Developmental Neurotoxicity of Pyrethroid Insecticides: Critical Review and Future Research Needs

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the worldwide insecticide market. Although their acute neurotoxicity to adults has been well characterized, information regarding the potential developmental neurotoxicity of this class of compounds is limited. There is a large age dependence to the acute toxicity of pyrethroids in which neonatal rats are at least an order of magnitude more sensitive than adults to two pyrethroids. There is no information on age-dependent toxicity for most pyrethroids. In the present review we examine the scientific data related to potential for age-dependent and developmental neurotoxicity of pyrethroids. As a basis for understanding this neurotoxicity, we discuss the heterogeneity and ontogeny of voltage-sensitive sodium channels, a primary neuronal target of pyrethroids. We also summarize 22 studies of the developmental neurotoxicity of pyrethroids and review the strengths and limitations of these studies. These studies examined numerous end points, with changes in motor activity and muscarinic acetylcholine receptor density the most common. Many of the developmental neurotoxicity studies suffer from inadequate study design, problematic statistical analyses, use of formulated products, and/or inadequate controls. These factors confound interpretation of results. To better understand the potential for developmental exposure to pyrethroids to cause neurotoxicity, additional, well-designed and well-executed developmental neurotoxicity studies are needed. These studies should employ state-of-the-science methods to promote a greater understanding of the mode of action of pyrethroids in the developing nervous system.

Pyrethroid insecticides have been used in agricultural and home formulations for more than 30 years and account for approximately one-fourth of the worldwide insecticide market (Casida and Quistad 1998). Currently, 16 pyrethroids are registered for use in the United States in a large variety of agricultural or consumer products (Bryant and Bite 2003). Often, pyrethroids are sold and/or used as mixtures containing a combination of two or more compounds (Farm Chemicals Handbook 1997). Exposure to pyrethroids has been widely documented in humans, including exposure of pregnant women, infants, and children (Berkowitz et al. 2003;Huedorf et al. 2004;Schettgen et al. 2002;Whyatt et al. 2002). Although the acute toxicity of these compounds to adults has been well characterized, the potential for developmental toxicity of pyrethroids is not well understood.
In the present review we focus on the potential for neurotoxicity after developmental exposure to pyrethroid insecticides. We also consider the current state and quality of scientific data that could be used to support risk decisions related to pyrethroid developmental and age-dependent neurotoxicity. Specifically, in this review we a) provide a brief overview of the toxicity of this class of compounds; b) review pyrethroid effects on voltage-sensitive sodium channels (VSSCs), a primary mode of action of pyrethroids; c) discuss the developmental profiles of VSSCs; d) provide examples of the results of perturbation of VSSCs during development by other insults; e) discuss the evidence for age-related sensitivity to this class of compounds; f ) summarize and critique studies of pyrethroid neurotoxicity after developmental exposure; and g) make recommendations regarding future research needs related to the developmental neurotoxicity of pyrethroids.
In addition to being important to scientists interested in characterizing the neurotoxicity of these compounds, this information will be useful when considering the scientific data needed to inform risk decisions related to pyrethroid insecticides. Under the Food Quality Protection Act (FQPA;1996), the U.S. Environmental Protection Agency (EPA) is required to include a default 10× safety factor (uncertainty factor) in risk decisions to protect against potentially greater sensitivity of developing individuals to toxic insult. This factor can be adjusted only if compelling scientific data exist regarding age-related differences in sensitivity. Furthermore, developing individuals must be considered under FQPA requirements for cumulative risk assessments (classes of compounds with the same mode of action). The quality of the scientific data used to support these and other risk decisions is an important component of scientifically based risk assessment. In addition, information regarding mode of action improves the scientific basis for risk decisions (Brock et al. 2003;Mileson et al. 1998;Sonich-Mullin et al. 2001), including those related to developmental neurotoxicity (Costa 1998;Tilson 2000aTilson , 2000b. The U.S. EPA has recently released the revised cumulative risk assessment of the organophosphate class of insecticides (U.S. EPA 2002) and has requested that registrants of these insecticides submit developmental neurotoxicity studies to the agency. In the near future, the U.S. EPA must consider developmental and cumulative risk for other classes of insecticides, including pyrethroids. Thus, in this review we focus on issues of mode of action and age-dependent and developmental neurotoxicity as related to risk decisions under the FQPA.

Overview of Pyrethroid Toxicity
compounds that retained the desirable insecticidal and toxicologic properties (Valentine 1990). All pyrethroids contain several common features: an acid moiety, a central ester bond, and an alcohol moiety (Figure 1). The acid moiety contains two chiral carbons; thus, pyrethroids typically exist as stereoisomeric compounds. Furthermore, some compounds also contain a chiral carbon on the alcohol moiety, which allows for three chiral carbons and a total of eight different stereoenantiomers. Pyrethroid insecticidal activity (Elliot et al. 1974), acute mammalian neurotoxicity (Gray and Soderlund 1985), and effects on VSSCs (Lund and Narahashi 1982) are stereospecific, indicating the presence of specific binding sites. For some compounds, several commercial products are available that differ in stereoisomer content. For example, allethrin is a mixture of all possible allethrin stereoisomers, d-allethrin contains only the 1R isomers, bioallethrin contains only the 1R-trans isomers, and S-bioallethrin is enriched in the S stereoisomer of the 1R-trans isomers ( Figure 2).
The acute mammalian neurotoxicity of pyrethroids has been well characterized, and several comprehensive reviews of pyrethroid toxicity, metabolism, and actions are available (Kaneko and Miyamoto 2001;Narahashi 2001;Ray 2001;Soderlund et al. 2002). Verschoyle and colleagues (Verschoyle and Aldridge 1980;Verschoyle and Barnes 1972) conducted structure-activity relationship studies with a series of pyrethroids and described two generalized syndromes after acute exposure. Based on toxic signs in the rat, pyrethroids have been divided into two types: a) compounds that produce a syndrome consisting of aggressive sparring, increased sensitivity to external stimuli, and fine tremor progressing to whole-body tremor and prostration (type I or T syndrome); and b) compounds that produce a syndrome consisting of pawing and burrowing, profuse salivation, and coarse tremor progressing to choreoathetosis and clonic seizures (type II or CS syndrome) (Verschoyle and Aldridge 1980). Analogous toxic signs have been observed in mice (Lawrence and Casida 1982;Staatz et al. 1982) and cockroaches (Gammon et al. 1981;Scott and Matsumura 1983). Structurally, a key difference between type I and type II pyrethroids is the absence or presence, respectively, of a cyano group at the α carbon of the 3-phenoxybenzyl alcohol moiety of the compound. Thus, the type I/II or T/CS nomenclatures are useful as general classification schemes and are widely used in the published literature. However, a few pyrethroids do not fit neatly into these schemes because they produce signs related to both syndromes (Verschoyle and Aldridge 1980; for review see Soderlund et al. 2002). Further, these schemes are based on doses of pyrethroids that cause overt neurotoxicity and thus may not apply to either low-dose or developmental exposures. Because it conveys useful structural information, in this review we use the type I/II classification system.

Effects of Pyrethroids on VSSCs
The primary mode of pyrethroid action in both insects and mammals is disruption of VSSC function. Perturbation of sodium channel function by pyrethroids is stereospecific (Lund and Narahashi 1982); those stereoisomers that are the most potent disruptors of VSSC function also have the most potent insecticidal or toxicologic activity (Ray 2001). Pyrethroids slow the activation, or opening, of VSSCs. In addition, they slow the rate of VSSC inactivation (or closing) and shift to more hyperpolarized potentials the membrane potential at which VSSCs activate (or open) (for review, see Narahashi 1996). The result is that sodium channels open at more hyperpolarized potentials (i.e., after smaller depolarizing changes in membrane potential) and are held open longer, allowing more sodium ions to cross and depolarize the neuronal membrane. In general, type II compounds delay the inactivation of VSSCs substantially longer than do type I compounds. Type I compounds prolong channel opening only long enough to cause repetitive firing of action potentials (repetitive discharge), whereas type II compounds hold open the channels for such long periods of time that the membrane potential ultimately becomes depolarized to the point at which generation of action potentials is not possible [depolarizationdependent block (Figure 3)]. These differences in prolongation of channel open times are hypothesized to contribute to the differences in the CS and T syndromes after exposure to type II and I pyrethroids, respectively (for review, see Ray 2001).
Mammalian VSSCs are composed of one α and two β subunits. Ten separate α subunits (Table 1; Ogata and Ohishi 2002) and four different β subunits (Isom 2002) have been identified and are expressed in a tissue-, region-, and time-specific manner. With one exception (the Na X subunit), α subunits all comprise VSSCs when expressed individually or with β subunits. The α subunit forms the pore of the channel and determines its major functional characteristics, whereas the β subunits are auxiliary proteins that influence gating properties, localization in the membrane, and interactions with cytoskeletal proteins (Isom 2001(Isom , 2002 diverse functional roles of VSSC, such as generating action potential spikes, amplifying subthreshold depolarizations, regulating repetitive firing and generating after-depolarizations, depend on the numerous potential combinations of α and β subunits (Ogata and Ohishi 2002). The types of VSSCs expressed in different regions, their relative sensitivity, and their functional role may all contribute to the manifestation of pyrethroid effects.

VSSC Heterogeneity and Pyrethroid Effects
All available evidence indicates that pyrethroids bind to the α subunit of the VSSC. Trainer et al. (1997) expressed only the Na v 1.2 α subunit in Chinese hamster ovary cells and found that the presence of the α subunit is sufficient for pyrethroids to produce their characteristic effects on sodium channel function in mammalian cells. This conclusion is supported by additional research demonstrating that pyrethroids alter currents produced by expression of Na v 1.2 (Smith and Soderlund 1998) or Na v 1.8 (Smith and Soderlund 2001) in oocytes in the absence of coexpression with β subunits. Interestingly, coexpression of the β1 subunit with Na v 1.2 increased the sensitivity of this channel compared with expression of Na v 1.2 alone (Smith and Soderlund 1998), indicating that the β subunit modulates the affinity of pyrethroid interaction with the channel. Mutations in the α subunit of both insects (Lee and Soderlund 2001;Smith et al. 1997) and mammals (Vais et al. 2000(Vais et al. , 2001Wang et al. 2001) alter the sensitivity of VSSCs to pyrethroid effects, supporting the conclusion that pyrethroids interact with the α subunit. The relative susceptibility of the 10 different VSSC α subunits to pyrethroids is not well understood. Differential sensitivity of VSSCs to pyrethroids was first reported by Tatebayashi and Narahashi (1994). In a comparison of tetramethrin effects on tetrodotoxinsensitive (TTX-S) versus -resistant (TTX-R) sodium channels in dorsal root ganglion neurons, TTX-R channels were demonstrated to be more sensitive to perturbation by tetramethrin (Tatebayashi and Narahashi 1994). However, TTX-R or TTX-S channels may arise from several different VSSC α subunits (Table 1). Although not all α subunits have been examined, differences in sensitivity to pyrethroids were reported after expression of different subunits in vitro (details provided in Table 1). For example, Na v 1.2 (Smith and Soderlund 1998) is sensitive to type II but not type I compounds, whereas Na v 1.8 (Smith and Soderlund 2001) is sensitive to both. Interactions of pyrethroids with other sodium channel α subunits have not been investigated to date. Importantly, the pyrethroid sensitivity of VSSC subunits and splice variants expressed during development has yet to be examined.

Developmental Expression of VSSC
VSSCs show complex regional and temporal ontogeny, which is briefly summarized in Table 1. In general, embryonically expressed forms of VSSCs are replaced by expression of adult forms as neurodevelopment proceeds. For example, high expression of Na v 1.3 during embryonic periods (Albrieux et al. 2004) diminishes as expression of Na v 1.2 increases in early postnatal periods in rodents (Felts et al. 1997), and expression of Na v 1.2 at immature nodes of Ranvier is replaced by Na v 1.6 as myelination proceeds (Boiko et al. 2001;Jenkins and Bennett 2002). Similar changes are observed with the β subunits, because β3 expression is replaced by β1 and β2 (Shah et al. 2001). Alternatively spliced forms of the VSSC subunits also contribute to developmental differences in expression because the Na v 1.2, Na v 1.3, and Na v 1.6 subunits all have splice variants that are expressed in rodents from embryonic through early postnatal ages (Gustafson et al. 1993;Plummer et al. 1997;Sarao et al. 1991). Given the previously reported differences in α subunit sensitivity to pyrethroids, the complex ontogeny of VSSC expression could result in altered sensitivity (either increases or decreases) of the developing nervous system to perturbation by various pyrethroids. In addition, understanding the timing and localization of expression of the most pyrethroid-sensitive VSSCs during neurodevelopment could help in understanding and explaining effects reported after developmental exposure. With respect to agedependent toxicity of pyrethroids, research to date indicates that toxicokinetic and not toxicodynamic factors account for differences in susceptibility between young and adult animals (Cantalamessa 1993;Sheets et al. 1994); however, toxicodynamic factors have not been systematically examined.

Disruption of VSSC Function and Expression during Development
Evidence from mutation and knockout models demonstrates that perturbation of VSSC function during development  These examples demonstrate the plausibility that perturbations in VSSC function by pyrethroids during development could result in adverse consequences in the developing nervous system. Knockout and mutant mouse models of sodium channel α subunits demonstrate varying degrees of adverse outcomes associated with loss or alteration of specific channel subunits. When mRNA for the Na v 1.2 subunit was reduced by approximately 85%, mice exhibited reduced levels of electrical excitability, had high levels of apoptotic neurons in the brainstem and cortex, and died from severe hypoxia within 1-2 days of birth (Planells-Cases et al. 2000). In contrast, mutation of the gene encoding the Na v 1.6 subunit resulted in development of hindlimb paralysis, skeletal muscle atrophy by postnatal day (PND)10, and death by PND20 (Porter et al. 1996). Atrophy was specific to muscle innervated by spinal and not oculomotor neurons (Porter et al. 1996). Finally, Na v 1.8 knockout mice survived to adulthood and exhibited normal behavior, although sensation of some types of noxious stimuli was lost or diminished (Akopian et al. 1999;Laird et al. 2002).
In humans, perturbation of nervous system development has been associated with altered VSSC structure or function. Recent advances in molecular genetics have identified in genes coding for VSSC subunits a number of mutations that result in neuronal hyperexcitability due to subtle changes in channel gating and inactivation (see Meisler et al. 2001, their Table 3). These mutations have been linked to various forms of epilepsy in humans, providing evidence that changes in VSSC function can give rise to clinically definable disease (Claes et al. 2001;Escayg et al. 2001;Meisler et al. 2002;Noebels 2002;Wallace et al. 2001). Mouse models expressing these mutant ion channels have been constructed, facilitating the study of these diseases Meisler et al. 2001). It is noteworthy that pyrethroids, like these mutations, alter VSSC activation, inactivation, and neuronal excitability. The mechanisms and magnitude of mutational versus pyrethroid effects are different, as would be the duration of effect (dependent on exposure for pyrethroids vs. permanent for mutations). Because of these differences, results from mutation and knockout models may not be predictive of developmental exposure to pyrethroids. Notably, potential interactions between pyrethroids and these mutations to VSSCs have not yet been examined.
Phenytoin, an anticonvulsant that blocks VSSCs as well as other ion channels (Catterall 1999), has been demonstrated to disrupt nervous system structure and function after developmental exposure (Adams et al. 1990). In humans, the use of anticonvulsants during pregnancy has been associated with a number of defects and malformations, which collectively are referred to as fetal hydantoin syndrome, and include microcephaly and intellectual impairment. Studies in animal models support the human findings Ohmori et al. 1997Ohmori et al. , 1999Schilling et al. 1999;Vorhees et al. 1995). Thus, developmental exposure to this drug, which acts on VSSCs, can produce significant alterations in nervous system structure and function. It should also be noted that, although phenytoin is used as an example, there are currently no data to suggest that developmental exposure to pyrethroids results in similar effects.

Age-Related Differences in Sensitivity to Pyrethroids
The magnitude of the age-related toxicity of pyrethroids appears to be much larger than for many other pesticide classes, but the number of studies is small. Whether this age-related neurotoxicity includes both type I and type II compounds is currently unclear. In neonatal versus adult rats, the acute lethality of the type II pyrethroid deltamethrin was 16-fold greater in young animals (Sheets et al. 1994). Concentration data indicate that the age dependency was due to lower metabolic capabilities in the young rats (Sheets et al. 1994). Similarly, the type II pyrethroid cypermethrin was 17-fold, and the type I pyrethroid permethrin was 6-fold more lethal in PND8 rats compared with adults; metabolic inhibitors were used to demonstrate that toxicokinetic factors were responsible for this age-dependent susceptibility (Cantalamessa 1993). In contrast, evidence has been presented that two type I pyrethroids, cismethin and permethrin, did not have any age-dependent toxicity (Sheets 2000).
Age-related sensitivity to pyrethroids may be influenced by dose. In a symposium report, Sheets (2000) argued that the age-dependent sensitivity of pyrethroids is apparent only at high acute doses. This report contained data suggesting a lack of age-dependent differences in the behavioral toxicity of type I and type II pyrethroids at doses below those causing overt toxicity. However, age-dependent differences in pyrethroid neurotoxicity have not been thoroughly studied at the lower end of the dose-response relationship (sublethal doses). The scientific basis for decisions related to the FQPA could be strengthened by additional studies comparing the relative susceptibility of differential sensitivity between young and adult animals, particularly at sublethal doses. For example, replication of Sheets's (2000) report and expansion to include additional compounds would provide useful information regarding sensitivity differences between developing and adult animals.

Pyrethroid Developmental Neurotoxicity Studies
A total of 22 studies were evaluated for this review (Tables 2-4), including 19 peer-reviewed publications (Table 2), unpublished studies (Muhammad and Ray, unpublished data; see  Figure 3. Pyrethroid effects on neuronal excitability. This schematic depicts pyrethroid effects on individual channels, whole-cell sodium currents, and action potentials. Depolarization opens VSSCs (top left) allowing sodium to enter the cell. To limit sodium entry and depolarization length, VSSCs inactivate and must return to a "resting" state before reopening. Pyrethroids inhibit the function of two different "gates" that control sodium flux through VSSCs (top right), delaying inactivation (indicated by double arrows between states) of the channel and allowing continued sodium flux (Open*). If sodium current through an entire cell is measured, depolarization leads to a rapidly inactivating current under normal circumstances (bottom left, Sodium current). Pyrethroid-modified VSSCs remain open when depolarization ends (bottom right, Sodium current), resulting in a "tail" current (the notch at the end of example currents). If membrane voltage is examined, depolarization under normal circumstances generates a single action potential (bottom left). VSSCs modified by type I compounds (bottom right, Action potential) depolarize the cell membrane above the threshold for action potential generation, resulting in a series of action potentials (repetitive firing). Type II compounds cause greater membrane depolarization, diminishing the sodium electrochemical gradient and subsequent action potential amplitude. Eventually, membrane potential becomes depolarized above the threshold for action potential generation (depolarization-dependent block).
Bayer AG (Table 4; Ivens et al., unpublished data; Jekat et al., unpublished data). The studies conducted by Muhammad and Ray (unpublished data) consisted of several similarly treated "cohorts" for both S-bioallethrin and deltamethrin. Rather than present the overall findings for each of these two compounds, the results of individual "cohorts" are summarized in Table 3 to provide more detailed information. Tables 2-4 contain a summary of important information from each study, including test compound/formulation, animal species, dosing period, and major findings. Because the vehicle used and route of exposure can have profound influence on the expression of pyrethroid neurotoxicity in adult rats (Crofton et al. 1995), this information is included as well.
Allethrin (in the form of allethrin, d-allethrin, bioallethrin, and S-bioallethrin) and permethrin are the only type I pyrethroids for which peer-reviewed studies of potential developmental neurotoxicity have been conducted. Of the type II compounds, results of developmental studies have been published for deltamethrin, cypermethrin, fenvalerate, and cyhalothrin, and data regarding the developmental neurotoxicity of cyfluthrin (Jekat et al., unpublished data) have been submitted to the U.S. EPA. Thus, no developmental neurotoxicity studies exist for many pyrethroids.
Rodents were the sole animal models used in these studies: 13 studies used rats and 9 studies used mice. No studies were conducted specifically to examine species differences, nor could any clear speciesdependent effects be discerned. The choice of rats or mice seemed to be based on a) previous use of that species in the laboratory or b) whether or not the study was designed to replicate (in whole or part) results published previously by other investigators. A systematic comparison of factors that underlie potential species differences in neurotoxic responses could provide useful information regarding the extrapolation of data from animals to humans. For example, Na v 1.3 expression in rodents appears to be primarily embryonic, yet in humans considerable expression in adults has been reported (Whitaker et al. 2000(Whitaker et al. , 2001. How this and other species differences influence neurotoxic responses has not been investigated. Several studies reported persistent changes in behavior and/or neurochemistry in animals examined long after exposure had stopped. Eriksson's group (Ahlbom et al. 1994;Eriksson and Fredriksson 1991;Eriksson et al. 1993;Eriksson and Nordberg 1990) has reported that mice exposed to pyrethroids during PND10-16 exhibit increased motor activity and lack of habituation, as well as changes in density of muscarinic acetylcholine receptor (mAChR) binding for as long as 5 months (Talts et al. 1998) after cessation of exposure. Given the short half-lives for pyrethroids (Anadón et al. 1991(Anadón et al. , 1996; for review, see Kaneko and Miyamoto 2001), these effects are likely due to exposure during development and not residual tissue concentrations of pyrethroids. Studies conducted by Eriksson and co-workers used bioallethrin and deltamethrin, which contain only two and predominantly one stereoisomer, respectively. Thus, effects can be ascribed to the compound that has insecticidal activity (vs. studies conducted with formulated products). In addition, dose-response relationships have been demonstrated for bioallethrin (Ahlbom et al. 1994), and the replication of effects,
There were several studies that examined both motor activity and mAChR expression after developmental exposure to pyrethroids. A summary of effects on these end points, independent of dose, exposure period, and other parameters, is provided in Table 5. In all of these studies, quinuclidinyl benzilate (QNB) binding was used to measure mAChR expression. QNB is a nonspecific antagonist for this receptor (Watling et al. 1995) and does not discriminate between mAChR subtypes (M1-M5). Measurement of QNB binding may in fact be one of the more comparable end points across these numerous studies. In addition, many but not all of these studies examined mAChR expression at PND17 and/or 4 months of age.
Comparison of pyrethroid effects on QNB binding across studies does not reveal clear trends in reported effects between laboratories. In preweanling animals, across all compounds and treatment protocols, QNB binding was reported to increase in six studies, decrease in two studies, and not change in four studies (Table 5). In cortical tissue, the data for PND17 are more consistent in that five of eight studies reported increases in mAChR expression. If only the various forms of allethrin are considered, four studies reported increases and two reported no change in QNB binding when measured on PND17. Persistent alterations in mAChR in adulthood after developmental exposure are less clear, with three studies reporting increases, three reporting decreases, and five reporting no change in QNB binding. Considering only allethrin forms again, QNB binding increased or decreased in two studies each and was unchanged in three studies.
Differences in a number of important variables may underlie some of the inconsistencies in QNB binding data. One difference is exposure route. Two studies used inhalation exposure (Ivens et al., unpublished data; Jekat et al., unpublished data), whereas exposure in the remainder of the studies was via oral gavage (Table 5). A comparison of effects in Tables 2-5 suggests that this is not a tenable explanation for these inconsistencies because results do not correlate to route. Another variable that differed between laboratories was the formulation of allethrin used. Allethrin, like all pyrethroids, exists as several different stereoisomers (Figure 2), and the insecticidal and toxic effects of pyrethroids are highly stereospecific. These studies employed allethrin formulations with differing contents of allethrin stereoisomers; two groups used d-allethrin (Ivens et al., unpublished data;Tsuji et al. 2002) Moniz et al., 1990 (no effect); culling + "cyhalothrin vehicle" activity in pup described but not even across studies (culled to 5, 6, and 8 pups/dam) Limitations: commercial product, unknown vehicle ("cyhalothrin vehicle") composition; dosing time frame not clear, but thought to be GD0-PND0 (Gomes et al. 1991a(Gomes et al. , 1991b Figure 3); differences in control testes descent day in Moniz et al. (1999) vs. Gomes et al. (1991Gomes et al. ( , 1991b bioallethrin (Eriksson group: Ahlbom et al. 1994;Ericksson and Fredriksson 1991;Eriksson and Nordberg 1990;Talts et al. 1998), and two used S-bioallethrin (Muhammad and Ray, unpublished data;Pauluhn and Schmuck 2003). Again, data in Table 5 suggest that this is not a tenable explanation because d-allethrin and bioallethrin result in either increases or no effects on mAChR binding. An additional variable in these data sets is the specific methods used in the competitive binding experiments. Competition experiments with carbachol were used in several studies to distinguish between high-and low-affinity QNB binding sites (Ahlbom et al. 1994;Eriksson and Fredriksson 1991;Eriksson and Nordberg 1990;Ivens et al., unpublished data;Jekat et al., unpublished data;Talts et al. 1998). Two studies (Ahlbom et al. 1994;Eriksson and Nordberg 1990) reported that bioallethrin increased the percentage of low-affinity binding sites in PND17 mice, an effect not reported in adult mice, despite changes in the density of muscarinic binding (Eriksson and Fredriksson 1991;Talts et al. 1998). Ivens et al. (unpublished data) did not find changes in the percentages of high-and low-affinity sites, even though they did report changes in the density of QNB binding sites in PND17 animals. In some cases, the relative proportion of high-and low-affinity sites was not investigated even though changes in density were reported (Muhammad and Ray, unpublished data). The ability to distinguish high-and low-affinity sites, and effects thereon, is dependent on the number of points included on the agonist competition curve. A smaller number of studies examined potential alterations in catecholaminergic systems. Both deltamethrin (Lazarini et al. 2001) and bioallethrin (Muhammad and Ray, unpublished data) were reported to increase 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the adult striatum after developmental exposure. However, developmental exposure to a commercial product containing fenvalerate had no effect on monoamine levels in the striatum (Moniz et al. 1999). Malaviya et al. (1993) reported that binding of 3 H-spiroperidol to striatal membranes from PND21 rats was decreased and increased, respectively, after gestational and lactational exposure to a commercial product containing fenvalerate, whereas binding was increased after only lactational exposure to a commercial product containing cypermethrin. Thus, similar to the muscarinic cholinergic system, the dopaminergic system may be affected by developmental exposure to pyrethroids, but studies examining this system have reported inconsistent results to date.
Eriksson and co-workers have consistently reported increased motor activity and a lack of habituation after exposure to pyrethroids (Ahlbom et al. 1994;Eriksson et al. 1993;Talts et al. 1998). A comparison of effects of pyrethroids on motor function between laboratories is not as consistent. Muhammad and Ray (unpublished data) observed effects on motor activity in some cohorts but not in others. After inhalation exposure to bioallethrin (Tsuji et al. 2002) or d-allethrin (Ivens et al., unpublished data), no effects on activity or habituation were reported. By contrast, inhalation exposure to cyfluthrin resulted in hyperactivity and decreased habituation in female mice (Jekat et al., unpublished data).
Several additional studies also examined other measures of open field or motor activity (Gomes et al. 1991a;Husain et al. 1992Husain et al. , 1994Lazarini et al. 2001). Reports of effects in these studies were also variable ( Table 2). The reasons for the discrepant nature of these findings are unknown.
A small number of studies tested cognitive functions (Table 2). Two studies reported that bioallethrin exposure during PND10-16 (via different routes) had no significant effect on performance in the Morris water maze at 5 (Talts et al. 1998) and 11 (Tsuji et al. 2002) months of age. Other studies reported decreases in avoidance and Y-maze learning (Aziz et al. 2001;Husain et al. 1994;Moniz et al. 1990) or no change in avoidance behavior (Gomes et al. 1991a). A major confounder in the Y-maze and avoidance studies is the use of commercial formulations rather than technical compound. comparisons (data sets) used for 0 effect on motor activity; altered statistical tests are sometimes unclear regional polyamine levels or confusing; not clear that litter is the statistical unit There are several common weaknesses in the developmental studies that temper the scientific strength of some individual reports, as well as the data set when taken as a whole. A key weakness is problematic statistical analyses. Most behavioral studies [with the exception of Ivens et al. (unpublished data), Jekat et al. (unpublished data), and Tsuji et al. (2002)] used multiple pups from the same litter without correction in the statistical analysis. The sampling of multiple pups from the same litter inflates the sample size and increases the probability of a type I statistical error (Abbey and Howard 1973;Holson and Pearce 1992;Muller et al. 1985;Reily and Meyer 1984). When biochemical end points were examined, statistical analyses often lacked robustness or, in some cases, were absent. In several studies examining receptor binding, results were compared (and significant differences found) using multiple Student's t-tests. Use of multiple t-tests can easily increase the probability of a type I error (Muller et al. 1985). These study designs should use statistical models that control for multiple comparisons (e.g., analysis of variance with appropriate post hoc test for comparisons of different group means). Metaanalyses or other statistical approaches to examine related data sets from the same and different laboratories could help strengthen conclusions when effect magnitude is small but have not been conducted to date.
An additional limitation common to these reports was a lack of tissue concentration data. None of the studies reported pyrethroid blood or brain concentrations from dams or pups. Such information would have greatly facilitated comparisons between studies and would also be useful to compare target tissue concentrations in the test species with exposure estimates in pregnant women (see Whyatt et al. 2002).
Lack of information about the stereoisomer composition and/or purity of the test compound was a serious confound in some reports. Such information is important to be able to compare studies generated in different laboratories, as discussed above for the different allethrin products. In addition, several studies Review | Developmental neurotoxicity of pyrethroids  used formulated products rather than purified compound (Aziz et al. 2001;Gupta et al. 1999aGupta et al. , 1999bHusain et al. 1992Husain et al. , 1994Malaviya et al. 1993). Formulated pesticide products typically contain solvents, emulsifying agents, petroleum distillates, and other "inerts" (Farm Chemicals Handbook 1997), many of which are known or suspected to have neurotoxic properties. Although use of formulated products may provide a more real-life exposure situation, lack of information on the content of proprietary formulations hampers comparisons between studies and often precludes attributing effects directly to the pyrethroid. Several other limitations should also be noted. The number of time points examined in these studies typically was three or fewer, one of which was often a measurement in adult animals. Considerable ontogeny of both behavioral responses as well as biochemical end points is well established. Thus, the tendency of most studies to examine a "snapshot in time" may miss important ontogenic shifts induced by these compounds. Dosing duration and age at exposure are two other important factors. Although a number of studies examined the period of PND10-15, the choice of dosing periods in the present studies was variable, and, to date, there has not been a systematic evaluation of potentially sensitive developmental periods. An additional consideration regarding dosing periods is the differential rates of neurodevelopment in rodents versus humans. Thus, studies such as those conducted by Whyatt et al. (2002) could potentially provide important information about exposure to the developing fetus. In addition, the effects of sex were not always considered in the present studies, with a few exceptions (e.g., Gomes et al. 1991b;Moniz et al. 1999). Also related to this topic is the relative distribution of males and females in a litter. In some cases, culling information was readily available; however, many studies provided no or insufficient information to evaluate this variable.
Although not necessarily a limitation, there is a significant conceptual gap between the variety of behavioral, biochemical, and physiologic end points studied to date (Tables 1-4). The relationships, if any, between these biochemical and behavioral changes have yet to be established. In addition, the relationship between the end points examined in the present studies and the major action of pyrethroids, disruption of VSSC function, is also unknown. Only one study to date has examined changes in VSSC expression (Muhammad and Ray, unpublished data). The relationship between biochemical alterations and pyrethroid-induced developmental neurotoxicity could be strengthened by better characterization of neurochemical mode(s) of action of pyrethroid neurotoxicity. Establishing mode-of-action pathways increases confidence that reported effects are the result of pyrethroid action, particularly when the magnitude of those effects is small.

Conclusions and Recommendations for Future Research
Several research needs in the area of developmental neurotoxicity are apparent from this review. These include additional information regarding potential differences underlying agedependent sensitivity to pyrethroids, clarification of changes in behavioral and biochemical end points, and linking these end points to VSSCs or other cellular targets. In considering these potential areas for future research, determining the priority of addressing different research questions often depends on individual perspectives. In this context, a different conceptual approach to conducting future including cannibalism; hypo-95.7% for S-bioallethrin); statistical Hypothermic, normothermic, thermic pups displayed reduced analysis using ANOVAs; randomized and hyperthermic groups motility; body weight gain selection of pups and dams for treatment PND10-17 was affected by groups from a pool. conditions of hypothermia, Limitations: pup mortality observed in hyperthermia; rectal temperature control, S-bioallethrin groups with no was affected by environmental information provided regarding number temperature, differences in of pups lost/cannibalized; replacement temperature between control pups came from a pool of pups that had and deltamethrin-treated animals been housed under "normal conditions," were present in hypothermic but which likely differed in temperature from not hyperthermic animals; group that lost pups (hypothermic pups); environmental temperature altered sample size for various end points is brain weight, with effects of difficult to determine; examined only S-bioallethrin and deltamethrin PND17 animals; not known if temperature observed only in hypothermic differences could contribute to long-term animals; both deltamethrin and changes in mAChR expression; S-bioallethrin decreased randomized assignment of pups to dams brain/body weight ratios in does not control for maternal effects; hypothermic animals; QNB did not demonstrate that typical p.o. binding: on PND17, mAChR dosing causes hypothermia; because of density was increased in both design of study (incomplete block design), sexes by S-bioallethrin in comparisons between vehicle and S-bioallethrin 0.7 mg/mL p.o.; 20% fat PND10-16 hypothermic and normothermic pyrethroid treatments cannot be made; emulsion (egg groups; no differences were study design was to compare effects of lecithin/peanut oil) observed in the hyperthermic different temperature conditions within Hypothermic, normothermic group or in the deltamethrin-these treatments and hyperthermic groups treated groups Abbreviations: 5HIAA, 5-hydroxyindoleacetic acid; 5HT, serotonin; AChE, acetylcholinesterase; ANOVA, analysis of variance; BBB, blood-brain barrier; BDNF, brain-derived neurotropic factor; B max , maximum number of binding sites; CTX, cortex; DA, dopamine; DMSO, dimethyl sulfoxide; EC, emulsifiable concentrate; GAP-43, growth-associated protein 43; GSH, glutathione; HP, hippocampus; HVA, homovanillic acid; i.p., intraperitoneal; LD 50 , dose lethal to 50%; LH, luteinizing hormone; MAO, monoamine oxidase; NA, noradrenaline; nAChR, nicotinic acetylcholine receptor; p.o., per os; STR, stratum. a Publications by the same group of authors are indicated by shading; in some cases, comments are made on groups of papers published by the same group of authors rather than on individual papers. b Not reported in original publication (Gupta et al. 1999a); data from A.K. Agarwal (personal communication). c Not reported in original publication (Tsuji et al. 2002); data from R. Tsuji (personal communication).  (Andersen and Dennison 2001) describe the relationships between different components of the continuum between exposure to and the adverse effects of a chemical (Figure 4). For example, such a model has recently been constructed for the developmental neurotoxicity of perchlorate (Jarabek et al. 2002). Mode-of-action models strengthen science in two important ways. First, the uncertainty regarding animal-tohuman extrapolations can be reduced if a toxicant's mode of action in an animal model Review | Developmental neurotoxicity of pyrethroids Environmental Health Perspectives • VOLUME 113 | NUMBER 2 | February 2005 (1:10) 40% fat emulsion and co-workers (see Table 2  is demonstrated to be relevant to humans (Cohen et al. 2004;Meek et al. 2003;Sonich-Mullin et al. 2001). Second, these models often provide insight into research needs by identifying data gaps and research needs. For pyrethroids, much of the future research needs can be described in the context of the type of data that would be useful in constructing a BBDR for this class of compounds, or for individual compounds within this class. A cornerstone of a BBDR model is a physiologically based pharmacokinetic (PBPK) model that describes the relationship between exposure and target tissue dose (Andersen and Dennison 2001). Additional pharmacokinetic information in animal models as well as additional pharmacokinetic and exposure information in humans is needed. For pyrethroids, this will involve defining the relationship between maternal and fetal compartments, and the involvement of oral (including lactation), inhalation, and dermal exposures to the newborn. Current data indicate that some exposure does occur to pregnant mothers, infants, and children, resulting in low internal doses (Berkowitz et al. 2003;Heudorf et al. 2004;Schettgen et al. 2002). However, insufficient information is available to adequately evaluate the range of internal doses of pyrethroids in humans. These data will be valuable in quantitative extrapolations of exposure from animals to humans (Andersen and Dennison 2001). Pharmacokinetic information is available comparing acute high-dose exposures in neonatal versus adult animals (Cantalamessa 1993;Sheets et al. 1994). However, only a limited number of compounds have been examined to date, and no information is available for ages before PND11.
Another component of a BBDR model is a physiologically based pharmacodynamic (PBPD) model (Andersen et al. 1992;Conolly 2002). PBPD models are quantitative models that describe the mode of action of a chemical. A benefit of PBPD models is identification of research gaps that are critical to link key events in the mode of action to adverse outcomes. Currently available studies of pyrethroid developmental neurotoxicity have examined a wide variety of end points but have not sought to link target tissue events (e.g., receptor activation, changes in ion channel function) to consequent biochemical, physiologic, or behavioral outcomes. Future studies need to target the large data gap between the target site (e.g., VSSCs) and adverse outcomes. For example, can the sequence of biochemical processes be described that, when perturbed by pyrethroids, result in changes in end points such as motor activity or mAChR binding? If changes in sodium currents alter neuronal firing rate, how does this then lead to alterations in neurodevelopment? Considerable information supports involvement of VSSCs in the mode of action of acute pyrethroid neurotoxicity, yet the potential role of VSSCs in developmental neurotoxicity of pyrethroids has not been examined. Future research on the developmental neurotoxicity of pyrethroids should endeavor to fill these research gaps. These studies must be designed and conducted  Abbreviations: 0, end point was examined and was not affected by treatment; CTX, cortex; HB, habituation; HP, hippocampus; MA, motor activity; ND, not determined; STR, striatum. a As measured by QNB binding. b Compounds are arranged in alphabetical order. c An increase in QNB binding was observed in one "cohort" but was not consistently observed in all "cohorts" in studies by this group. See Table 3

Data needs
Additional carefully designed/conducted studies to characterize adverse effects Studies designed to examine potential links between molecular targets and adverse effects following developmental exposure Figure 4. Major elements in a proposed BBDR model for pyrethroid neurotoxicity and research needs for the PBPK and PBPD components. Boxes with question marks indicate that the sequence of events between changes in the target and adverse effects has not been completely elucidated. so as to avoid the limitations mentioned in the preceding section. Such studies of the developmental neurotoxicity of these compounds can strengthen the scientific basis for risk decisions. The most efficient use of scientific resources will be to design those additional studies to fit into a BBDR scheme.