 1980; Lawrence 3.3.2. Regulatory neurotoxicity studies: results and Casida, 1982) because the dose is adminis- Of the pyrethroids that are registered for use in tered through the diet for an extended period (13 the U.S., acute regulatory neurotoxicity study re- weeks). However, comparison among subchronic sults are available for nine: four esters of primary studies is facilitated by the consistent use of di- alcohols (bifenthrin, S-bioallethrin, permethrin, etary administration over an extended period. and pyrethrum) and five compounds with an a- With sustained dietary exposure, the peak concen- cyanophenoxybenzyl moiety (beta-cyfluthrin, trations of pyrethroid in target neural tissues may lambda-cyhalothrin, cypermethrin, zeta-cyperme- be much lower, as the animal consumes the thrin and deltamethrin). In these studies the ef- treated feed ad libitum over the course of each fects of pyrethroids were transient, with peak day. Furthermore, administration may be self-lim- effects observed within hours of exposure and full iting, as the animal may eat less treated feed if the recovery within 1–14 days after treatment. Re- exposure produces systemic toxicity. sults of subchronic neurotoxicity studies are also It is important to determine whether the results available for six of the compounds that were of regulatory neurotoxicity studies identify syn- examined in acute studies: bifenthrin, beta- dromes of pyrethroid intoxication that are consis- cyfluthrin, cypermethrin, zeta-cypermethrin, tent with those described following intravenous deltamethrin, and permethrin. In these studies, administration in the same species (Verschoyle which involved dietary exposure ad libitum, the and Aldridge, 1980). One problem encountered in signs of pyrethroid intoxication generally per- such comparisons is the need to normalize de- sisted, but did not become more pronounced, with scriptions of the signs of intoxication observed continued treatment beyond 4 weeks of exposure. 16 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

Table 3 Agreement of regulatory acute neurotoxicology studies with syndromes of pyrethroid intoxication: non-cyano pyrethroids

Pyrethroid dose (mg/kg) Bifenthrin (75) S-Bioallethrin (90) Permethrin (150) Pyrethrum (200)

T-syndrome Aggressive sparring –a ––– Increased sensitivity to stimuli – – +b + Fine tremor ?c ??? Prostration – ––– Hyperthermia – + – + CS-syndrome Pawing and burrowing –––– Salivation – – ++ Coarse tremor ?c ?c ?c ?c Choreoathetosis – ––– Clonic seizures + – + – Abnormal hindlimb locomotion ++++ Hypothermia – –––

Based on syndromes described by Verschoyle and Aldridge (1980). a Not present. b Present. c The observations from these studies do not distinguish between fine and coarse tremor.

The results obtained for these nine compounds posture, clonic convulsions, and abdominogenital are summarized in the following sections. The staining. No effects were evident at the two lower signs of intoxication, obtained from both acute doses. In the subchronic study (Freeman, 1998), and subchronic studies, are grouped in Tables 3 bifenthrin was administered via the diet to male and 4 to indicate those consistent with the original and female Sprague–Dawley rats at dietary con- description of the T syndrome of intoxication and centrations of 50, 100 or 200 ppm (3, 7 and 13 those consistent with the original description of mg/kg per day, respectively). At the two higher the CS syndrome. In the case of permethrin and dietary levels, bifenthrin produced whole-body cypermethrin, the results of a published neurobe- tremors, twitching, decreased grip strength and havioral assessment in Long-Evans rats using a increased landing foot splay. Effects were not protocol similar to the FOB employed in regula- evident at the lowest dietary concentration. tory studies (McDaniel and Moser, 1993) are Bifenthrin, a pyrethroid with a unique non-cy- included for comparison in the discussion below ano alcohol moiety (Fig. 2), was not included in but are not incorporated into the comparisons either of the original studies that established the presented in Tables 3 and 4. T/CS classification of pyrethroid intoxication syn- dromes. The production of tremors in both acute 3.3.2.1. Bifenthrin. In the acute neurotoxicity and subchronic neurotoxicity does not assist the study of bifenthrin (Watt, 1998a), the FOB was classification of bifenthrin because the results performed at the time of peak effects, approxi- recorded in the FOB do not indicate whether the mately 6–8 h following the administration of 10, tremors were fine or coarse. Other effects of bifen- 35 or 75 mg/kg of the undiluted compound to thrin in these studies (e.g. splayed hindlimbs and male and female Sprague–Dawley rats. At the convulsions) are associated more closely with the highest dose, bifenthrin caused whole-body CS syndrome of intoxication, an unexpected re- tremors, twitching, staggered gait, uncoordinated sult for a pyrethroid that lacks the a-cyano-3-phe- movement/ataxia, splayed hindlimbs, abnormal noxybenzyl moiety. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 17

Table 4 Agreement of regulatory acute neurotoxicology studies with syndromes of pyrethroid intoxication: a-cyano pyrethroids

Pyrethroid dose (mg/kg)Beta-cyfluthrin Lambda-cyhalothrinCypermethrin Zeta-cypermethrin Deltamethrin (10)(35) (100) (50) (50)

T-Syndrome Aggressive sparring –a ––– – Increased sensitivity to –– – – +b stimuli Fine tremor – – ?c ?c ?c Prostration –––– – Hyperthermia ––––– CS-Syndrome Pawing and burrowing + – – –– Salivation + – + – + Coarse tremor – – ?c ?c ?c Cheoreoathetosis + –––+ Clonic seizures – ––++ Abnormal hindlimb –– ++ + locomotion Hypothermia + –––+

Based on syndromes described by Verschoyle and Aldridge (1980). a Not present. b Present. c The observations from these studies do not distinguish between fine and coarse tremor.

3.3.2.2. S-Bioallethrin. For the acute neurotoxicity tion. The tremor and hyperthermia produced by study of S-bioallethrin (Broadmeadow, 1997), the S-bioallethrin are consistent with the T syndrome FOB was performed approximately 30–60 min of intoxication, whereas the myoclonus, hunched after the administration of 5, 30 or 90 mg/kg in posture and splayed hindlimbs are consistent with corn oil to male and female Sprague–Dawley effects ascribed to the CS syndrome. rats. At the highest dose, S-bioallethrin produced tremor, hyperthermia, hunched posture, immobil- 3.3.2.3. Beta-cyfluthrin. For the acute neurotoxic- ity, head and/or body twitches (myoclonus), un- ity study of beta-cyfluthrin (Sheets et al., 1997), steady or crawling gait with splaying of the the FOB was performed approximately 3 h fol- hindlimbs, decreased landing foot splay and de- lowing the administration of 0.5, 2.0 or 10 mg/kg creased grip strength. Lower doses produced no of beta-cyfluthrin in Cremophor to male and fe- effects in either sex. male Fischer 344 rats. In rats that received the 10 Bioallethrin (a mixture of S-bioallethrin and its mg/kg dose, beta-cyfluthrin produced salivation, a-R epimer) was one of the compounds employed hypothermia, repetitive chewing and pawing in the initial description of the T syndrome of movements, gait incoordination, a flattened pos- pyrethroid intoxication following intravenous or ture, choreoathetosis, muscle fasciculations, de- oral administration to rats (Verschoyle and creased activity, impaired aerial righting, and Barnes, 1972) and was also classified as a Type I decreased response to stimuli. Effects at the next (T syndrome) compound upon intracerebral ad- lower dose were limited to a decreased approach ministration to mice (Lawrence and Casida, response, clear oral stain and decreased activity. 1982). In contrast, the results of the acute neuro- No effects were evident at the lowest dose of 0.5 toxicity study suggest a mixture of intoxication mg/kg. In the subchronic study (Sheets and signs that contains elements that are consistent Hamilton, 1997), beta-cyfluthrin was administered with both the T and CS syndromes of intoxica- via the diet to male and female Fischer 344 rats at 18 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 concentrations of 30, 125 and 400 ppm (2, 9 and oral discharge, abdominogenital staining, ataxia, 29 mg/kg per day, respectively). At the 400 ppm staggered gait, decreased locomotor activity, and dietary level, beta-cyfluthrin caused gait incoordi- mortality. No effects were evident at the lowest nation, repetitive chewing movements, hypother- dose of 30 mg/kg. For the subchronic study (Free- mia, increased reaction to stimuli, impaired aerial man, 1993b), cypermethrin was administered righting, and decreased grip strength. No effects through the diet to male and female Sprague– were evident at lower dietary concentrations. Dawley rats, at concentrations of 500, 1300 and Neither cyfluthrin nor beta-cyfluthrin were in- 1700 ppm (34, 86 and 111 mg/kg per day, respec- cluded in the group of compounds forming the tively). At the 1700-ppm dietary level, cyperme- basis of the T/CS classification of intoxication thrin caused tremor, ataxia, splayed hindlimbs, syndromes. However, as a member of the a-cy- abnormal posture, staggered gait, and impaired ano-3-phenoxybenzyl subfamily of pyrethroids aerial righting. Splayed hindlimbs and staggered and a close structural relative of cypermethrin (see gait were also evident at the 1300-ppm dietary Fig. 2), cyfluthrin might be hypothesized to cause level, but no effects were seen at 500 ppm. In a the CS syndrome. The combination of beta- published study (McDaniel and Moser, 1993), cyfluthrin-induced effects (i.e. salivation, hy- cypermethrin was administered by gavage in corn pothermia, repetitive pawing movements and oil to Long-Evans hooded rats at doses of 20, 60 choreoathetosis) found in the acute and sub- and 120 mg/kg, and the animals were evaluated chronic neurotoxicity studies are generally consis- using an FOB at 1.5 and 3 h following treatment. tent with the CS syndrome of intoxication. Results of this study that are generally consistent with those of the regulatory acute neurotoxicity studies include salivation, urination, ataxia and 3.3.2.4. Lambda-cyhalothrin. In the acute neuro- tremors. However, the absence of a staggering toxicity study with lambda-cyhalothrin (Barmmer, gait and the presence of a flattened body posture 1999), the FOB was performed approximately 7 h with splayed limbs, increased landing footsplay following the administration of 2.5, 10 or 35 and choreoathetosis in the latter study are not mg/kg in corn oil to Alderley Park rats. At the 35 consistent with the findings of the regulatory neu- mg/kg dose, this compound caused decreased ac- rotoxicity study. tivity, ataxia, reduced stability, tiptoe gait, de- Both the 1R,trans,aRS and 1R,cis,aRS isomers creased landing foot splay and decreased tail flick of cypermethrin were previously identified as pro- response. Some signs were also evident at 10 ducing the CS syndrome of intoxication in rats mg/kg dose but not at the lowest dose level. and mice (Verschoyle and Aldridge, 1980; a Cyhalothrin, another member of the -cyano-3- Lawrence and Casida, 1982). Among the signs of phenoxybenzyl structural subfamily (see Fig. 2), intoxication found in the regulatory neurotoxicity was not included among the compounds on which studies, the presence of splayed hindlimbs is con- the T/CS classification syndrome is based. The sistent with the CS syndrome. Otherwise, the signs effects observed in the acute neurotoxicity study that are used to distinguish the CS syndrome of with lambda-cyhalothrin are not specific to either intoxication were generally absent from both reg- syndrome of intoxication. Thus, these results do ulatory neurotoxicity studies. The signs of intoxi- not support inclusion of this pyrethroid into ei- cation reported by McDaniel and Moser (1993) ther category. are more consistent with the CS syndrome than those of the regulatory studies. The basis for this 3.3.2.5. Cypermethrin. In the acute neurotoxicity difference in outcome is not known. Whereas the study with cypermethrin (Freeman, 1993a), the vehicle and source of test substance were the same FOB was performed at approximately 4 h follow- in both studies, there are also many differences ing the administration of the compound at 30, 100 between the studies (e.g. animal strain, dose selec- or 200 mg/kg in corn oil to male and female tion, observation time points and other differ- Sprague–Dawley rats. Animals that received ences in study design) that could have contributed doses of 100 or 200 mg/kg exhibited salivation, to the differences in neurotoxic signs. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 19

3.3.2.6. Zeta-cypermethrin. For the acute neuro- tened posture with limbs extended, clonic and toxicity study of zeta-cypermethrin (Watt, 1998b) tonic convulsions, tremor, biting or gnawing the the FOB was performed at the time of peak cage, eyelid ptosis, decreased reaction to removal effects, approximately 4–6 h following the admin- and handling, lacrimation, decreased arousal, al- istration of 10, 50 or 250 mg/kg of undiluted ternating limb movements with wave-like move- compound to male and female Long-Evans rats. ments of the abdomen (choreoathetosis), There was one death at the highest dose. At the hindlimbs splayed or dragging, decreased re- two highest dose levels, zeta-cypermethrin caused sponse to stimuli, increased auditory startle re- tremors, staggered gait, ataxia, loss of muscle sponse, impaired aerial righting response, reduced control, splayed hindlimbs, immobility, an im- forelimb grip strength, impaired rotorod perfor- paired righting response and convulsions. No ef- mance, decreased fore- and hindlimb extensor fects were evident at the lowest dose of 10 mg/kg. strength, decreased hindlimb foot splay, hypother- For the subchronic study (Freeman, 1999), zeta- mia, and mortality. At the next lower dose, the cypermethrin was administered through the diet only evidence of toxicity was salivation and im- to male and female Long-Evans rats, at concen- paired mobility. No effects were observed at the trations of 75, 400 and 750 ppm (5, 29 and 51 lowest dose in both sexes. In the subchronic study mg/kg per day, respectively). At the 750-ppm (Nemec, 1998b), deltamethrin was administered in dietary level, zeta-cypermethrin caused decreased the diet to male and female Sprague–Dawley rats, activity, increased tail flick latency, and increased at concentrations of 50, 200 and 800 ppm (4, 15 landing foot splay. Decreased activity was the and 56 mg/kg per day, respectively). At the 800 only evidence of toxicity at the next lower dose ppm dietary level, deltamethrin caused impaired and no effects were observed at the lowest dose in mobility and gait (rocking, lurching or swaying; both sexes. walking with hindlimbs splayed; walking on tip- Zeta-cypermethrin, the 1RS,cis,trans,aS isomer toes) and postural changes (body dragging with of cypermethrin, contains four of the eight iso- hindlimbs splayed or dragging), hypersensitivity mers present in fully racemic cypermethrin includ- to noise, impaired aerial righting reflex, reduced ing both of the isomers (e.g. the 1R,trans,aS and grip strength, decreased hindlimb extensor 1R,cis,aS isomers) expected to exhibit high mam- strength, piloerection, convulsions, popcorn malian toxicity (see Table 2). Because other iso- seizures, and mortality. There were no treatment- meric mixtures of cypermethrin containing the related neurobehavioral findings at lower dietary two most active isomers were identified as causing concentrations. the CS syndrome in rats and mice (Verschoyle The signs of intoxication observed with and Aldridge, 1980; Lawrence and Casida, 1982), deltamethrin following either oral or intravenous zeta-cypermethrin would be expected to produce administration formed the basis of the original the CS syndrome under similar assay conditions. description of the CS intoxication syndrome Among the findings of the regulatory neurotoxic- (Barnes and Verschoyle, 1974). Several of the ity studies the presence of splayed hindlimbs and signs observed in the regulatory neurotoxicity convulsions are consistent with the CS-type syn- studies (e.g. salivation, convulsions, choreoatheto- drome but other findings are not specific to either sis, hypothermia and hindlimb splay) are also syndrome of intoxication. consistent with the production of the CS syn- drome by deltamethrin. Since the FOB does not 3.3.2.7. Deltamethrin. In the acute neurotoxicity indicate whether the tremor caused by study of deltamethrin (Nemec, 1998a), the FOB deltamethrin was fine or coarse, it is not known was performed approximately 3 h following the whether this finding is consistent or inconsistent administration of 5, 15 or 50 mg/kg of with the CS classification. Many other effects of deltamethrin in corn oil to male and female deltamethrin at the highest exposure levels (e.g. Sprague–Dawley rats. Animals treated with 50 decreased arousal, impaired righting, reduced grip mg/kg of deltamethrin exhibited salivation, a flat- strength) are more consistent with overt toxicity 20 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 at a lethal dose level, rather than evidence of a ated with the CS syndrome. In contrast, the specific syndrome of intoxication. findings in the published neurotoxicity study (Mc- Daniel and Moser, 1993) are generally more con- 3.3.2.8. Permethrin. For the acute neurotoxicity sistent with the production of the T syndrome. study (Freeman, 1993c), the FOB was performed The basis for this difference in outcome is not approximately 12 h following the administration known but, as with the different studies of cyper- of 10, 150 or 300 mg/kg of permethrin in corn oil methrin (see Section 3.3.2.5) may lie in differences to male and female Sprague–Dawley rats. At in animal strain, dose, and observation criteria doses of 150 and 300 mg/kg permethrin caused between the studies. salivation, tremor, splayed hindlimbs, abnormal posture, staggered gait, decreased grip strength, 3.3.2.9. Pyrethrum. For the acute neurotoxicity exaggerated reaction to sound, exaggerated study of pyrethrum (Hermansky and Hurley, hindlimb flexion, convulsions, and mortality. No 1993), the FOB was performed at approximately 3 treatment-related effects were evident at the low- h following the administration of 40, 125 or 400 estdoseof10mg/kg. In a published behavioral mg/kg in males and 20, 63 or 200 mg/kg in neurotoxicity study (McDaniel and Moser, 1993), females. Doses were delivered in corn oil to permethrin was administered by gavage in corn Sprague–Dawley rats. At the 200 and 400 mg/kg oil to Long-Evans hooded rats, at doses of 25, 75 dose levels pyrethrum produced salivation, and 150 mg/kg, and the animals were evaluated tremor, hyperthermia, urogenital wetness, an ex- using an FOB at 2 and 4 h following treatment. aggerated startle response, decreased grip strength Results that are consistent with the acute regula- and hindlimb splay, and mortality. Tremor was tory study included tremor, chromodacryorrhea, the only effect evident at the next lower dose level decreased grip strength and an exaggerated startle and there were no effects evident in either sex at response. However, the absence of salivation, the lowest dose. splayed hindlimbs and convulsions and the pres- Pyrethrum was identified as producing the T ence of aggressive sparring in the latter study were syndrome of intoxication in rats (Verschoyle and inconsistent with the findings of the regulatory Barnes, 1972) and mice (Lawrence and Casida, acute neurotoxicity study. In the subchronic neu- 1982). However, the results of these neurotoxicity rotoxicity study (Freeman, 1993d), permethrin studies suggest a mixed type of effect that is not was administered through the diet to male and consistent with either classic syndrome of intoxi- female Sprague–Dawley rats, at concentrations of cation. Tremor and hyperthermia are consistent 250, 1500 and 2500 ppm (18, 101 and 170 mg/kg with the T syndrome, whereas salivation and per day, respectively). At the 1500 and 2500 ppm hindlimb splay are more characteristic of the CS dietary levels permethrin produced tremor, syndrome. splayed hindlimbs, abnormal posture, a staggered gait, and decreased grip strength. There were no 3.3.3. Results of other beha6ioral neurotoxicity effects at the lowest dose of 250 ppm. studies Various isomers and isomer mixtures of perme- thrin were identified as causing the T syndrome of 3.3.3.1. Effects on motor acti6ity. Crofton and intoxication in rats and mice (Verschoyle and Reiter (1984, 1988a,b) compared the effects of Aldridge, 1980; Lawrence and Casida, 1982). nine commercial or experimental pyrethroids (cis- However, the results of the regulatory neurotoxic- methrin, cyfluthrin, cypermethrin, deltamethrin, ity studies suggest a mixed type of effect that is fenvalerate, flucythrinate, fluvalinate, permethrin, not consistent with either classic syndrome of and RU 266074) on motor activity in rats. Com- intoxication. The tremor may be consistent with the T-type syndrome, whereas salivation, splayed 4 The difluoro analog of deltamethrin: [S]-a-cyano-3-phe- hindlimbs, exaggerated hindlimb flexion and con- noxybenzyl-3-(2,2-difluorovinyl)-2,2-dimethylcyclopropanecar- vulsions are signs that are more commonly associ- boxylate. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 21 pounds were administered orally in corn oil and did not affect latency and also, unlike any other motor activity was assessed in a figure-eight maze compound tested, increased sensitization to back- at the time of peak effect (1.5–2 h after dosing as ground acoustical stimuli. Among the three com- defined in these studies). All nine compounds pounds not formally classified according to the caused a dose-dependent decrease in motor activ- T/CS taxonomy of intoxication syndromes, ity, and no differences in this response were noted cyfluthrin and flucythrinate decreased the ampli- between compounds identified by Verschoyle and tude and increased the latency of acoustic startle Aldridge (1980) as producing the T syndrome of responses in a manner similar to cypermethrin intoxication (cismethrin, permethrin) and those and deltamethrin, whereas fluvalinate had no ef- producing the CS syndrome (cypermethrin, fect on any measure of the acoustic startle re- deltamethrin, fenvalerate and RU 26607). The sponse. In another study (Hijzen and Slangen, potency of deltamethrin in these assays varied 1988), cypermethrin, fenfluthrin, and permethrin more than 200-fold, depending on the vehicle and increased the amplitude of the acoustic startle route employed (Crofton et al., 1995). Other stud- response in rats, whereas deltamethrin decreased ies of the effects of deltamethrin on motor activity the amplitude of this response. The effects of employing different experimental protocols pro- permethrin and deltamethrin in this study are duced contradictory results. A single oral dose of generally consistent with the findings of Crofton deltamethrin administered to mice caused a reduc- and Reiter (1984, 1988a,b), but the increase in tion in wheel-running behavior (Chanh et al., startle amplitude caused by cypermethrin is an 1984), a result consistent with the effects of unexpected and contradictory finding. pyrethroids on motor activity summarized above (Crofton and Reiter, 1984, 1988a,b). In contrast, 3.3.3.3. Effects on conditioned beha6ior. Intraperi- daily oral administration of deltamethrin to rats toneal administration of allethrin, deltamethrin, for 15 days caused an increase in spontaneous fenvalerate or permethrin to rats caused a dose- motor activity measured on the day following the dependent reduction in the frequency of a previ- last dose (Husain et al., 1996). ously-learned behavior (i.e. bar-pressing reinforced by food) (Bloom et al., 1983; Stein et 3.3.3.2. Effects on acoustic startle response. al., 1987). Oral administration of either cyperme- Crofton and Reiter (1984, 1988a,b) also assessed thrin or permethrin to rats caused similar reduc- the effects of nine pyrethroids (cismethrin, tions in food-reinforced learned behavior (Peele cyfluthrin, cypermethrin, deltamethrin, fenvaler- and Crofton, 1987). Daily oral administration of ate, flucythrinate, fluvalinate, permethrin, and deltamethrin to rats for 15 days also reduced ethanomethrin) on the acoustic startle response of learning and memory measured in a Y-maze using rats. Compounds were administered orally in corn a negatively reinforced visual discrimination re- oil and the latency to onset and peak amplitude of sponse (Husain et al., 1996). startle responses to standard acoustical stimuli were assessed 1.5–2 h after dosing. Compounds 3.3.4. Summary identified by Verschoyle and Aldridge (1980) as The regulatory neurotoxicity studies summa- producing the T syndrome of intoxication (cis- rized here provide a unique opportunity to com- methrin, permethrin, and ethanomethrin) in- pare the actions of different pyrethroids creased the amplitude of the startle response determined under standardized experimental con- without affecting its latency, whereas compounds ditions. Each of these pyrethroids produced a classified as producing the CS syndrome of intox- distinct combination of neurobehavioral effects at ication (cypermethrin, deltamethrin and fenvaler- the highest dose tested. Moreover, comparisons of ate) produced varied effects. Cypermethrin and the findings for any given pyrethroid between deltamethrin decreased the amplitude and in- acute and subchronic studies and of the results for creased the latency of the startle response, two isomeric compositions of cypermethrin that whereas fenvalerate increased the amplitude and were examined independently illustrate the consis- 22 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 tency and reproducibility of the signs of intoxica- literature describe studies that involved a variety tion derived from the FOB. However, a detailed of routes and schedules of administration, test assessment of how closely these pyrethroids com- methods, and assay endpoints. Whereas, these pare with each other or with either classical syn- studies identify additional endpoints for the be- drome of intoxication (i.e. T or CS) is limited by havioral effects of pyrethroids that are not explic- several factors, including: a lack of concurrence itly included in the FOB employed in regulatory between the signs reported in intravenous toxicity studies, they are of limited value in delineating studies (Verschoyle and Aldridge, 1980) with ob- similarities and differences in the behavioral ef- servations used to characterize the effects in the fects of different pyrethroids. regulatory studies by the oral route (e.g. whether tremor was coarse or fine and the nature of 3.4. Paresthesia postural changes or gait abnormalities); differ- ences in dose selection; and the absence of data The experience of workers involved in the han- for several registered pyrethroids. dling of technical or formulated pyrethroids, ei- Despite these limitations, it is possible to reach ther during manufacture or use, provides insight some conclusions from these results. For example, into the effects of pyrethroids on humans follow- it appears that two of the pyrethroids examined in ing cutaneous exposure. A large body of occupa- these studies (deltamethrin and cyfluthrin) pro- tional exposure data, encompassing published duced signs of intoxication that conform generally clinical reports and unpublished reports obtained to the CS syndrome, three compounds (perme- from industrial sources, has been assembled in thrin, S-bioallethrin, pyrethrum) produced a mix- two review articles (Vijverberg and van den ture of signs associated with the T and CS Bercken, 1990; Clark, 1995). The most frequently- syndromes, and three (bifenthrin, cyhalothrin, reported symptom in worker exposure studies was cypermethrin) produced signs of intoxication that paresthesia, which was characterized by numb- did not clearly correspond to either syndrome. It ness, itching, burning, or tingling of the skin is noteworthy that none of the compounds iden- following dermal exposure to a pyrethroid. These tified previously as producing the T syndrome sensations generally occurred in the absence of (permethrin, S-bioallethrin, pyrethrum) caused erythema, edema, vesiculation, or other signs of signs of intoxication in regulatory neurotoxicol- overt skin irritation and were usually limited to ogy studies that were exclusively correlated with the directly exposed areas of the skin. Pyrethroid- the T syndrome. These results show that the T/CS induced paresthesia was transient and reversible classification system of intoxication syndromes is within hours after exposure, but in some instances not adequate to describe the various com- it lasted for up to 48 h. binations of effects seen with these compounds In studies of workers who experienced cuta- following oral administration. Additional studies neous sensations following pyrethroid exposure, with other pyrethroids, including the remaining no clinical signs of acute pyrethroid intoxication registered compounds for which comparable were observed (Vijverberg and van den Bercken, data are not currently available, would serve to 1990; Clark, 1995). In addition, no exposure-re- clarify these relationships further. Finally, it is lated differences were detected in hematology also clear from these studies that the effects of parameters or in heart, lung, liver, kidney, or each of these pyrethroids is reversible following nervous system function in these individuals. acute exposure, and that these compounds cause Further, electrophysiological assessment of pe- limited cumulative toxicity following sustained ex- ripheral nerve function did not detect any abnor- posure. malities related to occupational exposure to In contrast to the highly comparable experi- pyrethroids. mental design employed for regulatory neurotoxi- Reports of occupational exposure are sup- city studies of different pyrethroids, reports of the ported and amplified by the results of studies with behavioral effects of pyrethroids in the published human volunteers and experimental animals. In D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 23 human volunteers, the effects of four pyrethroids 4. Toxicokinetics and metabolism (cypermethrin, fenvalerate, flucythrinate and per- methrin) were evaluated by application to the 4.1. Introduction earlobe (Flannigan and Tucker, 1985). In this study, permethrin caused the least pronounced A large body of published literature describes and flucythrinate the most pronounced paresthe- the toxicokinetics and metabolic fate of sia, whereas cypermethrin and fenvalerate were pyrethroid insecticides in rodents and other ani- intermediate and approximately equal in effect. In mal species, and a more limited literature consid- each case, paresthesia developed within 30 min of ers the fate of pyrethroids in humans. Whereas exposure, peaked by 8 h, and dissipated by 24–32 much of the published literature on pyrethroid h after exposure. metabolism appeared prior to 1985, additional In studies with guinea pigs, the onset of abnor- information on the fate of pyrethroids, particu- mal sensation caused by six pyrethroids (cyperme- larly those introduced since 1985, is available thrin, deltamethrin, esfenvalerate, fenvalerate, from studies submitted to the EPA in support of flucythrinate and permethrin) was judged by an petitions for registration. The published literature increase in scratching, licking or biting behavior is summarized in several review articles at the site of dermal application (Cagen et al., (Miyamoto, 1976; Ruzo and Casida, 1977; Hut- 1984). The onset of symptoms usually occurred son, 1979; Casida and Ruzo, 1980; Gray and within 1 h after application. However, permethrin Soderlund, 1985; Bradbury and Coats, 1989; elicited a qualitatively lower overall response than Soderlund, 1992; Clark, 1995). A recent compre- the other five compounds tested, which were ap- hensive summary of the metabolic fate of proximately equal in effect under these experi- pyrethroids (Roberts and Hutson, 1999) includes mental conditions. The latency and duration of extensive bibliographies of the published literature the behavioral response were affected by the for- describing the metabolic fate of all currently regis- mulation employed for any single pyrethroid, but tered pyrethroids. The sections that follow briefly the magnitude of the response was independent of summarize the toxicokinetics and metabolic fate formulation. of pyrethroids based on these reviews from the The results of worker exposure data coupled perspective of the role of toxicokinetics and with the results of controlled experiments with metabolism in acute neurotoxicity. human volunteers and animals show that pares- thesia appears to be an exclusively local effect. It 4.2. Toxicokinetics occurs only at the site of dermal exposure, is not correlated with the appearance of a rash or other 4.2.1. Absorption, distribution and excretion signs of classical skin irritation, and is not associ- documented in in 6i6o metabolism studies ated with any signs of systemic intoxication. Studies designed to elucidate the metabolic fate Based on these observations and the proposed of numerous radiolabeled pyrethroids in animals origins of paresthesia in the sensory nervous sys- (Roberts and Hutson, 1999) show that these com- tem (Rizzo et al., 1996), pyrethroid-induced pares- pounds are rapidly and extensively absorbed from thesia has been postulated to be a direct the gastrointestinal tract following oral adminis- excitatory effect of pyrethroids on small sensory tration. The influence of administration vehicle on nerve fibers in the skin rather than a response due acute oral toxicity (see Table 1) implies that the to classical skin irritation. We conclude from vehicle affects the rate or extent of gastrointestinal these findings that paresthesia is a transient, local absorption, but there are no published studies effect of pyrethroids that is limited to the site of designed specifically to address this issue. In con- exposure. Paresthesia is therefore distinct from trast, pyrethroids are poorly absorbed following the signs of systemic intoxication and is therefore dermal exposure. A comparative study of the not relevant to cumulative risk assessments based gastrointestinal and dermal absorption of radiola- on systemic intoxication. beled trans- and cis-phenothrin in rats (Kaneko et 24 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 al., 1981) showed that at least 96% of an oral dose vehicles (Ruzo et al., 1979). In contrast to the was excreted after 6 days, whereas at least 78– strong correlation between brain levels and toxic 85% of the dermally-applied doses was recovered effects, blood levels of cismethrin and unabsorbed from the surface of the skin and deltamethrin in rats were poorly correlated with 3–17% of the doses was recovered in the excreta signs of intoxication (White et al., 1976; Gray et after 6 days. The administration vehicle also al., 1980; Rickard and Brodie, 1985). greatly influenced dermal absorption: absorption Following a single oral dose, pyrethroids are of phenothrin isomers was 3-fold greater, based rapidly taken up by the blood and distributed on the proportion of phenothrin-derived radiocar- throughout the body. This behavior is exemplified bon recovered in the excreta, when presented in by a balance and tissue retention study conducted an emulsifiable concentrate formulation than with cypermethrin (Crawford et al., 1981). In this when applied as a dust formulation. Pyrethroids study, blood levels of both the cis and trans are anticipated to be efficiently absorbed from the isomers of cypermethrin peaked by the 1st day respiratory tract following inhalation (Clark, after treatment and had declined to very low 1995). There are no published studies that directly levels by 8 days after treatment. A similar pattern compare the efficiency of absorption by this route of distribution for both isomers was found with with gastrointestinal absorption, but the similarity other internal tissues examined (liver, kidney, of the LD50 values calculated for deltamethrin muscle, brain) and for the trans isomer in fat. The following inhalation exposure to the values ob- only residue that was significantly higher was that tained following oral administration (Kavlock et of the cis isomer in fat, which was found to be al., 1979) implies that pyrethroids are readily ab- mostly unmetabolized cis-cypermethrin and was sorbed from the respiratory tract. eliminated from this tissue with an estimated half- The influence of pyrethroid toxicokinetics on life of 12 days, a value similar to the half-lives the availability of parent compound at sites in the estimated for the much lower residues of this central nervous system, and therefore on the ex- compound in kidney and liver. This pattern of pression of acute toxicity, is further demonstrated internal distribution is similar to that found for in studies correlating brain levels of cismethrin resmethrin (Miyamoto et al., 1971; Ueda et al., and deltamethrin in rats and mice with the onset 1975), permethrin (Gaughan et al., 1977), fen- of specific signs of acute intoxication. Administra- propathrin (Crawford and Hutson, 1977), tion of cismethrin to rats either orally or by deltamethrin (Ruzo et al., 1978; Cole et al., 1982), intravenous injection at different environmental fenvalerate (Ohkawa et al., 1979; Lee et al., 1985), temperatures (White et al., 1976; Gray et al., and tralomethrin (Cole et al., 1982). The preferen-

1980) yielded acute LD50 values ranging from 4.5 tial distribution and greater persistence of to \1000 mg/kg. However, administration of pyrethroids in fat was a common feature of these equitoxic doses, regardless of the route and studies, but the magnitude of these residues rela- amount of compound administered, resulted in tive to administered dose varied with the the production of characteristic brain concentra- pyrethroid and isomer examined. tions that were associated with the onset of Following daily oral doses, pyrethroids achieve tremors (3 nmol/g tissue) and with severe approximately steady-state levels in internal tis- tremors and death (12 nmol/g). Administration sues within a few days. These levels persist of deltamethrin to rats identified threshold con- throughout the dosing period and then decline centrations in the brain associated with salivation when daily administration ceases. This behavior is (0.1–0.2 nmol/g), tremor (0.2–0.35 nmol/g), and exemplified in a study of the bioaccumulation and choreoathetosis (0.4–1 nmol/g) (Rickard and persistence in rats of radiolabeled cypermethrin Brodie, 1985). A similar brain threshold concen- administered at 2 mg/kg per day for 70 days tration (1 nmol/g) for deltamethrin toxicity in (Rhodes et al., 1984). Cypermethrin-derived ra- mice was obtained following administration of diocarbon levels in the liver, kidney, and blood equitoxic oral or intraperitoneal doses in various increased rapidly during the 1st week of dosing D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 25 and then more slowly during the next 9 weeks, so 4 h after dosing. Both pyrethroids were cleared that levels at the end of 10 weeks were approxi- slowly from plasma with elimination half-lives of mately twice those at the end of 1 week. Radio- 12.4 (permethrin) and 38.5 h (deltamethrin). For carbon levels in fat and skin also increased rapidly both compounds, maximum concentrations in during the 1st week; in skin, there was no further various elements of the central and peripheral accumulation during the following 9 weeks, nervous system were higher than plasma concen- whereas in fat radiocarbon levels continued to trations and declined with half-lives similar to increase during the 2nd week and then were stable those measured for plasma. for the remainder of the study. At the end of the 10-week dosing period, tissue levels were 0.5– 4.2.3. Toxicokinetics in humans 1.0 mg cypermethrin equivalents/g tissue in blood, Few studies are available that describe the toxi- liver, kidney, and skin and 5 mg/g in fat. Upon cokinetic behavior of pyrethroids in humans. the cessation of dosing, radioactivity was elimi- Studies using an in vitro model of penetration nated rapidly from blood, liver and kidney, reach- through human skin (Shehata-Karam et al., 1988) ing background levels by 28 days after the showed that the penetration of fenvalerate was termination of dosing. Radioactivity levels de- much slower than that of carbofuran, parathion, clined more slowly from fat and skin; analysis of or lindane. Another study described the adminis- the cypermethrin isomer content of fat samples at tration of cypermethrin orally or dermally to hu- various times after the end of dosing showed that man volunteers followed by quantitation of the rate of elimination of the trans isomer from metabolites excreted in the urine (Woollen et al., fat was 5-fold faster than that of the cis isomer. 1992). This study documented the much greater Following single oral doses, most pyrethroids absorption of cypermethrin by the oral route ( are rapidly excreted in the urine and feces. Typi- 36% of the administered dose) than the dermal cally, \90% of the administered dose is excreted route (1.2% of the administered dose) based on within a week after treatment (Roberts and Hut- quantitation of the excreted metabolites in urine son, 1999). The rate of excretion and the distribu- samples. Additional studies of cypermethrin and tion of pyrethroid derived radiolabel between cyfluthrin in human volunteers exposed orally or urine and feces varies with the compound and dermally (Eadsforth and Baldwin, 1983; Eads- isomer examined and the position of the radiola- forth et al., 1988; Ku¨hn et al., 1999) further bel within the molecule. In particular, radiolabel document the rapid elimination of cypermethrin derived from the cyano group of a-cyano-3-phe- metabolites in the urine following exposure. In a noxybenzyl esters is very slowly eliminated from study of pest control operators receiving occupa- the body (Crawford et al., 1981) because this tional exposure to cypermethrin or cyfluthrin, the moiety is cleaved during ester hydrolysis and con- mean elimination half-lives for cyfluthrin and verted to thiocyanate, which has a pattern of cypermethrin metabolites in the urine were found disposition and elimination very different from to be 5.52 and 8.09 h, respectively (Ku¨hn et al., pyrethroids and their component acid and alcohol 1999). moieties (Ruzo et al., 1978; Ohkawa et al., 1979). A case of intentional ingestion of an emul- sifiable concentrate formulation of permethrin 4.2.2. Toxicokinetic models for pyrethroids provided an opportunity to assess the clearance of Two published studies have modeled the toxi- trans- and cis-permethrin from the blood during cokinetic behavior of permethrin (Anadon et al., the individual’s recovery from intoxication (Go- 1991) and deltamethrin (Anadon et al., 1996) toh et al., 1998). Serum concentrations of perme- following single oral doses to rats. For both com- thrin peaked 3–4 h after ingestion and then pounds, the kinetics of absorption, distribution declined. Levels of the trans isomer were below and elimination were fitted to a two-compartment the limits of detectability within 25 h after expo- open model. Pyrethroid absorption was relatively sure, whereas cis-permethrin was still present at rapid, so that plasma levels were maximal within detectable levels 10 days after exposure. The dif- 26 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 ferential persistence of the cis and trans isomers is these initial products of hydrolytic or oxidative consistent with toxicokinetics and metabolic fate attack involves conjugation with amino acids, of cis and trans permethrin in animal studies. sugars, sugar acids, or sulfate prior to excretion. In vivo metabolism studies are supported by stud- 4.3. Metabolism ies employing in vitro preparations from mam- malian tissues, which identify tissues having high 4.3.1. Pathways of pyrethroid metabolism levels of pyrethroid-metabolizing activity and the The metabolic fate of most pyrethroids in ani- enzyme activities catalyzing the principal bio- mals is described by the generalized pathway transformation pathways for pyrethroids. shown in Fig. 5. The initial biotransformation of the parent compound results from attack by either 4.3.2. Pyrethroid metabolism in humans esterases at the central ester bond or cytochrome Information on the metabolic fate of P450-dependent monooxygenases at one or more pyrethroids in humans is limited to a small num- sites in the acid or alcohol moieties. Although ber of studies involving volunteers or persons most oxidative pathways yield hydroxyester exposed to pyrethroids in the course of their metabolites, oxidative ester cleavage may be a occupations. For workers exposed to allethrin minor pathway for some pyrethroids. The relative (Leng et al., 1999) and volunteers or workers importance of initial hydrolytic or oxidative at- exposed to permethrin, cypermethrin, cyfluthrin, tack varies from compound to compound and or deltamethrin (Eadsforth and Baldwin, 1983; from isomer to isomer for each pyrethroid. Fol- Eadsforth et al., 1988; Woollen et al., 1992; Leng lowing ester cleavage, primary alcohol moieties et al., 1996; Llewellyn et al., 1996; Ku¨hn et al., (e.g. 3-phenoxybenzyl alcohol) undergo further 1999), the metabolites identified in urine samples oxidation via the aldehyde to the corresponding were consistent with the metabolic pathways for carboxylic acids, whereas a-cyano-substituted al- these compounds identified in rodents. cohols lose cyanide non-enzymatically to form the corresponding aldehyde. A portion of these ester 4.3.3. Toxicological significance of pyrethroid cleavage products may be hydroxylated, and a metabolism portion of the hydroxylated ester metabolites may Two lines of evidence suggest that the first be hydrolyzed, thereby producing hydroxylated metabolic attack on a pyrethroid, whether hy- ester cleavage products. Further metabolism of drolytic or oxidative, generally achieves detoxica- tion of the parent compound. First, the acute toxicity of pyrethroids to mammals can be en- hanced in several cases by compounds that inhibit liver esterases and monooxygenases (Soderlund and Casida, 1977; Casida et al., 1983; Gray and Soderlund, 1985). Second, the metabolites derived from the ester cleavage products of permethrin, which include compounds that are also major metabolites of cypermethrin, deltamethrin, fen- propathrin, and fenvalerate, are generally of equal or lower acute toxicity to mammals than the parent pyrethroids (Gaughan et al., 1977). These two considerations imply that metabolism effec- tively limits the expression of the acute toxicity of pyrethroids in animals. Fig. 5. Generalized pathway for the metabolism of pyrethroids There is little published evidence implicating in mammals by hydrolytic ([H]), oxidative ([O]) and conjuga- pyrethroid metabolites as causative agents for tion ([C]) reactions. chronic or long-term effects of pyrethroid admin- D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 27 istration. A notable exception involves the 2R,aS Section 3.3.2.7). Plasma levels of both permethrin isomer of fenvalerate, which lacks the appropriate and deltamethrin declined to 510% of peak lev- stereochemistry in the acid moiety to confer high els by 48 h after treatment (Anadon et al., 1991, insecticidal activity or mammalian toxicity (see 1996). These findings are in good agreement with Fig. 4). This fenvalerate isomer is implicated as the reversibility of acute intoxication by the causative agent of microgranulomatous le- pyrethroids following sublethal oral doses (see sions in various tissues, following a stereospecific Section 3.3). transesterification reaction that results in the for- The metabolic ability of pyrethroids, which re- mation of a cholesterol ester conjugate of the 2R sults from attack by both esterases and cy- isomer of the fenvalerate acid moiety (Kaneko et tochrome P450-dependent monooxygenases, al., 1986; Okuno et al., 1986). A subsequent in insures that accumulation and persistence in lipid- vitro study confirmed the stereospecific transester- rich tissues is not excessive and that pyrethroids ification reaction responsible for the formation of are cleared from the body over a period of days to the cholesterol ester of the 2R enantiomer of the weeks after the cessation of exposure. The signifi- fenvalerate acid moiety and identified analogs of cance of pyrethroid metabolism as a limiting fac- fenvalerate that yielded cholesterol ester products tor in the expression of acute toxicity is evident in in vitro, but did not identify transesterification the synergism of pyrethroid intoxication by com- products arising from the pyrethroids phenothrin, pounds that inhibit biotransformation enzymes. cyphenothrin, and fenpropathrin (Kaneko et al., The products of pyrethroid biotransformation 1988). typically exhibit lower acute toxicity than the parent pyrethroids, thereby implying that any 4.4. Summary acute hazard associated with these compounds is conferred by the toxicological properties of the The disposition of pyrethroids in animals is parent material. Finally, with the notable excep- governed by two properties shared by all insecti- tion of one isomer of fenvalerate having low cides of this class: their high degree of lipophilic- insecticidal activity and mammalian toxicity, the ity and their susceptibility to metabolic attack at metabolites of pyrethroids are not implicated as multiple sites. The lipophilicity of pyrethroids fa- causative agents for chronic or long-term effects. vors absorption in the gastrointestinal and res- piratory tracts but limits absorption through the skin and also confers preferential distribution into 5. Physiological and neurochemical indices of lipid-rich internal tissues, including body fat and intoxication elements of the central and peripheral nervous system. Administration of pyrethroids at doses that The time course of pyrethroid distribution is cause overt signs of acute intoxication produces a well-correlated with the onset and duration of the variety of effects that are detected using physio- signs of intoxication that are observed in acute logical and biochemical assays. The neurophysio- neurotoxicity studies. In the case of permethrin, logical, neurochemical, and cardiovascular effects the peak plasma concentration following oral ad- associated with acute pyrethroid intoxication are ministration was achieved 4 h after dosing (Ana- summarized in the following sections. don et al., 1991), suggesting that concentrations in plasma reach their peak in advance of the time to 5.1. Neurophysiology peak effect (12 h) found in acute neurotoxicity studies (see Section 3.3.2.8). In the case of Electroencephalographic studies of altered deltamethrin, the peak plasma concentration was brain function during pyrethroid intoxication are achieved 2 h after oral treatment (Anadon et al., limited to studies of two pyrethroids, deltamethrin 1996) and the time to peak effect in acute neuro- and cypermethrin. Studies of the action of toxicity studies was 3 h after treatment (see deltamethrin, involving both conscious and anes- 28 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 thetized animals, showed altered patterns of corti- threshold of hair follicle receptors and produced cal and subcortical spiking, suppression of evoked spontaneous activity in some sensory cells and responses in the cortex, and an increase in caudate repetitive firing in afferent sensory nerves follow- neuronal activity (Ray and Cremer, 1979; Ray, ing electrical stimulation (Carlton, 1977). In the 1980). These findings were interpreted as evidence cat, cis-permethrin but not deltamethrin enhanced of a primary action of deltamethrin on the ex- both spontaneous and evoked activity in the sci- trapyramidal motor system of the rat. Daily in- atic nerve (Staatz-Benson and Hosko, 1986). Both traperitoneal administration of cypermethrin to cismethrin and deltamethrin increased the ex- rats, followed by electroencephalographic record- citability of rat tail nerve preparations (Parkin ing for 30 min, identified episodes of paroxysmal and Le Quesne, 1982; Takahashi and Le Quesne, epileptic activity that appeared on the 1st or 2nd 1982). Pyrethroids classified as producing the T day of cypermethrin administration that increased syndrome of intoxication (e.g. cismethrin, progressively in number and duration throughout fenfluthrin) and those classified as producing ele- the 10-day period (Conde´s-Lara et al., 1999). ments of both the T and CS syndromes (cyphe- Electrophysiological assays of specific neuronal nothrin, fenpropathrin) produced excitatory pathways have also been employed to assess the effects on sensory and motor neurons in the rat impact of systemically-administered pyrethroids trigeminal reflex pathway, but five compounds on the normal function of elements of the central classified as producing the CS syndrome (fluoro- nervous system. Several studies have documented cyphenothrin, cyhalothrin, cyfluthrin, the enhancement by a variety of pyrethroids of deltamethrin and fenvalerate) did not cause repet- synaptic inhibition in the hippocampal dentate itive discharges in this neuronal pathway at doses gyrus of the rat brain (Gilbert et al., 1989; Joy et that caused repetitive activity in skeletal muscle al., 1989, 1990; Joy and Albertson, 1991). In these (Forshaw and Ray, 1986; Wright et al., 1988). studies, pyrethroids classified as causing the T syndrome of intoxication (e.g. cismethrin, 5.2. Neurochemistry fenfluthrin) produced a transient enhancement of inhibition, whereas compounds causing the CS Pyrethroid-dependent neurotransmitter release syndrome (e.g. deltamethrin, fenvalerate) pro- from presynaptic nerve terminals in the brain was duced a more persistent enhancement of inhibi- first documented in rats treated with deltamethrin tion and compounds classified as producing (Aldridge et al., 1978). Deltamethrin treatment elements of both the T and CS syndromes (e.g. resulted in a significant decrease in acetylcholine cyphenothrin, fenpropathrin) gave effects of inter- levels in whole brain and, most significantly, in mediate duration. The enhancement of inhibition the cerebellum. In contrast, cismethrin produced by pyrethroids in this system was interpreted to be no significant reduction in acetylcholine levels the result of excitatory presynaptic effects on in- (Aldridge et al., 1978). Other effects of hibitory interneurons in the dentate gyrus. In pyrethroids in the cerebellum include assays with spinal cord preparations, cismethrin deltamethrin- and cypermethrin-induced increases increased the excitability of both monosynaptic in cyclic guanosine monophosphate levels and polysynaptic spinal reflexes in the rabbit (Aldridge et al., 1978; Lock and Berry, 1981; (Carlton, 1977) and enhanced dorsal root poten- Brodie and Aldridge, 1982; Brodie and Opacka, tials in the rat (Smith, 1980), whereas cis-perme- 1987) and enhanced calcium-dependent neuro- thrin and deltamethrin both increased the transmitter release by fenvalerate from rabbit stri- excitability of spinal neurons in both the rat and atal slices but not from hippocampal slices (Eells the cat (Staatz-Benson and Hosko, 1986). and Dubocovich, 1988). Pyrethroids also in- Electrophysiological assays have also revealed creased the levels of some amino acid neurotrans- effects of systemically-administered pyrethroids mitters and metabolites of monoamine on elements of the peripheral nervous system. In neurotransmitters in the brain. At doses that pro- the rabbit, cismethrin lowered the response duced tremor in rats, permethrin increased the D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 29 levels of aspartate in the brainstem and striatum, Forshaw and Bradbury, 1983). At doses that pro- the levels of glutamate in the brain stem, the levels duced choreoathetosis in rats, deltamethrin in- of serotonin metabolites in the hypothalamus, creased plasma noradrenaline and adrenaline to brain stem, hippocampus, and brain stem, and the levels as much as 100-fold higher than controls levels of dopamine metabolites in the striatum (Cremer and Seville, 1982). Although this level of (Hudson et al., 1986). Similarly, allethrin, cyper- plasma catecholamine could be responsible for methrin, fenvalerate, and permethrin increased some cardiovascular effects, studies with isolated the levels of dopamine metabolites in the striatum hearts in vitro showed that deltamethrin increased (Doherty et al., 1986b). Finally, recent studies aortic output and mean systolic pressure follow- have documented marked alterations in the levels ing pretreatment with reserpine, a catecholamine of endogenous polyamines (spermine, spermidine, reuptake blocker and anti-adrenergic tranquilizer putrescine), associated principally with Purkinje (Forshaw and Bradbury, 1983). This direct posi- neurons in the rat cerebellum, that are correlated tive inotropic effect of deltamethrin on the heart with changes in locomotive and aggressive behav- muscle has also been reported in anesthetized iors following deltamethrin exposure (Husain et al., 1992, 1994, 1996). dogs (Navarro-Delmasure, 1981) and in isolated Selective effects of pyrethroids on the cerebel- guinea pig atrial muscle (Berlin et al., 1984). In lum were also found in assays of brain glucose contrast to deltamethrin, treatment with cis- levels during intoxication. Rat brain glucose levels methrin resulted in no cardiovascular alterations and brain blood flow were elevated in all brain in intact or pithed animals and was without an areas except the cerebellum during writhing fol- effect on isolated heart preparations (Ray, 1982a; lowing a lethal dose of deltamethrin. In contrast, Cremer et al., 1983; Forshaw and Bradbury, glucose levels in the cerebellum were depleted, 1983). Although cismethrin caused an approxi- indicating a high level of glucose utilization in this mately 10-fold increase in plasma catecholamines region (Cremer et al., 1980, 1983). Cypermethrin (10% of the response level induced by produced similar increases in cerebellar glucose deltamethrin), this increase was correlated to levels during writhing but resulted in elevated physiological responses that occurred due to in- cerebellar lactate concentrations during both creased physical activity of the animal during the writhing and salivation (Lock and Berry, 1981). tremor syndrome that was produced following Brain blood flow was also increased significantly exposure (Cremer and Seville, 1982; Ray, 1982a). by cismethrin but, except for the cerebellum, this Blood glucose and lactate levels in rats were increase was eliminated by blocking cortical va- increased following treatment with deltamethrin sodilation by atropine (Cremer et al., 1983). The (Ray and Cremer, 1979; Cremer and Seville, 1982; atropine-insensitive component of blood flow ap- Ray, 1982a) and cypermethrin (Lock and Berry, peared to be due to increased cerebellar neuronal 1981), particularly once choreoathetosis was es- activity caused by cismethrin (Cremer et al., tablished. Mephenesin pretreatment, which elimi- 1983). nated deltamethrin-induced choreoathetosis, protected pithed rats from the increase in blood 5.3. Cardio6ascular effects glucose concentration, but similarly treated intact rats, still elicited this increase (Bradbury et al., Lethal doses of deltamethrin produced an in- creased arterial blood pressure level in writhing 1983). Thus, only part of the increased blood adult rats (Ray and Cremer, 1979; Ray, 1982a; glucose level produced by deltamethrin can be Bradbury et al., 1983). In pithed rats, in which the attributed to the physiological responses to central nervous system no longer controls cardio- choreoathetosis and there is a portion of this vascular function, deltamethrin increased mean increase that is a result of an action of the and differential arterial blood pressure, pulse pyrethroids on supraspinal centers in the central pressure, and heart rate (Bradbury et al., 1983; nervous system (Bradbury et al., 1983). 30 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

5.4. Summary also been investigated, and some of the effects identified in these studies may be relevant to the The studies summarized in this section docu- neurotoxic actions of at least some pyrethroids. ment the neuroexcitatory effects of pyrethroids The literature in these areas has been covered in that are associated with the onset of signs of acute comprehensive review articles (Soderlund and intoxication. These studies also provide evidence Bloomquist, 1989; Bloomquist, 1993a; Clark, that pyrethroid-dependent alterations in nerve 1995; Bloomquist, 1996). In the sections that fol- function encompass both central and peripheral low, we summarize information on the action of elements of the nervous system. Although pyrethroids on voltage-sensitive ion channels, lig- pyrethroid action does not appear to be restricted and-gated ion channels, and other neuronal target to any particular region of the nervous system, sites. We then propose criteria to assess the toxi- some regions appear to be more sensitive than cological significance of putative target sites and others. In particular, alterations in normal brain provide a critical evaluation of the toxicological function and neurochemistry that are associated importance of the putative target sites discussed in with intoxication are most commonly noted in the this section. cerebellum. The responses of the central and peripheral 6.2. Actions of pyrethroids on 6oltage-sensiti6e nervous system to pyrethroids, in themselves, ion channels provide little insight into the mechanisms of pyrethroid neurotoxicity. The observed neuroexci- 6.2.1. Voltage-sensiti6e sodium channels tatory effects are consistent with the modification of cellular excitability through an action on 6.2.1.1. Function, structure and pharmacology. voltage-dependent ion channels. Whereas such an Voltage-sensitive sodium channels mediate the effect could also indirectly account for the en- transient sodium permeability of the cell mem- hanced neurotransmitter release and turnover as- brane that is associated with the production of sociated with intoxication, the latter effects could action potentials in vertebrate and invertebrate also arise from actions of pyrethroids on presy- nerves and in vertebrate skeletal and cardiac mus- naptic elements that are specifically involved in cle. Voltage-sensitive sodium channel components neurotransmitter release. The studies summarized from electric eel electroplax, mammalian brain, here do not, however, provide evidence for an and mammalian skeletal muscle have been solubi- indirect neuroexcitatory effect of pyrethroids re- lized and purified (Catterall, 1992; Kallen et al., sulting from an interference with inhibitory 1993; Catterall, 1995, 1996). A large glycosylated neurotransmission. a subunit (260 kDa) is a common feature of purified channels; in addition, two smaller sub- b b   units ( 1 and 2; 33 and 36 kDa) are associ- 6. Mechanisms of toxicity ated with sodium channels purified from the brain and from skeletal muscle. Reconstitution experi- 6.1. Introduction ments with purified rat brain channel components a b show that incorporation of the and 1 subunits The action of pyrethroids on specific molecular into phospholipid membranes in the presence of targets in mammals has been investigated using brain lipids or brain phosphatidylethanolamine is both electrophysiological and biochemical tech- sufficient to produce channels that mimic the niques. The strong evidence implicating the functional properties of sodium channels in native voltage-sensitive sodium channel as the principal membranes. site of insecticidal action of pyrethroids has led to Knowledge of the primary structure of mam- extensive studies of the action of pyrethroids on malian sodium channels has been advanced mammalian sodium channels. However, actions greatly over the past 15 years by the cloning and of pyrethroids on numerous other systems have comparative sequence analysis of genes encoding D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 31

Fig. 6. Dendrogram depicting the evolutionary relationships among identified rat sodium channel a subunit isoforms calculated from published amino acid sequences; the sensitivity of isoforms to TTX, where known, is indicated. sodium channel subunits. Sodium channel a sub- sively in both functional assays and radioligand units in the nervous system and in skeletal and binding experiments to characterize the pharma- cardiac muscle are encoded by a family of highly cological properties of sodium channels. The ac- homologous but distinct genes. The mouse and tion of these agents has led to the human genomes contain at least 10 sodium chan- pharmacological identification of numerous bind- nel a subunit genes that are conserved in structure ing domains for natural and synthetic toxicants and genetic localization (Fig. 6) (Plummer and on the sodium channel. Meisler, 1999). Sodium channel heterogeneity in mammals is further increased by alternative 6.2.1.2. Electrophysiological studies of pyrethroid mRNA splicing of some isoforms (Sarao et al., action on sodium channels. The actions of 1991; Schaller et al., 1992; Gustafson et al., 1993). pyrethroid insecticides on sodium channels in in- The structural diversity of sodium channel a sub- vertebrate and vertebrate nerve preparations have unit isoforms and splice variants is the basis for been widely documented over the past four the well characterized functional and pharmaco- decades and are extensively and critically summa- logical diversity of sodium channels expressed in rized in numerous reviews (Soderlund and different mammalian tissue types and at different Bloomquist, 1989; Vijverberg and van den stages of development (Mandel, 1992). The het- Bercken, 1990; Narahashi, 1992; Bloomquist, erogeneity of sodium channel a subunits in mam- 1993a; Clark, 1995; Soderlund, 1995; Bloomquist, mals, and their distribution in both nerve and 1996; Narahashi, 1996). Briefly, intracellular mi- muscle, contrasts with the situation in insects, croelectrode recordings of action potentials which appear to have only a single sodium chan- demonstrate that pyrethroids produce a range of nel a subunit gene that codes for physiologically effects on nerve excitability depending on the and pharmacologically important sodium chan- structure of the pyrethroid employed (Lund and nels and is expressed exclusively in the nervous Narahashi, 1983; Vijverberg et al., 1983). Natural system (Hong and Ganetzky, 1994). pyrethrins and a structurally heterogeneous group The central importance of sodium channels in of synthetic pyrethroids lacking the a-cyano-3- the function of excitable cells is evident in the phenoxybenzyl alcohol moiety are characterized number and diversity of neurotoxicants and drugs by the induction of long trains of action poten- that act at the sodium channel, producing a vari- tials (‘burst discharges’) following a single stimu- ety of effects on channel function and neuronal lus with little or no effect on resting potential. In excitability. These agents have been used exten- contrast, pyrethroids that contain the a-cyano-3- 32 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 phenoxybenzyl alcohol moiety typically do not Patch clamp studies of the action of pyrethroids produce repetitive firing but instead block the on single sodium channels are limited to a much action potential upon repeated nerve stimulation smaller group of compounds. Tetramethrin, a and depolarize the resting membrane potential. compound known to produce burst discharges in Examination of a wide variety of pyrethroid intact nerves and transient sodium current prolon- structures has also identified compounds with in- gation in voltage clamp experiments, increased the termediate effects on neuronal excitability; these mean open time of individual sodium channels in compounds typically produce bursts of action po- patch clamp experiments approximately 10-fold tentials of declining amplitude followed, after (Yamamoto et al., 1983). In contrast deltamethrin repetitive stimulation, by nerve block. and fenvalerate, compounds known to produce Effects of pyrethroids on sodium channel func- use-dependent block of intact nerves and persis- tion that underlie these effects on nerve excitabil- tent sodium current prolongation in patch clamp ity have been elucidated using voltage and patch experiments, increased mean open times of indi- clamp techniques. Under voltage clamp, neuroac- vidual sodium channels in voltage clamp studies tive pyrethroids prolong the deactivation of up to 200-fold and produced channels that re- sodium channels, which is evident as the produc- mained open at the end of the depolarizing pulse tion of a slowly-decaying sodium tail current that (Chinn and Narahashi, 1986; Holloway et al., flows following a depolarization–repolarization 1989). Recordings of single sodium channels re- cycle (Narahashi, 1992, 1996). In most prepara- vealed that deltamethrin also delayed channel opening in response to a voltage pulse (Chinn and tions, pyrethroids also retard the closing (inactiva- Narahashi, 1986). These findings have been inter- tion) of sodium channels during a depolarizing preted as evidence that pyrethroids stabilize multi- pulse. Voltage-clamp experiments show that the ple channel states, slowing the transitions between apparently divergent effects of pyrethroids on them. nerve excitability observed in intracellular mi- Voltage and patch clamp experiments have also croelectrode recordings are the result of similar been employed to assess the combined effects of effects on sodium channels (Lund and Narahashi, two different pyrethroids on sodium channels. In 1983; Vijverberg et al., 1983). Compounds that voltage clamp experiments, initial exposure of produce burst discharges in intact nerves produce dorsal root ganglion neurons to either fenvalerate tail currents that decay rapidly, whereas com- (Song et al., 1996) or deltamethrin (Tabarean and pounds that produce use-dependent block of ac- Narahashi, 1998) produced characteristic modifi- tion potentials produce extremely persistent tail cations of sodium currents, including the induc- currents that exhibit little or no decay for several tion of slowly-decaying tail currents. Subsequent seconds after repolarization and persist for several application of tetramethrin to these preparations minutes. The differences in tail current kinetics antagonized the effects of fenvalerate or between these groups of compounds are conceptu- deltamethrin, so that the persistent tail current ally consistent with their different effects on associated with these compounds disappeared and evoked action potentials: transient prolongation was replaced by the rapidly-decaying tail current of sodium channel inactivation and deactivation typical of the action of tetramethrin in this exper- results in a depolarizing after-potential and repeti- imental system. Subsequent patch clamp studies tive firing, whereas persistent prolongation of of the action of tetramethrin and deltamethrin at sodium inactivation and deactivation produces single sodium channels (Motomura and Nara- slow depolarization of the cell membrane and hashi, 2001) confirmed the results of voltage concomitant block of the action potential. It is clamp studies by documenting the displacement of also noteworthy that pyrethroids identified as deltamethrin by subsequently applied te- having intermediate behavior in intracellular tramethrin. Interestingly, this study found no evi- recordings produce tail currents under voltage dence of the coexistence of tetramethrin- and clamp conditions with intermediate decay kinetics. deltamethrin-modified channels in recordings D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 33 from a single membrane patch. The results of structure–toxicity relationships. The enhancement these studies yield two important and provocative of both veratridine-dependent sodium uptake into conclusions. First, pyrethroids that produce quali- brain synaptosomes and BTX-B binding to brain tatively divergent kinetic modifications of sodium sodium channels is stereospecific for deltamethrin channels exert their effects either at a shared and the neurotoxic isomers of cypermethrin and binding site or at sites that are allosterically cou- fenvalerate (Ghiasuddin and Soderlund, 1985; pled, so that occupancy of one site precludes Brown et al., 1988; Rubin et al., 1993). However, occupancy of the second. Second, for the combi- structural analogs of deltamethrin and cyperme- nations of pyrethroids that have been examined, thrin lacking the a-cyano substituent were much effects of two different pyrethroids on both less effective in these assays. macroscopic sodium currents and individual The results of these studies implied the exis- sodium channels are antagonistic rather than ad- tence of a pyrethroid binding site on sodium ditive or synergistic. channels, designated Site 6 by Lombet et al. (1988), that is distinct from the sites labeled by 6.2.1.3. Biochemical studies of pyrethroid action on other radioligands and allosterically coupled to sodium channels. Biochemical studies of the ac- the veratridine/batrachotoxin site. Initial attempts tions of pyrethroids on sodium channels have to label this site with a pyrethroid radioligand provided important information on the effects of were unsuccessful (Soderlund et al., 1983; Lombet these insecticides on channels in the mammalian et al., 1988) due to the extreme lipophilicity and central nervous system. Two types of experiments modest potency of the available pyrethroid radi- have been employed: studies of the effects of oligands. More recent studies using a more potent pyrethroids and other sodium channel-directed experimental pyrethroid as a radioligand have toxins on radiosodium uptake into synaptic vesi- demonstrated high-affinity saturable binding to cles, and studies of the effects of pyrethroids on brain sodium channels that exhibits the allosteric the binding of radioligands that label the sodium coupling to other binding domains predicted by channel. Pyrethroids alone do not affect ra- previous studies (Trainer et al., 1997). However, diosodium uptake into brain synaptosomes, but the utility of this ligand for the detailed character- they enhance sodium uptake that is stimulated by ization of pyrethroid binding is still limited by its the alkaloid neurotoxins veratridine or batra- extreme lipophilicity, which produces high levels chotoxin (Ghiasuddin and Soderlund, 1985; of unsaturable, non-specific binding to cell mem- Soderlund et al., 1987; Bloomquist and Soder- branes and limits the sensitivity of the assay. lund, 1988). Pyrethroids also enhance the binding It is not clear from the available data whether a of [3H]batrachotoxinin A-20-a-benzoate (BTX-B), single binding site on any given sodium channel an analog of batrachotoxin, to brain sodium mediates the action of pyrethroids on that chan- channels (Brown et al., 1988; Lombet et al., 1988). nel. Molecular genetic studies of pyrethroid resis- Pyrethroid-dependent enhancement of sodium up- tance in insects (see Section 2.2), take into presynaptic and postsynaptic nerve ter- electrophysiological studies with dorsal root gan- minals is also indirectly evident in assays of the glion neurons, and biochemical studies using sodium-dependent components of neurotransmit- mammalian brain sodium channels are consistent ter release (Brooks and Clark, 1987; Eells and with the existence of a single binding site per Dubocovich, 1988), depolarization of synapto- channel protein associated with insecticidal activ- somes (Eells et al., 1992, 1993), and ity and mammalian toxicity. However, studies of phosophoinositide turnover in brain synaptoneu- the interactions of the isomers of tetramethrin rosomes (Gusovsky et al., 1989). with sodium channels in squid giant axon prepa- Structure–activity relationships for the action rations suggested the existence of multiple binding of pyrethroid isomers on rat and mouse brain sites with specificity for different isomers includ- sodium channels in sodium uptake and radioli- ing the 1S isomers that lack insecticidal activity gand binding assays are in general agreement with (Lund and Narahashi, 1982). Also, kinetic studies 34 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 of the effects of deltamethrin on the binding of These findings imply that pyrethroids affect multi- BTX-B to mouse brain sodium channels docu- ple sodium channel isoforms, including those not ment the allosteric antagonism of the effects of expressed in the mammalian central nervous sys- deltamethrin by its a-R epimer, which lacks tem, and that these isoforms may vary in their demonstrable mammalian toxicity (Rubin et al., relative sensitivity to different pyrethroids. 1993). Whereas these studies imply the existence The clearest evidence of differential sensitivity of one or more additional sites on sodium chan- to pyrethroids between sodium channel isoforms nels that are capable of binding inactive isomers, is found in the responses of the tetrodotoxin the toxicological significance of such sites is not (TTX)-sensitive and TTX-resistant sodium chan- established. nel populations in dorsal root ganglion cells to pyrethroids. The TTX-resistant current in these 6.2.1.4. Differential pyrethroid sensiti6ity of sodium cells is much more sensitive than the TTX-sensi- channel isoforms. Most of what is known about tive current to allethrin (Ginsburg and Narahashi, the actions of pyrethroids on mammalian sodium 1993), tetramethrin (Tatebayashi and Narahashi, channels has been learned using neuronal tissue 1994; Song and Narahashi, 1996) and preparations, which are now known to express deltamethrin (Tabarean and Narahashi, 1998). multiple sodium channel a subunit isoforms. As a It is now possible to examine the functional and result, the action of pyrethroids has not been pharmacological properties of individual cloned correlated with the expression of identified sodium channel isoforms by expressing them in sodium channel isoforms in these tissues. How- unfertilized oocytes of the frog Xenopus lae6is. ever, a limited number of physiological studies This model expression system synthesizes sodium suggest that sodium channel isoforms expressed in channel proteins from cloned cDNA injected into various mammalian tissues exhibit differential the nucleus or synthetic messenger RNA injected sensitivity to pyrethroids. Deltamethrin, but not into the cytoplasm and inserts functional channels cismethrin or permethrin, has a direct positive into the cell membrane (Lester, 1988). Because inotropic effect on the mammalian heart in addi- oocytes do not normally express endogenous tion to an indirect effect mediated by the stimula- sodium channels, the properties of the expressed tion of catecholamine release (Forshaw and channels can be investigated using electrophysio- Bradbury, 1983; Berlin et al., 1984; Daly et al., logical techniques (typically voltage or patch 1987). Five a-cyano pyrethroids (fluorocyphe- clamp) without interference from endogenous nothrin, cyhalothrin, cyfluthrin, deltamethrin, fen- sodium currents (Stu¨hmer, 1988). valerate) that produce the CS syndrome of Initial experiments using cloned sodium chan- poisoning in the rat (Verschoyle and Aldridge, nel isoforms in the Xenopus oocyte expression 1980) also cause repetitive action potentials in system examined the sensitivity of the rat brain directly-stimulated mammalian skeletal muscle IIa sodium channel isoform (Auld et al., 1988), but do not cause repetitive discharges evoked by which is abundantly expressed in the adult brain. sensory stimulation in the trigeminal reflex path- Expression of the rat brain IIa sodium channel a way (Forshaw and Ray, 1986; Forshaw et al., subunit, either alone or in combination with the b 1987; Wright et al., 1988). In contrast, three com- rat 1 subunit, produced functional sodium chan- pounds (cismethrin, fenfluthrin, NRDC 108) that nels in oocytes that were sensitive to modification produce the T syndrome are inactive on skeletal by [1R,cis,aS]-cypermethrin (Smith and Soder- muscle but are active on sensory and motor neu- lund, 1998). These studies demonstrated that the ronal elements of the rat trigeminal reflex path- pyrethroid-binding site was intrinsic to the a sub- b way (Forshaw and Ray, 1986; Wright et al., unit. However, coexpression with the 1 subunit 1988). Two pyrethroids classified as intermediate increased the apparent affinity of rat brain IIa with respect to signs of intoxication (cyphe- channels to pyrethroids more than 20-fold, thus nothrin, fenpropathrin) are active on both nerve implying an allosteric effect of coassembly with b and muscle in these assays (Wright et al., 1988). the 1 subunit on the pyrethroid binding site. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 35

b a Even in the presence of the 1 subunit, rat brain the selective effects of -cyano compounds on IIa channels exhibited only modest sensitivity to muscle action potentials observed in electrophysi- cypermethrin and deltamethrin. The potency of ological assays (Forshaw and Ray, 1986; Forshaw cypermethrin and deltamethrin in these assays et al., 1987; Wright et al., 1988). was similar to the potency of RU39568, a struc- The actions of pyrethroids on the rat brain IIa turally-related a-cyano pyrethroid, as a displacer channel isoform were compared to those on the of the specific binding of a tritiated pyrethroid rat SNS/PN3 sodium channel isoform, which is radioligand to rat brain IIa sodium channels ex- preferentially expressed in the peripheral nervous pressed in Chinese hamster ovary cells (Trainer et system and is distinguished by its high level of al., 1997). resistance to TTX (Akopian et al., 1996; The effects of deltamethrin and related com- Sangameswaran et al., 1996). In contrast to rat pounds on rat brain IIa sodium channels exhib- brain IIa sodium channels, rat SNS/PN3 sodium ited the stereospecificity predicted by channels were highly sensitive to pyrethroids rep- structure–toxicity relationships. Only resenting both the T (cismethrin) and CS (cyper- deltamethrin itself and its 1R,trans,aS isomer, methrin) syndromes of intoxication (Smith and which is neurotoxic to rats (Verschoyle and Soderlund, 2001). The threshold cypermethrin Aldridge, 1980), were effective in modifying cur- concentration for the modification of SNS/PN3 rents carried by rat brain IIa sodium channels, channels was approximately 60-fold lower than whereas other isomers having the 1S or aR the threshold concentration for cypermethrin-de- configurations, which have very low acute toxic- pendent modification of rat brain IIa channels. ities, were inactive at the highest concentrations The biophysical properties, TTX resistance, and attainable in this assay system (Smith and Soder- pyrethroid sensitivity of the SNS/PN3 sodium lund, 1996). channel isoform expressed in oocytes suggest that Rat brain IIa channels expressed in oocytes this isoform carries the TTX-resistant, pyrethroid- were completely insensitive to several pyrethroids sensitive sodium current found in dorsal root that produce the T syndrome (cismethrin, ganglion neurons (Ginsburg and Narahashi, 1993; fenfluthrin, tetramethrin) at the maximum nomi- Tatebayashi and Narahashi, 1994; Tabarean and nal concentrations of pyrethroid that were attain- Narahashi, 1998). able in this assay system (]250 mM) (Smith and Soderlund, 1998). The complete insensitivity of 6.2.2. Voltage-sensiti6e calcium channels rat brain IIa channels to these compounds is surprising in view of the well-established neuro- 6.2.2.1. Function, structure, classification and phar- toxic effects produced by these pyrethroids upon macology. Voltage-sensitive calcium channels are direct injection into the brain (Gray and Soder- ubiquitous in excitable membranes and modulate lund, 1985). It is therefore likely that the central a wide range of cellular events including the re- neurotoxic effects of these pyrethroids, as well as lease of neurotransmitters and other secretions, other compounds that produce the T syndrome, metabolic adjustments, cell proliferation, contrac- are mediated by actions on one or more molecular tion, and control of gene expression. Voltage-sen- targets that are expressed in the central nervous sitive calcium channels are heterooligomeric system other than the brain IIa sodium channel complexes comprised of several structurally dis- isoform. similar subunits. Purification of calcium channels Preliminary experiments with rat skeletal mus- from vertebrate skeletal muscle has identified five a a b g d a cle (rSkM1) isoform suggest that this channel, like subunits ( 1, 2, , and ) with the large 1 the rat brain IIa channel isoform is also insensi- subunit forming the ion-conducting pore of the tive to several non-cyano compounds and mod- channel. Molecular cloning studies have shown a a estly sensitive to [1R,cis, S]-cypermethrin and that calcium channel 1 subunits are members of a deltamethrin (T.J. Smith and D.M. Soderlund, multigene family that is evolutionarily related to unpublished results). This result is consistent with the voltage-sensitive sodium channel gene family. 36 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

There also are multiple isoforms of the b subunit pletely blockled only the T-type calcium current that differentially alter the biophysical properties and resulted in a significant slowing of the dias- a of calcium channels when co-expressed with a 1 tolic depolarization in the latter two-thirds of the subunit. The structural basis of calcium channel pacemaker’s action potential in heart muscle diversity is still not completely understood, and (Hagiwara et al., 1988). Tetramethrin also abol- the relationship between calcium currents ished the T-type calcium currents in intestinal recorded from native channels and currents smooth muscle cells from guinea pig in a dose recorded from expressed channel subunit genes in dependent manner but had no effect on L-type various combinations is not well established (Con- currents (Yabu et al., 1989). Despite the lack of ley and Brammar, 1999). effect of deltamethrin and fenvalerate on T- and Several classes of calcium channels have been L-type currents in neuroblastoma cells, these com- identified based on their biophysical, electrophysi- pounds have been reported to block calcium cur- ological and pharmacological properties. An im- rents in guinea pig olfactory cortex slices portant biophysical distinction is that certain (Narahashi, 1987). channels are activated by only small depolariza- Two calcium currents (putatively associated tions (i.e. low-voltage activated), whereas others with L- and T-type calcium channels, respectively) are activated only in the presence of larger depo- recorded from house fly thoracic neurons exhibit larizations (high-voltage activated). The high a differential response to deltamethrin (Duce et voltage-activated channels are further subdivided al., 1999). Deltamethrin treatment resulted in a on the basis of tissue distribution and biophysical significant hyperpolarizing shift in the voltage de- and pharmacological properties into five classes pendence of activation of the T-type current but (L-, N-, Q-, P-, and R-types) whereas the low- had no effect on the L-type current. This hyper- voltage activated channels are represented by only polarizing shift was similar to the effect caused by one biophysical and pharmacological class (T- deltamethrin on the TTX-sensitive sodium current type). Comparison of the deduced amino acid recorded from the same preparation. Addition- a sequences of numerous cloned 1 subunits has ally, deltamethrin increased the amplitude or fre- resulted in a molecular classification that divides quency of the spontaneous oscillations of internal these subunits into 10 structural subgroups. Cor- Ca2+ and enhanced the amplitude of depolariza- relation of structural subgroups with functional tion-induced increases in Ca2+. These actions channel types reveals that L- and T-type channels were not altered by the presence or absence of exhibit the greatest molecular diversity, whereas TTX but were blocked by verapamil, a pheny- N-, P- and Q-type channels appear more homoge- lalkylamine calcium channel antagonist. neous at the level of primary structure (Conley and Brammar, 1999). 6.2.2.3. Effects of pyrethroids on neurotransmitter release in 6itro. The release of neurotransmitters is 6.2.2.2. Electrophysiological studies of pyrethroid a highly organized event dependent on external action on calcium channels. Patch clamp tech- calcium influx via voltage-sensitive calcium chan- niques detect distinct L- and T-type calcium cur- nels associated with the plasma membrane of the rents in mouse neuroblastoma (N1E-115) cells. In presynaptic nerve terminals (Llinas, 1982). N-type these cells, the non-cyano pyrethroid tetramethrin calcium channels appear to play a dominant role blocked 75% of the T-type calcium current but in depolarization-dependent release of neurotrans- only 30% of the L-type current (Yoshii et al., mitter, but P-/Q- and T-type calcium channels are 1985). In contrast, the a-cyano pyrethroids now known to be involved in neurotransmitter deltamethrin and fenvalerate had no effect on release and also function in excitation-secretion either calcium current. Similar results were ob- coupling and spontaneous firing activity, respec- tained in assays of the T- and L-type calcium tively (Conley and Brammar, 1999). currents in single isolated sino-atrial node cells of The first in vitro findings that established the rabbit heart. In this study, tetramethrin com- direct action of pyrethroids on the spontaneous D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 37 release of neurotransmitters were obtained using voltage-sensitive sodium channels. In another isolated presynaptic nerve terminals (synapto- study, deltamethrin displaced 3H-verapamil (a somes) from guinea pig cortex (Nicholson et al., phenethylamine calcium channel blocker of L- 1987). Both deltamethrin and permethrin in- and T-type calcium channels) binding to rat brain creased spontaneous release but permethrin was synaptic membranes (Kadous et al., 1994). much less potent than deltamethrin. The sponta- neous release of neurotransmitter was only par- 6.2.2.5. The Paramecium model: a study of tially blocked by tetrodotoxin and was partially pyrethroid action in the absence of 6oltage-sensiti6e dependent upon external calcium. A series of 25 sodium channels. Deltamethrin and resmethrin are pyrethroids also increased the spontaneous release highly toxic to Paramecium tetraurelia, an organ- of neurotransmitters from rat brain synaptosomes ism that does not possess voltage-sensitive sodium (Doherty et al., 1986a, 1987). Release promoted channels (Clark et al., 1995). In behavioral by pyrethroids was only partially abolished by bioassays, deltamethrin-treated P. tetraurelia ex- tetrodotoxin or by substituting choline for hibited an increase in backward swimming (Clark sodium, indicating an action at a site other than et al., 1995), a well-characterized avoidance re- the sodium channel. Fenvalerate, cypermethrin, sponse controlled by calcium influx via T-type and deltamethrin also increased the spontaneous, calcium channels associated with the ciliary mem- calcium-dependent release of dopamine and brane (Ehrlich et al., 1984, 1988). The enantiomer acetylcholine from rabbit striatal brain slices of deltamethrin, which has very low insecticidal (Eells and Dubocovich, 1988). This spontaneous activity and mammalian toxicity, had no signifi- release was concentration-dependent and was spe- cant effect on either mortality or avoidance be- cific for the neurotoxic isomers of these havior. Pawn mutants, which lack a functional pyrethroids. Fenvalerate, however, had no effect calcium channel, were unaffected by deltamethrin. on the spontaneous release of neurotransmitters Intracellular electrophysiological recordings of from hippocampal brain slices, indicative of a whole P. tetraurelia showed that 10−9 M regional sensitivity difference to pyrethroids. deltamethrin resulted in membrane destabiliza- A more sensitive and marked enhancement of tion, increased spontaneous action potentials, pyrethroid-induced neurotransmitter release is ev- repetitive discharges, and membrane depolariza- ident following nerve terminal depolarization. tion. These and other recent findings establish Treatment with deltamethrin, cypermethrin and that deltamethrin acts as a T-type calcium channel fenvalerate greatly enhanced a calcium-dependent agonist in P. tetraurelia (Symington et al., 1999). neurotransmitter release following the depolariza- tion of rat brain synaptosomes by potassium 6.2.3. Voltage-sensiti6e chloride channels (Brooks and Clark, 1987). The deltamethrin-in- duced release was highly correlated with calcium 6.2.3.1. Function, structure and pharmacology of uptake and only partially blocked by TTX (Clark 6oltage-sensiti6e chloride channels. Voltage-sensi- and Brooks, 1989), but release was completely tive chloride channels are a structurally diverse blocked by D595, a potent phenethylamine cal- and widely-distributed group of cell membrane cium channel blocker (Brooks and Clark, 1987). proteins (Jentsch, 1994; Gelband et al., 1996). Molecular cloning has revealed two structural 6.2.2.4. Biochemical studies of pyrethroid action on classes of voltage-sensitive chloride channels that calcium channels. Several pyrethroids have been are distinct from each other and structurally unre- reported to inhibit the binding of a tritiated L- lated to the voltage sensitive cation (e.g. sodium type calcium channel blocker, [3H]nimodipine, to and calcium) channel family (Jentsch, 1996). The rat brain synaptosomes (Ramadan et al., 1988a). CLC class of chloride channels, encoded by a However, the affinity for pyrethroids in this assay multi-gene family, are involved in the stabilization was low so that the concentration required for of membrane resting potential, transepithelial inhibition exceeded that necessary to modify transport, and cell volume regulation in virtually 38 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 all cell types (Jentsch, 1996). A second structural deltamethrin and cypermethrin but not cismethrin class of voltage-dependent chloride channel (Forshaw et al., 1993; Ray et al., 1997). proteins is exemplified by the cystic fibrosis transmembrane conductance regulator (CFTR), a 6.2.3.3. Action of pyrethroids: pharmacological cell membrane protein that regulates chloride con- studies in 6i6o. Studies in vivo of the interactions ductance in epithelial cells that was first identified between pyrethroids and agents known to act at as the molecular locus of genetic defects that voltage-sensitive chloride channels provide further cause cystic fibrosis (Gelband et al., 1996; Jentsch, insight into the involvement of this target in 1996). pyrethroid intoxication. These experiments in- The pharmacology of voltage-dependent chlo- volved co-administration of deltamethrin with ride channels is poorly characterized due to the ivermectin (which is known to activate voltage- lack of high-affinity ligands specific for these sensitive chloride channels and have limited access proteins (Gelband et al., 1996). Many compounds to the central nervous system), pentobarbital (a that affect or bind to voltage-dependent chloride barbiturate that selectively activates voltage-sensi- channels, such as the convulsant t-butylbicy- tive chloride channels), or phenobarbital (which clophosphorotrithioate (TBPS), certain cyclodiene exhibits sedative effects typical of barbiturates insecticides, avermectins, and barbiturates, also without activating voltage-sensitive chloride chan- act at the GABA receptor–chloride ionophore nels) (Ray et al., 1999; Forshaw et al., 2000). complex (Schwartz et al., 1984; Abalis et al., 1985; Intraperitoneal pretreatment of rats with iver- Payne and Soderlund, 1991; Bloomquist, 1993b). mectin reduced the degree of salivation caused by The existence of pharmacological crosstalk be- subsequent intravenous treatment with tween different chloride channel types complicates deltamethrin and also reduced the incidence of pharmacological discrimination between GABA- deltamethrin mortality and the motor signs of gated and voltage-sensitive chloride channels. deltamethrin intoxication at this dose, but iver- mectin only affected salivation at a lower dose of 6.2.3.2. Actions of pyrethroids: electrophysiological deltamethrin (Ray et al., 1999; Forshaw et al., studies in 6itro. Studies of the action of cismethrin 2000). Ivermectin also reduced the severity of the and deltamethrin on skeletal muscle showed that direct effects of deltamethrin on skeletal muscle deltamethrin but not cismethrin increased muscle excitability in urethane-anesthetized rats (Ray et membrane resistance, which was suggested to re- al., 1999; Forshaw et al., 2000). sult from a block of the chloride permeability of In parallel experiments, pentobarbital signifi- the muscle membrane (Forshaw et al., 1987). A cantly antagonized the motor signs of intoxication subsequent study (Forshaw and Ray, 1990) confi- and reduced the degree of mortality in rmed the effect of deltamethrin but not cismethrin deltamethrin-treated rats but was less effective on the input resistance of rat diaphragm skeletal than ivermectin in reducing salivation (Ray et al., muscle fibers and showed that a reduction in 1999; Forshaw et al., 2000). In contrast, an equi- extracellular chloride ion concentration prevented sedative dose of phenobarbital (which lacks the the effects of deltamethrin. This study also docu- selectivity of pentobarbital for voltage-sensitive mented similar effects of deltamethrin on the in- chloride channels) reduced the number of deaths put resistance of rat vagus nerve preparations that in deltamethrin-treated rats but did not signifi- were prevented by low extracellular chloride or cantly affect salivation or the motor signs of treatment of the preparation with ivermectin, intoxication (Ray et al., 1999; Forshaw et al., which activates neuronal voltage-dependent chlo- 2000). These studies were also extended to exam- ride channels (Abalis et al., 1986a). Patch-clamp ine the effects of barbiturates on intoxication by studies of single neuronal voltage-dependent chlo- cismethrin, a pyrethroid without demonstrable ef- ride channels in excised membrane patches from fects on voltage-sensitive chloride channels in N1E-115 neuroblastoma documented the block- vitro (see Section 6.2.3.2). Pentobarbital did not ade of single channel conductance by affect either the motor signs or the number of D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 39 deaths in rats treated intravenously with cis- chloride channel, which include naturally occur- methrin, but phenobarbital produced a significant ring compounds such as picrotoxinin and syn- reduction in lethality under these conditions (Ray thetic insecticides such as the chlorinated et al., 1999). These authors concluded that the cyclodienes (e.g. dieldrin, endosulfan) effects of ivermectin and pentobarbital on the (Bloomquist, 1998) and phenylpyrazoles (e.g. signs of deltamethrin intoxication reflected a spe- fipronil) (Hosie et al., 1995), act as convulsants by cific antagonism of the action of deltamethrin on blocking the inhibitory effects of GABA and pro- voltage-sensitive chloride channels, whereas the ducing an indirect augmentation of excitatory effects of pentobarbital and phenobarbital on the neurotransmission. lethality of both deltamethrin and cismethrin were ascribed to central effects on neuronal excitability 6.3.1.2. Action of pyrethroids: biochemical and that were not specifically attributable to an action physiological e6idence.Thefirst report of an inter- on chloride channels (Ray et al., 1999). action of pyrethroids on GABA receptors showed the stereospecific inhibition by deltamethrin but 6.3. Actions of pyrethroids on ligand-gated ion not its non-toxic a-R epimer of the binding of channels [3H]dihydropicrotoxinin to the convulsant (chlo- ride channel) site of rat brain GABA receptors 6.3.1. GABA receptors (Leeb-Lundberg and Olsen, 1980). The subse- quent development of [35S]TBPS as an improved 6.3.1.1. Function, structure, and pharmacology of radioligand for the chloride channel site of GABA GABA receptors. The GABA (g-aminobutyric receptors led to the further documentation of the acid) receptor–chloride ionophore complex is an interaction of pyrethroids with the convulsant/ important mediator of inhibitory neurotransmis- chloride channel site (Lawrence and Casida, 1983; sion in the mammalian nervous system (Macdon- Casida and Lawrence, 1985). These studies, em- ald and Olsen, 1994). Release of GABA by ploying 37 pyrethroids, documented the inhibition presynaptic nerve terminals activates a chloride of [35S]TBPS binding by the toxic isomers of four channel on the postsynaptic membrane, leading to pyrethroids containing the a-cyano-3-phenoxy- hyperpolarization of the postsynaptic nerve termi- benzyl moiety (cypermethrin, deltamethrin, fen- nal and thus requiring an increase in the amount valerate and fluvalinate) and by isomer mixtures of excitatory input into that terminal in order to of two other a-cyano compounds (cyphenothrin excite the postsynaptic neuron. GABA receptors and fenpropathrin) but not by the non-toxic iso- are pentameric complexes composed of members mers of a-cyano compounds or by any of a large group of structurally related subunit pyrethroids lacking the a-cyano substituent. How- proteins that comprise several structural classes ever, the original claims for absolute stereospecifi- (designated a, b, g, d, o,andr) with as many as city in these assays were contradicted by the six isoforms in each class (Barnard et al., 1998). subsequent finding that the 1S,cis,aS isomer of Many GABA receptors in the vertebrate CNS are cypermethrin, which has low acute toxicity to formed from two a subunits, two b subunits, and mammals, produced significant inhibition of one g subunit, but the existence of multiple iso- [35S]TBPS binding to rat brain GABA receptors forms of these subunits, plus the incorporation of (Seifert and Casida, 1985). The structure–activity subunits from other classes in some receptor sub- correlations for pyrethroid-dependent inhibition types, results in a high level of molecular diversity of [35S]TBPS binding led to the widely-recognized among GABA receptors. hypothesis that a-cyano pyrethroids caused the GABA receptors are important target sites for CS intoxication syndrome by an action at the the action of several classes of therapeutic drugs GABA receptor–ionophore complex (Lawrence and toxicants (Johnston, 1996). Ethanol, barbitu- and Casida, 1983). rates, and benzodiazepine anxiolytics allosterically Further analysis of the action of pyrethroids on modify the action of GABA. Blockers of the GABA receptors was undertaken using assays of 40 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

GABA receptor function. Several studies of the principal mediators of excitatory transmission in effects of pyrethroids on GABA-stimulated chlo- the central nervous system and are an important ride-36 uptake into brain vesicles confirmed the insecticide target site. Nicotinic acetylcholine recep- action of neurotoxic isomers of a-cyano tors are pentameric structures that form a relatively pyrethroids as antagonists at mammalian brain non-selective cation channel that is opened by the GABA receptors (Bloomquist and Soderlund, binding of acetylcholine to the receptor. Nicotinic 1985; Abalis et al., 1986b; Bloomquist et al., 1986; acetylcholine receptor subunits are encoded by a Ramadan et al., 1988c). However, the inhibition of gene family and are related structurally to GABA chloride uptake in these studies was typically in- receptor subunits (Vafa and Schofield, 1998). complete at maximally effective pyrethroid concen- trations. Also, the incomplete stereoselectivity of 6.3.2.2. Actions of pyrethroids: biochemical and pyrethroid action on GABA receptors in these electrophysiological studies. An effect of assays was inconsistent with the profound pyrethroids on nicotinic acetylcholine receptors stereospecificity of pyrethroid toxicity. For exam- was initially suggested by a study showing the 3 ple, the enantiomer of deltamethrin, which is at inhibition of [ H]perhydrohistrionicotoxinin (H12- least 500-fold less toxic than deltamethrin, was only HTX) to a site associated with the channel of the 10-fold less effective as an inhibitor of GABA-de- nicotinic acetylcholine receptor of electric eel elec- pendent chloride uptake (Bloomquist and Soder- troplax membranes (Abbassy et al., 1982). Further lund, 1985; Bloomquist et al., 1986). studies with a range of pyrethroids showed that

Electrophysiological assays have been employed pyrethroids inhibited both the binding of H12-HTX to assess the relative sensitivity of GABA receptors to electroplax membrane receptors and receptor- and voltage-sensitive sodium channels expressed in regulated calcium uptake into electroplax mem- the same cell or neuronal pathway to pyrethroids. brane vesicles (Abbassy et al., 1983a,b). Several GABA receptors in cultured dorsal root ganglion non-cyano pyrethroids (allethrin, pyrethrins, te- neurons were much less sensitive to the actions of tramethrin, and resmethrin) were significantly more deltamethrin than were the populations of voltage- potent inhibitors of H12-HTX binding in these sensitive sodium channels expressed in the same assays than permethrin and several a-cyano cells (Ogata et al., 1988). Also, electrophysiological pyrethroids (cypermethrin, fenvalerate, fluvalinate, recordings from defined GABAergic pathways in deltamethrin, and fenpropathrin). However, the the rat hippocampus (Gilbert et al., 1989; Joy et al., potencies of selected pyrethroids as inhibitors of

1989, 1990; Joy and Albertson, 1991) showed that H12-HTX binding were poorly correlated with their the effects of pyrethroids were consistent with an potencies as inhibitors of receptor-regulated cal- augmentation of inhibition, possibly as a result of cium uptake. Further, the inhibition of receptor- sodium channel-mediated presynaptic excitation of regulated calcium uptake by allethrin and GABAergic neurons, rather than the antagonism of fenvalerate could not be reproduced in assays of inhibition that is typical of established blockers of receptor-activated sodium uptake (Sherby et al., the GABA-gated chloride channel. 1986). Finally, studies of acetylcholine-gated cur- rents in voltage-clamped N1E-115 neuroblastoma 6.3.2. Nicotinic acetylcholine receptors cells showed that [1R,cis]-cyphenothrin, [1R,cis]- fenfluthrin, and [1S,cis]-fenfluthrin blocked both 6.3.2.1. Structure and function of nicotinic acetyl- acetylcholine- and serotonin-induced currents choline receptors. The nicotinic acetylcholine recep- through effects on separate receptors for tor is the mediator of fast excitatory neurotrans- these ligands, but the concentration required to mission at vertebrate neuromuscular junctions. produce such effects was in excess of the concentra- Nicotinic acetylcholine receptors are also present in tions of [1R,cis]-cyphenothrin and the neurotoxic the vertebrate central nervous system, but their role 1R,cis isomer of fenfluthrin to modify sodium in central neurotransmission is less clearly defined. channel function in the same cells (Oortgiesen et al., In insects, nicotinic acetylcholine receptors are the 1989). D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 41

6.3.3. Excitatory glutamate receptors tory glutamate receptors in this preparation (Narahashi, 1987). 6.3.3.1. Structure, function and di6ersity of gluta- mate receptors. In the vertebrate central nervous 6.4. Actions of pyrethroids on other biochemical system, excitatory glutamate receptors mediate targets the fast synaptic action of L-glutamate, a major excitatory neurotransmitter (Sprengel and See- 6.4.1. Peripheral-type benzodiazepine receptors burg, 1995). These complexes form relatively non- selective cation-selective channels that are 6.4.1.1. Function and pharmacology of peripheral- permeable to calcium and sodium, and are essen- type benzodiazepine receptors. Benzodiazepines ex- tial components of synaptic function and plastic- ert their therapeutic effects primarily as a result of ity in the brain. Disruptions of glutamatergic their actions on the GABA receptor complex (see neurotransmission is involved in excitotoxicity Section 6.3.1.1). However, studies of the binding that is associated with local brain damage due to of the benzodiazepine radioligand [3H]diazepam deficient blood flow. Glutamate receptors are also in rat tissue preparations identified high affinity implicated in epileptic discharge and play an im- binding sites in peripheral, non-neuronal tissues portant role in long-termed potentiation. that lacked GABA receptors (Braestrup and Excitatory glutamate receptor-channel com- Squires, 1977). Subsequent studies showed that plexes are heteromultimers formed from subunits this second class of benzodiazepine binding sites that are structurally related to subunits of nico- (called ‘peripheral-type benzodiazepine receptors’) tinic acetylcholine and the GABA receptors could be distinguished from benzodiazepine bind- (Sprengel and Seeburg, 1995). Excitatory gluta- ing sites associated with the GABA receptors by mate receptor-channel complexes are classified on its pharmacological profile, tissue distribution, the basis of their agonist pharmacology into five and subcellular localization (Zisterer and major classes: the N-methy-D-aspartate; kainate; Williams, 1997). In contrast to diazepam, which a-amino-3-hydroxy-5-methyl-4-isoxazole propi- binds to both classes of benzodiazepine binding onate; 2-amino-4-phosphonobutanoate and trans- sites with relatively high affinity, the ligands Ro5- 1-amino-cyclopentyl-1,3-decarboxylate receptors. 4864 and PK11195 specifically label the periph- Of these, the N-methyl-D-aspartate receptors eral-type benzodiazepine receptor. form ion channels that are primarily permeable to The structure and function of the peripheral- calcium, whereas the other glutamate receptor type benzodiazepine receptor remain incompletely classes form channels that preferentially transport characterized (Zisterer and Williams, 1997). This sodium ions. receptor is associated with the mitochondrial outer membrane and appears to be associated 6.3.3.2. Action of pyrethroids on glutamate recep- with the voltage-dependent anion channel tors. The initial evidence for an action of (‘porin’). Although, a variety of physiological and pyrethroids on glutamate receptors was obtained biochemical effects have been attributed to pe- in binding assays with [3H]kainate, which labels ripheral-type benzodiazepine receptor ligands one class of glutamate receptors. In these studies, (Zisterer and Williams, 1997), the most relevant both cis-permethrin and deltamethrin inhibited from the perspective of this review are the convul- [3H]kainate binding in mouse brain preparations sant and proconvulsant effects of Ro5-4864 and (Staatz et al., 1982). Allethrin, deltamethrin and the specific antagonism of these effects by fenvalerate also blocked N-methy-D-aspartate-de- PK11195 (Benavides et al., 1984). pendent calcium currents in rat hippocampal and neocortical neurons (Frey and Narahashi, 1990). 6.4.1.2. Actions of pyrethroids: biochemical studies. In contrast to these findings, assays with guinea Biochemical evidence for the action of a-cyano pig olfactory cortex slices did not detect any pyrethroids on GABA receptors (see Section effects of deltamethrin or fenvalerate on excita- 6.3.1.2) led several investigators to explore the 42 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 effects of pyrethroids on the binding of benzodi- teristic signs of pyrethroid-induced intoxication azepine radioligands in brain preparations. Initial (Devaud and Murray, 1986). PK11195, which an- studies with [3H]Ro5-4864, performed under the tagonizes the proconvulsant activity of Ro5-4864 assumption that this ligand labeled a site associ- (Benavides et al., 1984), also antagonized the pro- ated with the GABA receptor complex, showed convulsant effects of deltamethrin and perme- that both deltamethrin and the insecticidal and thrin, thereby implicating an action of pyrethroids neurotoxic 1R,cis,aS isomer of cypermethrin were at the peripheral-type benzodiazepine receptor. A potent inhibitors of Ro5-4864 binding in rat brain second study by the same research group (Devaud synaptic membranes, whereas fenvalerate, perme- and Murray, 1988) extended these observations to thrin, allethrin, and the remaining seven isomers include five additional non-cyano pyrethroids (al- of cypermethrin were all at least 15-fold less po- lethrin, cismethrin, kadethrin, resmethrin and te- tent inhibitors of Ro5-4864 binding (Lawrence et tramethrin) and the 1R,cis and 1S,cis isomers of al., 1985). Two subsequent studies examined a permethrin. All of the compounds tested except total of 15 pyrethroids as inhibitors of [3H]Ro5- allethrin and [1S,cis]-permethrin were active as 4864 binding to rat brain preparations (Devaud proconvulsants in this assay. Moreover, PK11195 and Murray, 1988; Ramadan et al., 1988b). These also antagonized the proconvulsant effects of cis- studies confirmed that pyrethroids inhibited the methrin. In contrast to these findings, a subse- binding of this ligand with IC50 values ranging quent study by other researchers (Gilbert et al., from 40 nM to \100 mM. Most of the a-cyano 1990) did not confirm the high potency of either pyrethroids examined were more potent than the cismethrin or deltamethrin assays of proconvul- non-cyano pyrethroids in these studies, but this sant activity using the pentylenetetrazole or amyg- generalization did not extend to fenvalerate and dala kindling seizure models. fluvalinate. These studies also confirmed that only the 1R,cis,aS isomer of cypermethrin was an ef- 6.4.2. Mediators of ion channel and receptor fective inhibitor and that the insecticidal and neu- regulation rotoxic 1R,cis isomer of permethrin was much more effective than the inactive 1S,cis isomer. 6.4.2.1. Biochemistry of ion channel and receptor regulation. Neuronal excitability, both within and 6.4.1.3. Action of pyrethroids: pharmacological between neurons, is tightly regulated. Ion chan- studies in 6i6o. Studies with rats in vivo explored nels and neurotransmitter receptors undergo the possible actions of pyrethroids at the phosphorylation and dephosphorylation by peripheral-type benzodiazepine receptor using protein kinases and phosphatases, respectively, the potentiation of pentylenetetrazole-induced and interact with G protein subunits (Catterall, seizures as an experimental model system. Initial 1997; Conley and Brammar, 1999). Kinases, phos- studies showed that [1R,cis,aS]-cypermethrin, phatases, and G proteins are regulated in turn by deltamethrin, fenvalerate, and permethrin acted as pathways that involve diacylglycerols, inositol proconvulsants in this system, producing a dose- phosphates and intracellular calcium. These dependent lowering of the dose of pentylenetetra- events mediate ion channel and receptor up- and zole required to induce seizures, but the non-toxic down-regulation, modify the kinetics of voltage- enantiomer of [1R,cis,aS]-cypermethrin was com- sensitive channels, and regulate receptor desensi- pletely ineffective (Devaud and Murray, 1986). tization to maintain the integrity of signaling The actions of pyrethroids in these experiments pathways in the nervous system. Agents that were similar to the proconvulsant (seizure-potenti- modify these regulatory pathways exert both di- ating) effects of Ro5-4864 in rodents (Benavides rect and indirect effects on ion channel and recep- et al., 1984). The maximal pyrethroid-dependent tor function. reduction in the pentylenetetrazole seizure threshold occurred at intraperitoneal doses of 6.4.2.2. Effects of pyrethroid action on ion channel pyrethroid that were too low causing the charac- and receptor regulation. Several studies describe D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 43 effects of pyrethroids on protein phosphorylation 6.4.3. Mitochondrial electron transport patterns in invertebrate nerve and mammalian Mitochondrial electron transport is central brain preparations (Clark, 1986; Gusovsky et al., to the energy metabolism of all eukaryotic 1986; Matsumura, 1986; Gusovsky et al., 1988; cells. The literature describing the action of Matsumura, 1988; Ishikawa et al., 1989; Mat- pyrethroids on mitochondrial respiration is sumura et al., 1989; Clark and Matsumura, 1991; sparse. Allethrin has been shown to perturb the Enan and Matsumura, 1991; Kanemoto et al., light scattering properties of mouse liver 1992; Enan and Matsumura, 1993). These results mitochondria and increase mitochondrial respira- are supported by studies documenting effects of tion (Settlemire et al., 1974). Also, fenvalerate pyrethroids on second messenger pathways that affected oxygen consumption in fish (Reddy et al., 1992) and cypermethrin (Ghosh et al., 1989) in- regulate kinase and phosphatase activity. For ex- hibited fish liver mitochondrial enzymes ample, deltamethrin stimulated phosphatidylinosi- involved in cellular respiration. Recently, perme- tol turnover in guinea pig brain and resulted in thrin and cyhalothrin were shown to be increased levels of diacylglycerols and inositol potent inhibitors of respiratory complex I, the polyphosphates (Gusovsky et al., 1986; Gusovsky rotenone sensitive complex, from rat liver mito- and Daly, 1988; Gusovsky et al., 1989). Other chondria (Gassner et al., 1997). Assays with sub- experiments using rat brain slices showed that mitochondrial particles demonstrated that −10 deltamethrin at concentrations as low as 10 M complex I was the sensitive site within the respira- increased the levels of inositol polyphosphate for- tory chain having the greatest sensitivity to mation, increased intracellular free calcium, in- pyrethroids. creased calcium/diacylglycerol-dependent kinase activity and specific protein phosphorylations re- 6.5. Critical e6aluation of putati6e mechanisms of lated to neurotransmitter release (Enan and Mat- action sumura, 1993). Most interesting, however, was the increased recruitment of calcium diacylglycerol- / 6 dependent kinase to the plasma membrane frac- 6.5.1. Criteria for the assessment of rele ant mechanisms of toxicity tion versus the cytosol in the presence of deltamethrin, cypermethrin, fenvalerate and al- lethrin. In Paramecium, it has been recently estab- 6.5.1.1. Functional relationship to pyrethroid intox- lished that deltamethrin significantly and ication. A fundamental criterion for assessing the stereospecifically increased the G protein bg sub- relevance of any proposed mechanism of unit-dependent phospholipase C activity in ciliary pyrethroid action is whether or not this mecha- membrane vesicles, as determined by increased nism is consistent with the signs of pyrethroid inositol polyphosphate production (Symington et intoxication. The acute effects of pyrethroids in mammals are neuroexcitatory, involving enhanced al., 1999). sensitivity to external stimuli, tremor, and convul- Pyrethroids also bind directly to the bg subunits sions. When pyrethroids are administered intrac- of G proteins (Rossignol, 1991a). A photoreactive erebrally or intravenously, these signs develop fenvalerate analog that possessed insecticidal ac- very rapidly. Pyrethroid intoxication is therefore tivity labeled a 36-kDa protein that was identified likely to occur by mechanisms that mediate a b as the subunit of G protein. Labeling was direct, excitatory effect on the nervous system. In g increased by GTP- S, a non-hydrolyzable analog contrast, mechanisms that result in a decrease in b g a of GTP that persistently liberates / and sub- neuronal excitation, or those which involve units, and treatment with the photoreactive fen- slowly-developing but longer-term changes in neu- valerate analog resulted in the dissociation of the ronal excitability, are not consistent with the signs G protein subunits as measured by ADP-ribosyla- and time course of acute intoxication by tion of the a subunit (Rossignol, 1991b). pyrethroids. 44 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

6.5.1.2. Structure–acti6ity relationships. The struc- available to membrane-bound targets in the ner- ture and three-dimensional configuration of vous system. For in vitro studies, concentrations pyrethroids are critical determinants of both in- of applied pyrethroid are difficult to compare secticidal activity and acute toxicity to mammals. from system to system due to the nature of the In particular, pyrethroid action is highly biological preparation used, the co-solvents em- stereospecific: typically, mixtures of four or eight ployed to achieve an aqueous ‘solution’ of optical and geometrical isomers of a pyrethroid pyrethroid, and the method and duration of contain only one or two active isomers, and the pyrethroid exposure. remaining isomers have toxicities so low that they Despite these difficulties, considerations of po- are difficult to measure accurately. The stereospe- tency may assist the evaluation of toxicological cific effects of pyrethroids are not explained by relevance of candidate pyrethroid target sites in differential detoxication and are observed upon three ways. Actions of pyrethroids on specific direct introduction of pyrethroids into the verte- targets identified in in vivo experiments may be brate central nervous system. We therefore con- considered to be potentially toxicologically rele- clude that toxicologically relevant sites of vant if they can be demonstrated to occur at doses pyrethroid action must exhibit the stereospecific- of pyrethroids that produce signs of intoxication. ity that is evident in pyrethroid intoxication. Al- In most cases, evidence for such effects is based though stereospecificity is an essential criterion on the pharmacological antagonism of one or for toxicological relevance, it is not the sole crite- more signs of pyrethroid intoxication by agents rion: whereas all toxicologically relevant sites having defined modes of action. Alternatively, must be stereospecific, not all stereospecific ac- toxicological relevance may be established in ex tions of pyrethroids are necessarily toxicologically vivo experiments, in which the disruption of a relevant. candidate target is documented by biochemical or Other structure–activity relationships evident physiological techniques in vitro following expo- in pyrethroid intoxication are less absolute than sure of the animal at a toxicologically relevant those based on stereochemistry and must be used pyrethroid dose in vivo. For in vitro experiments, with greater caution in evaluating proposed mech- the usefulness of potency as a criterion of toxico- anisms of action. The division of pyrethroids into logical relevance is limited to those systems in two broad structural and functional classes (a- which two putative target sites can be assayed and non-cyano pyrethroids) has received wide simultaneously in the same preparation. If one acceptance in the literature and is supported to a such target can be established by other criteria to degree by the pharmacological and toxicological be toxicologically relevant, then the relative sensi- properties of these two groups of compounds. tivity of the second candidate target may provide Candidate sites of pyrethroid action (e.g. voltage- insight into the probability that an effect at that sensitive chloride channels) that are preferentially site contributes meaningfully to intoxication. sensitive to a subset of pyrethroids, and which also conform to the other criteria for toxicological 6.5.2. E6aluation of putati6e mechanisms of relevance, may play a role in the divergent signs toxicity of pyrethroid intoxication. 6.5.2.1. Voltage-sensiti6e sodium channels. 6.5.1.3. Potency. The criterion of potency is par- Voltage-sensitive sodium channels are well-estab- ticularly difficult to apply to a class of compounds lished as the principal site of insecticidal action of such as the pyrethroids, which are extremely pyrethroids, and many lines of evidence implicate lipophilic and preferentially associate with biolog- voltage-sensitive sodium channels as an important ical membranes and other lipid-rich physiological site of pyrethroid action in mammals. The effects compartments. For in vivo studies, these proper- of pyrethroids on sodium channel function are ties render circulating levels of pyrethroid unreli- consistent with a mechanism involving enhanced able as indices of the amount of compound neuronal excitation, and these effects also exhibit D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 45 the stereospecificity anticipated for a toxicologi- sodium channels. However, a direct effect of cally relevant effect. Moreover, the differences pyrethroids on currents carried by N-type calcium observed in the modification of sodium channels channels has not been reported. Pyrethroid ac- by a- and non-cyano pyrethroids are consistent tions in these assays exhibit appropriate stereospe- with the differences in the disruption of nerve cificity for the isomer pairs that have been action potentials caused by members of these two examined. Electrophysiological and biochemical structural groups and with the production of CS evidence suggests that L-type calcium channels and T syndromes of intoxication in vivo. How- are relatively insensitive to pyrethroids, but other ever, the results of biophysical experiments sug- types of calcium channels have not been investi- gest that the division of a- and non-cyano gated for their sensitivity to pyrethroids. Al- a pyrethroids into two distinct functional classes though numerous calcium channel 1 subunits represents an oversimplification, because biophys- have been cloned, only some of these have been ical studies identify a spectrum of pyrethroid ef- expressed functionally and the assays of fects on sodium channel function that are pyrethroids on identified calcium channel iso- correlated with the production of signs of poison- forms, analogous to assays with cloned sodium ing that are intermediate between the T and CS channel isoforms, have not been performed. syndromes. Although an action of pyrethroids on N-type The emerging picture of mammalian voltage- calcium channels is consistent with the massive sensitive sodium channels as products of a multi- neurotransmitter release caused by pyrethroid in- gene family complicates the interpretation of the toxication in vivo, there is no evidence from phar- action of pyrethroids on sodium channels. Lim- macological studies in vivo that illuminates the ited evidence from physiological experiments in role of calcium channels in intoxication. However, vitro and from assays with cloned sodium channel the observation of effects of pyrethroids on cal- isoforms suggest that different sodium channel cium channels in neurotransmitter release assays isoforms vary in their sensitivity to pyrethroids as in vitro suggests that effects on calcium channels a class as well as to compounds representative of may contribute to intoxication. the a- and non-cyano structural classes. However, information on the relative sensitivity of sodium 6.5.2.3. Voltage-sensiti6e chloride channels.The channel isoforms to pyrethroids is incomplete, action of pyrethroids on voltage-sensitive chloride making it impossible at present to identify those channels has been postulated to be an effect lim- isoforms that are most sensitive to pyrethroids as ited to compounds in the a-cyano structural a class and those with differential sensitivity to group, but this conclusion is based on experi- pyrethroids that might contribute to the produc- ments with deltamethrin as the sole example of tion of different signs of intoxication in vivo. the a-cyano class and cismethrin as the sole exam- ple of the non-cyano class. The blockade of 6.5.2.2. Voltage-sensiti6e calcium channels. A sub- voltage-dependent chloride channels by stantial body of evidence from electrophysiologi- deltamethrin would contribute to the excitation of cal and biochemical studies in vitro shows that nerve and muscle in combination with other neu- pyrethroids affect calcium channel function. roexcitatory effects and is therefore consistent Pyrethroids are antagonists of T-type calcium with the overall neuroexcitatory actions of channels in vertebrate tissues and agonists of T- pyrethroids. At present, the stereospecificity of the type channels in insect muscle and in the ciliary action of pyrethroids on this target is not known. membrane of P. tetraurelia. Actions of Additional evidence for the toxicological rele- pyrethroids on N-type calcium channels also are vance of pyrethroid actions on voltage-sensitive implicated indirectly by the finding that chloride channels comes from pharmacological pyrethroid-stimulated neurotransmitter release is studies in vivo. Both ivermectin and pentobarbi- not completely abolished in some studies under tal, which selectively activate voltage-dependent experimental conditions that eliminate effects on chloride channels, antagonized the signs of 46 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 deltamethrin intoxication and reduced the inci- the CS syndrome was similar to the signs of dence of lethality at the deltamethrin doses em- intoxication produced by the intracerebral admin- ployed but did not similarly affect cismethrin istration of picrotoxinin (Lawrence and Casida, intoxication (Ray et al., 1999; Forshaw et al., 1982). The ability of diazepam, a benzodiazepine 2000). The effects of these agents on salivation known to act at GABA receptors, to increase the and the motor signs of intoxication caused by latency of the CS syndrome and to antagonize the deltamethrin appeared to be due to antagonism of acute intracerebral toxicity of deltamethrin and the action of deltamethrin at peripheral and cen- the neurotoxic isomer of fenvalerate more than tral nervous system chloride channels, respec- 10-fold was also interpreted as evidence for an tively. However, the protection against lethality action at GABA receptors in the production of afforded by pentobarbital could not be distin- the CS syndrome (Gammon et al., 1982). How- guished from the similar effects of phenobarbital, ever, the relatively low potency and incomplete which is a poor activator of chloride channels, stereospecificity of pyrethroids as GABA receptor and may therefore result from the overall depres- antagonists in functional assays do not support an sant actions of barbiturates at other sites. Despite action at the GABA receptor as a significant the limited number of pyrethroids used in these target site involved in the production of the CS studies, the results suggest that actions at voltage- intoxication syndrome. sensitive chloride channels may contribute to some of the signs of intoxication associated with 6.5.2.5. Acetylcholine receptors. The possible in- the CS poisoning syndrome. volvement of acetylcholine receptors in pyrethroid intoxication is based on the ability of certain 6.5.2.4. GABA receptors. A large body of bio- pyrethroids, principally non-cyano compounds, to chemical evidence documents the ability of a-cy- inhibit the binding of the ligand H -HTX to ano pyrethroids to bind to and block GABA 12 acetylcholine receptors in eel electroplax prepara- receptors in mammalian brain preparations. tions. Analysis of the effect of pyrethroids on Blockade of GABA receptors is an indirect neu- acetylcholine receptors and other ligand-gated roexcitatory effect, involving the removal of in- cation channels in cultured N1E-115 neuroblas- hibitory neuronal input, and is the established toma cells revealed that receptor blockade, the mode of action for convulsants such as picrotox- effect predicted from binding studies, was not inin. From a functional point of view, an action appropriately stereospecific and also was not spe- on GABA receptors is therefore consistent with cific for acetylcholine receptors. Moreover, block- the neuroexcitatory signs of pyrethroid intoxica- ade of cholinergic neurotransmission would not tion in vivo. The action of pyrethroids on GABA cause or contribute to neuroexcitation. We con- receptors is somewhat stereoselective for neuro- clude that the action of pyrethroids on nicotinic toxic isomers of a-cyano compounds but does not acetylcholine receptors in vitro is not likely to be exhibit the absolute stereospecificity predicted by relevant to intoxication in vivo. structure–toxicity relationships. In experimental systems where effects on GABA receptors and sodium channels can be assayed in the same 6.5.2.6. Excitatory glutamate receptors. The evi- preparation, GABA receptor blockade is not ob- dence for an action of pyrethroids on glutamate served at concentrations of pyrethroid that dis- receptors is very limited and somewhat con- rupt sodium channel function. tradictory. In the absence of structure–activity There is only a limited amount of information data correlating effects on glutamate receptors in correlating the in vitro biochemical effects of vitro with intoxication in vivo or pharmaco- pyrethroids on GABA receptors with intoxication logical evidence of glutamate receptor effects in in vivo. The initial description of the T and CS intoxication, we conclude that a toxicologically intoxication syndromes following intracerebral relevant effect of pyrethroids on this target is administration of pyrethroids to mice noted that unlikely. D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 47

6.5.2.7. Peripheral-type benzodiazepine receptors. peripheral-type benzodiazepine receptor rather A variety of pyrethroids, including both non- and than at the GABA receptor. We consider that a-cyano compounds, inhibit the binding of the some pyrethroids may act as proconvulsants at ligand Ro5-4864 to peripheral-type benzodi- peripheral-type benzodiazepine receptors at toxi- azepine receptors. Given that Ro5-4864 is a con- cologically relevant doses, but the relationship of vulsant, competitors for this site are also such an effect to the primary neuroexcitatory potentially convulsants, although there is no in actions of pyrethroids and its contribution to the vitro assay for peripheral-type benzodiazepine re- signs of pyrethroid intoxication remain to be ceptor function. For the isomer pairs that have elucidated. been examined, the inhibition of Ro5-4864 binding exhibits a degree of stereospecificity that is consis- 6.5.2.8. Ion channel and receptor regulation. Several tent with the stereospecificity of pyrethroid intoxi- lines of evidence point to the ability of pyrethroids cation. to modify second messenger pathways involved in Evidence for the involvement of effects on pe- ion channel and neurotransmitter receptor regula- ripheral-type benzodiazepine receptors in intoxica- tion. Moreover, pyrethroids appear to exert spe- tion comes from pharmacological studies in vivo. cific effects on the pattern of protein Pyrethroids at doses below those that produce phosphorylation in neurons. Although some of signs of intoxication potentiate the convulsant these effects represent neuronal responses to the actions of pentylenetetrazole in a manner similar neuroexcitatory actions of pyrethroids rather than to Ro5-5864, and these proconvulsant effects are primary mechanisms of toxicity, others may reflect blocked by PK11195, a specific antagonist of Ro5- a more specific action of pyrethroids. In view of 4864 at the peripheral-type benzodiazepine recep- the direct modulation of N-, P-, and T-type cal- tor. These results provide evidence for the cium channels by bg subunits of G proteins, the interaction of pyrethroids with the peripheral-type binding of pyrethroids to the bg subunit implied benzodiazepine receptor, but this effect cannot be by the results of photoaffinity labelling experi- a primary mechanism of intoxication because it ments could result in effects on a number of occurs at pyrethroid doses that are without effect macromolecules, including sodium and calcium outside of the proconvulsant experimental model. channels. At present, these effects are limited to Moreover, it should be noted that the potent observations from in vitro experiments and are not proconvulsant actions of cismethrin and directly correlated with pyrethroid intoxication in deltamethrin could not be reproduced by other vivo. researchers. Despite the contradictory nature of the results 6.5.2.9. Mitochondrial electron transport. The evi- obtained in vivo, it is possible that the action of dence for an action of pyrethroids on mitochon- pyrethroids at the peripheral-type benzodiazepine drial electron transport is very limited. However, receptor contributes to pyrethroid intoxication by the syndromes of pyrethroid intoxication in vivo enhancing convulsions arising due to neuroexcita- are distinct from the well-characterized toxic ac- tory effects at other target sites. Some support for tions of respiratory inhibitors and uncouplers. We this interpretation comes from the ability of di- therefore conclude that a toxicologically important azepam to increase the latency of pyrethroid intox- action of pyrethroids on this target is unlikely. ication (Gammon et al., 1982). This effect was originally interpreted as evidence for an effect of pyrethroids at GABA receptors; however, di- 7. Conclusions azepam binds with high affinity to both GABA receptors and peripheral-type benzodiazepine re- 7.1. Pyrethroid toxicity ceptors in rodents. Thus, the effects of diazepam on the latency of pyrethroid intoxication may Most pyrethroids are moderately toxic (EPA more plausibly result from antagonism at the Category II) to mammals. The principal effects 48 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 observed following acute or subchronic exposure tivity or cause pyrethroid-like mammalian of mammals to pyrethroids as a class are various toxicity. signs of excitatory neurotoxicity. In contrast, there is no evidence that other effects of 7.2. Syndromes of pyrethroid intoxication pyrethroids, involving either acute effects on other organ systems or chronic effects, are characteristic Historically, pyrethroids were grouped into two of pyrethroids as a class (Clark, 1995). Therefore, subclasses (commonly designated ‘Type I’ and consideration of common mechanisms among ‘Type II’) based on chemical structure and the pyrethroids is appropriately focused on those production of either the T (tremor) or CS mechanisms that are responsible for the acute (choreoathetosis with salivation) intoxication syn- neurotoxicity of these compounds. drome following intravenous administration to Physiological and neurochemical studies of rats or intracerebral administration to mice. Al- pyrethroid-intoxicated animals confirm that acute though, this classification system is widely recog- pyrethroid intoxication is associated with altered nized and employed, it has several shortcomings nerve function, principally involving neuroexcita- for the identification of common toxic effects tory effects, in the brain, spinal cord, and ele- among pyrethroids. First, this classification did ments of the peripheral nervous system. not include several pyrethroids that are currently Although, differences in sensitivity between brain registered for use in the United States. Because regions are evident, there is no single region of the the production of the T and CS syndromes was nervous system that is the locus of pyrethroid not unambiguously correlated with chemical intoxication and identifies a mechanism of toxic structure, the extension of this taxonomy to com- action. pounds not included in the original studies would Despite their lipophilicity, pyrethroids do not require empirical evaluation of each compound by bioaccumulate in lipid-rich tissues because they methods comparable to those employed in the are readily metabolized by hydrolases and cy- classification studies. Second, the T and CS cate- tochrome P450-dependent monooxygenases in the gories identified in these studies were not absolute liver and other tissues. The metabolites of and mutually exclusive, because some compounds pyrethroids are typically of lower toxicity were identified that exhibited elements of both than the parent compound and are readily ex- syndromes. Third, the T and CS syndromes were creted. The toxicokinetic profiles of parent defined using routes of exposure (i.e. intravenous pyrethroids following single oral doses are consis- or intracranial) that are of limited relevance to tent with the onset and duration of signs of risk assessment for human exposure to pyrethroid intoxication. residues. Finally, the diversity of intoxication Structure–activity relationships for both signs found in acute and subchronic neurotoxicity the insecticidal activity and the acute mammalian studies, which involve oral administration, are toxicity of pyrethroids show that activity is not congruent with those that form the basis of determined by the overall shape, three- the historical classification of intoxication syn- dimensional configuration, and physical proper- dromes. ties of the parent compound. The structural diversity of commercially used pyrethroids 7.3. Mechanisms of pyrethroid toxicity illustrates that requirement for pyrethroid-like ac- tivity can be met by a variety of chemical struc- A large body of published literature describes tures. Unlike organophosphate or methyl- the actions of pyrethroids in vitro on a variety of carbamate insecticides, which contain a specific putative biochemical and physiological target chemical moiety that both defines each class sites. These studies vary in technical quality, chemically and functions as a toxophore, depth, and relevance to an understanding of the there is no single essential chemical moiety that is toxic actions of pyrethroids in animals. Our criti- required to obtain pyrethroid-like insecticidal ac- cal analysis identified four target sites that merit D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 49 consideration as sites of toxic action of mains even if one only considers voltage-sensitive pyrethroids. First, there is ample evidence that sodium channels as sites of action, but it is exacer- voltage-sensitive sodium channels, the sites of in- bated by possible involvement of other function- secticidal action of pyrethroids, are also impor- ally and pharmacologically distinct targets in tant target sites in mammals. Mammals, unlike pyrethroid intoxication at the level of the whole insects, have multiple sodium channel isoforms animal (e.g. voltage-sensitive calcium and chloride that vary in their biophysical and pharmacologi- channels). cal properties, including their sensitivity to some Second, pyrethroids differ not only in their or all pyrethroids. Therefore, it is not appropriate relative potency but also in the qualitative nature to consider ‘the mammalian sodium channel’ as a of their effects at some targets. Considering single, pharmacologically homogeneous target site voltage-sensitive sodium channels as an example for pyrethroids. Moreover, where the combined target for which extensive data exist, the pro- actions of different pyrethroids on sodium chan- foundly different lifetimes of the pyrethroid- nels have been examined, these effects are not modified open states caused by compounds such additive and may be antagonistic. There is also experimental evidence for the ac- as tetramethrin (short) or deltamethrin (very long) tion of pyrethroids at three other sites that may produce profoundly different effects (burst dis- contribute to intoxication in vivo. Pyrethroids charges or use-dependent block, respectively) at have been shown to act on some isoforms of the the level of the intact nerve. There are no data voltage-sensitive calcium channel, an effect that available to determine whether equivalent frac- may contribute to the release of neurotransmitters tional occupancy of binding sites by compounds that is associated with pyrethroid intoxication. such as tetramethrin and deltamethrin produces Blockade of voltage-sensitive chloride channels by an equivalent disruption of nerve function given some pyrethroids is associated with the produc- the divergent qualitative effects of these com- tion of salivation, a hallmark of the CS intoxica- pounds on any single isoform of the voltage-sensi- tion syndrome, and may also contribute to tive sodium channel and the divergent enhanced excitability in the central nervous sys- consequences for nerve function that ensue. tem. Effects on peripheral-type benzodiazepine Third, an implicit assumption of cumulative receptors are unlikely to be a principal cause of risk assessment is that the effects of two com- pyrethroid intoxication but may contribute pounds acting by the same mechanism at the same to or enhance convulsions caused by actions at macromolecular target are, at a minimum, addi- other target sites. In contrast, other putative tive or perhaps synergistic. In the case of the target sites for pyrethroid action that have been action of pyrethroids on sodium channels, there is identified in in vitro experiments (e.g. GABA experimental evidence that binary interactions are receptors, nicotinic acetylcholine receptors) do not potentially subadditive or antagonistic, so that the appear to play a major role in pyrethroid intoxi- presence of a second compound displaces or can- cation. cels the action of a previously administered compound. 7.4. Implications for cumulati6e risk assessment We conclude from these considerations that simple additivity models to assess the risks of 7.4.1. Risk of exposure to multiple pyrethroids The cumulative risk assessment of pyrethroids combined exposure to multiple agents are not is complicated by several factors. First, the exis- appropriate to the pyrethroids. Such models do tence of multiple target sites with differing phar- not account for the multiplicity of targets, the macological profiles raises the question of which qualitative differences that distinguish the actions target should be employed in the assessment of of different pyrethroids at some targets, and the the effects of multiple compounds. This problem potential for non-additive effects of two or more of multiple, pharmacologically distinct targets re- compounds at the same target. 50 D.M. Soderlund et al. / Toxicology 171 (2002) 3–59

7.4.2. Risk of combined exposure to pyrethroids vivo. A similar situation exists for nicotinic acetyl- and other classes of pesticides choline receptors, which are sites of action of Because human exposure to pyrethroids occurs chloronicotinoid insecticides (e.g. imidacloprid) in the context of concurrent exposure to other (Nagata et al., 1998). Although pyrethroids dis- environmental chemicals and pharmaceutical place the binding of a ligand that labels nicotinic agents, it is necessary to consider the possibility acetylcholine receptors, there is no evidence that that pyrethroids may share common toxic effects this effect is correlated with a change in receptor and mechanisms with other chemically- and func- function or is related in any way to intoxication in tionally-unrelated substances. Some of the puta- vivo. These considerations suggest that tive sites of action for pyrethroids are known sites pyrethroids do not act at either GABA or nico- of action of other substances. tinic acetylcholine receptors in a manner that Voltage-sensitive sodium channels are targets would cause them to be grouped on the basis of for the bioactivation product of the novel insecti- common mechanism with other insecticides that cide indoxacarb and for experimental pyrazoline are known to act at either of these targets. insecticides that are part of the same structure– We conclude from the foregoing that the puta- activity group (McCann et al., 2001). These com- tive sites of action of pyrethroids that are most pounds block ion transport through sodium likely to be involved in the production of acute channels, an action that is antagonistic to the neurotoxic effects are not shared with other prolongation of sodium channel opening that is classes of pesticides. Therefore, the effects of caused by pyrethroids (Payne et al., 1998; Wing et pyrethroids at these sites, to the extent that such al., 1998). Since indoxacarb and pyrethroids pro- effects are actually involved in pyrethroid toxicity, duce different and opposite effects on sodium are not relevant to cumulative risk assessments channels that are mediated by different binding involving other pesticide classes. sites on the sodium channel protein (Soderlund and Knipple, 1995), they therefore do not merit grouping on the basis of a common mechanism. Acknowledgements A similar situation exists in the case of voltage- sensitive chloride channels, which are important The preparation of this review was supported target sites for avermectin insecticides. Aver- by the Pyrethroid Working Group, a consortium mectins activate voltage-sensitive chloride chan- of firms (Aventis CropScience, Bayer Corpora- nels (Abalis et al., 1986a; Payne and Soderlund, tion, DuPont Crop Protection, FMC Corpora- 1991), an effect that is opposite to the channel- tion, Syngenta Crop Protection, Inc., and Valent blocking effect found for some pyrethroids. More- Corporation) that market pyrethroid-based insec- over, an avermectin analog has been shown to ticide products in the United States. We thank the antagonize in vivo some of the effects of following persons for their contributions during pyrethroids that are thought to be mediated by an the preparation of this review: Terry Fico (for- action on voltage-sensitive chloride channels (For- merly of FMC Corporation); Joel Kronenberg shaw et al., 2000). Because avermectins and (formerly of AgrEvo USA Company); Stephen pyrethroids produce different and opposite effects Longacre (FMC Corporation); Jane McCarty on chloride channels, they should not be grouped (FMC Corporation); William Salminen (formerly based on their actions at this target. of FMC Corporation); and Ashley Wickrama- GABA receptors are target sites for several ratne (formerly of Syngenta). insecticides, including chlorinated cyclodienes (e.g. endosulfan) and phenylpyrazoles (e.g. fipronil) (Bloomquist, 1993b, 1996, 1998). Al- References though pyrethroids bind to and affect GABA receptors in vitro, we conclude that these effects Abalis, I.M., Eldefrawi, A.T., Eldefrawi, M.E., 1986a. Actions g are not likely to be relevant to intoxication in of avermectin B1a on the -aminobutyric acidA receptor D.M. Soderlund et al. / Toxicology 171 (2002) 3–59 51

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