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. Key words: age-dependent toxicity, biologically based dose--response model, developmental neurotoxicity, mode of action, physiologically based pharmacokinetic model, pyrethroid, risk assessment, voltage-sensitive sodium channel. doi:10.1289/ehp.7254 available via http://dx.doi.org/[Online 14 October 2004]

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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; el) 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 10x 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 2000a, 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

The pyrethroid class of insecticides was derived from natural compounds (the pyrethrins) isolated from the Chrysanthemum genus of plants (Casida 1980). Although natural pyrethrins do have insecticidal activity, they also are inherently unstable when exposed to light. Therefore, the pyrethrin structure was modified to produce more stable 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 1 R-trans isomers (Figure 2).

[FIGURES 1-2 OMITTED]

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 [alpha] 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 [depolarization-dependent 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).

[FIGURE 3 OMITTED]

Mammalian VSSCs are composed of one [alpha] and two [beta] subunits. Ten separate [alpha] subunits (Table 1; Ogata and Ohishi 2002) and four different [beta] subunits (Isom 2002) have been identified and are expressed in a tissue-, region-, and time-specific manner. With one exception (the [Na.sub.X] subunit), 0t subunits all comprise VSSCs when expressed individually or with [beta] subunits. The [alpha] subunit forms the pore of the channel and determines its major functional characteristics, whereas the [beta] subunits are auxiliary proteins that influence gating properties, localization in the membrane, and interactions with cytoskeletal proteins (Isom 2001, 2002). The diverse functional roles of VSSC, such as generating action potential spikes, amplifying sub-threshold depolarizations, regulating repetitive firing and generating after-depolarizations, depend on the numerous potential combinations of [alpha] and [beta] 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 [alpha] subunit of the VSSC. Trainer et al. (1997) expressed only the [Na.sub.v]1.2 [alpha] subunit in Chinese hamster ovary cells and found that the presence of the [alpha] 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.sub.v]1.2 (Smith and Soderlund 1998) or [Na.sub.v]1.8 (Smith and Soderlund 2001) in oocytes in the absence of coexpression with [beta] subunits. Interestingly, coexpression of the [beta]1 subunit with [Na.sub.v]1.2 increased the sensitivity of this channel compared with expression of [Na.sub.v]1.2 alone (Smith and Soderlund 1998), indicating that the [beta] subunit modulates the affinity of pyrethroid interaction with the channel. Mutations in the [alpha] subunit of both insects (Lee and Soderlund 2001; Smith et al. 1997) and mammals (Vais et al. 2000, 2001; Wang et al. 2001) alter the sensitivity of VSSCs to pyrethroid effects, supporting the conclusion that pyrethroids interact with the [alpha] subunit.

The relative susceptibility of the 10 different VSSC [alpha] 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 [alpha] subunits (Table 1). Although not all [alpha] 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.sub.v]1.2 (Smith and Soderlund 1998) is sensitive to type II but not type I compounds, whereas [Na.sub.v]1.8 (Smith and Soderlund 2001) is sensitive to both. Interactions of pyrethroids with other sodium channel [alpha] 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.sub.v]1.3 during embryonic periods (Albrieux et al. 2004) diminishes as expression of [Na.sub.v]1.2 increases in early postnatal periods in rodents (Felts et al. 1997), and expression of [Na.sub.v]1.2 at immature nodes of Ranvier is replaced by [Na.sub.v]1.6 as myelination proceeds (Boiko et al. 2001; Jenkins and Bennett 2002). Similar changes are observed with the [beta] subunits, because [beta]3 expression is replaced by [beta]1 and [beta]2 (Shah et al. 2001). Alternatively spliced forms of the VSSC subunits also contribute to developmental differences in expression because the [Na.sub.v]1.2, [Na.sub.v]1.3, and [Na.sub.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 [alpha] 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 age-dependent 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 impairs nervous system structure and function. Several examples are discussed below for illustrative purposes. 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 [alpha] subunits demonstrate varying degrees of adverse outcomes associated with loss or alteration of specific channel subunits. When mRNA for the [Na.sub.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.sub.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.sub.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 (Kearney et al. 2001; 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 (Hatta et al. 1999; Ohmori et al. 1997, 1999; Schilling 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 Table 3), and regulatory studies provided by 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 species-dependent 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.sub.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, 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 (Anadon et al. 1991, 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, both behavioral and biochemical, within this laboratory has been consistent over several studies. Others have also reported persistent changes in behavior and/or biochemistry, including learning (Moniz et al. 1990), motor activity (deltamethrin only; Husain et al. 1992), sexual behavior (Lazarini et al. 2001), mAChR binding (Aziz et al. 2001; Malaviya et al. 1993), and blood-brain barrier permeability (Gupta et al. 1999a).

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), one used 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. Studies conducted by the group at Bayer (Ivens et al., unpublished data; Jekat et al., unpublished data) used seven different concentrations of carbachol, whereas studies conducted by Eriksson's group (Ahlbom et al. 1994; Eriksson and Fredriksson 1991; Eriksson and Nordberg 1990) used 18 concentrations of carbachol (Eriksson P, personal communication). This information was typically not available to evaluate and may account for some reported differences, because use of too few points may preclude detection of changes in the low-affinity site. Overall, the data across laboratories indicate that changes in QNB binding may not be a robust response to developmental exposure to pyrethroids and that conditions may need to be more carefully controlled in order to observe changes.

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 [sup.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. 1992, 1994; Lazarini 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.

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 used formulated products rather than purified compound (Aziz et al. 2001; Gupta et al. 1999a, 1999b; Husain et al. 1992, 1994; Malaviya 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 age-dependent 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 research may improve the resulting data's usefulness for the purpose of risk decisions.

Biologically based dose-response (BBDR) models (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-to-human extrapolations can be reduced if a toxicant's mode of action in an animal model 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.

[FIGURE 4 OMITTED]

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, ch2anges 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 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.

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Timothy J. Shafer, (1) Douglas A. Meyer, (2) and Kevin M. Crofton (1,2)

(1) Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; (2) Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Address correspondence to T.J. Sharer, Neurotoxicology Division, MD B105-05, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank D. Ray (Medical Research Council, UK) and Bayer CropScience for graciously making available unpublished data for this review; L. Sheets (Bayer CropScience) and S. Padilla (U.S. EPA) for comments on a previous version of the manuscript; and J. Harrill (University of North Carolina at Chapel Hill) and J. Havel (CSC Corporation) for their assistance with figures and graphics.

Preparation of this document has been funded wholly by the U.S. Environmental Protection Agency. This document has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

The authors declare they have no competing financial interests.

Received 14 May 2004; accepted 14 October 2004.

Figure 2. Eight possible stereoisomers of allethrin (A-H). The inset
lists allethrin-containing products and the stereoisomer content of
each.

Common name           Proprietary name            Isomer composition

Allethrin        Pynamin, Pyresyn, Pyresin,
                   Allycancerin, Pallethrin          All isomers
d-Allethrin      trans-d-Allethrin, Pynamin           A, B, C, D
Bioallethrin     d-trans-Allethrin, +-trans-
                   Allethrin, Depallethrin               A, B
S-Bioallethrin   Esbiol, Espallethrin, d-trans-
                   Allethrin, d-Allethronyl             A only

Table 1. Sodium channel [alpha] subunit nomenclature and effects of
pyrethroids. (a)

                                                               TTX
[alpha] subunit                 Older names                sensitivity

[Na.sub.v] 1.1     Rat I, HBSCI, GPBI,                        TTX-S
                   SCN1A
[Na.sub.v] 1.2     Rat II, HBSCII, HBA                        TTX-S
[Na.sub.v] 1.3     Rat III                                    TTX-S
[Na.sub.v] 1.4     SkM1, [micro]1                             TTX-S
[Na.sub.v] 1.5     SkM2, H1                                   TTX-R
[Na.sub.v] 1.6     NaCh6, PN4, Scn8a,                         TTX-S
                   Cerlll
[Na.sub.v] 1.7     NaS, hNE-NA, PN1                           TTX-S
[Na.sub.v] 1.8     SNS, PN3, NaNG                             TTX-R
[Na.sub.v] 1.9     NaN, SNS2, PN5,                            TTX-R
                   NaT, SCN12A
[Na.sub.x]         [Na.sub.v] 2.1, [Na.sub.v] 2.3 Na-G,         ?
                   SCL11

                          Tissue
[alpha] subunit         expression

[Na.sub.v] 1.1     CNS, PNS, Purkinje, HP
                   pyramidal cells, spinal
                   motor neurons, somatic
                   localization
[Na.sub.v] 1.2     CNS, forebrain, substantia
                   nigra, HP mossy fibers, CB
                   molecular layer, axonal
                   localization
[Na.sub.v] 1.3     CNS and DRG
[Na.sub.v] 1.4     Skeletal muscle
[Na.sub.v] 1.5     Uninnervated skeletal
                   muscle, heart, brain
[Na.sub.v] 1.6     CNS, DRG (all diameter
                   neurons), node of
                   Ranvier-peripheral nerve
[Na.sub.v] 1.7     DRG (all diameter neurons)
                   CNS, Schwann cells
[Na.sub.v] 1.8     DRG (small diameter
                   neurons)
[Na.sub.v] 1.9     DRG (small diameter
                   neurons)
[Na.sub.x]         Heart, uterus, skeletal
                   muscle, astrocytes, DRG

[alpha] subunit          Developmental expression

[Na.sub.v] 1.1     Not detected in HP during development,
                   detectable in CB Purkinje cells at PND15,
                   detected at PND2 in SC; strong
                   expression in motor neurons (b)
[Na.sub.v] 1.2     In HP, increase between GD17 and PND30,
                   in CB granule cells on PND15 and Purkinje
                   cells on PND2; detected at all ages in SCI
                   Splice variant expressed during
                   development (c)
[Na.sub.v] 1.3     HP expression at GD17, increasing at PND2,
                   then decreasing to barely detectable at PND30.
                   Detected at GD17 in CB neuroepithelium,
                   decreasing thereafter, similar in SC (b);
                   developmentally regulated splice variant'
[Na.sub.v] 1.4     Increases with age (f)
[Na.sub.v] 1.5     mRNA expressed in rat PNOQ limbic
                   structures and medulla, expressed in fetal
                   and adult human brain (h)
[Na.sub.v] 1.6     Truncated form expressed from GD12 to
                   PND7, full-length mRNA expression is slight
                   at GD14 and increases with age (j)
[Na.sub.v] 1.7     All DRG neurons at PND2, increased during
                   development (b)
[Na.sub.v] 1.8     Expression beginning at GD15 with adult
                   levels by PND7; largely in unmyelinated
                   C-fibers (j)
[Na.sub.v] 1.9     Expression beginning at GD17 with adult
                   levels by PND7; largely in unmyelinated
                   C-fibers (j)
[Na.sub.x]         Transient between PND2 and 15 in HP;
                   peak expression at PND2 in CB, SC;
                   large DRG neurons, GD17 to PND30 (b)

[alpha] subunit            Effect of pyrethroids

[Na.sub.v] 1.1     Not tested to date
[Na.sub.v] 1.2     Cypermethrin-induced tail currents
                   detectable at > 30 nM in rat 1.2 (adult
                   splice variant) co-expressed with
                   (3, subunits; reported insensitive to
                   permethrin or cismethrin (d)
[Na.sub.v] 1.3     Not tested to date
[Na.sub.v] 1.4     Only slightly modified by 10 [micro]M
                   deltamethrin when expressed in
                   HEK 293t cells (g)
[Na.sub.v] 1.5     Not tested to date
[Na.sub.v] 1.6     Not tested to date
[Na.sub.v] 1.7     Not tested to date
[Na.sub.v] 1.8     Sensitive to both cismethrin and
                   cypermethrin at thresholds of 500 nM
                   and 30 nM, respectively (k)
[Na.sub.v] 1.9     Not tested to date
[Na.sub.x]         Not tested to date

Abbreviations: CB, cerebellum; CNS, central nervous system; DRG,
dorsal root ganglion; GD, gestation day; HP, hippocampus; PND,
postnatal day; PNS, peripheral nervous system; SC, spinal cord;
TTX, tetrodotoxin; TTX-R, TTX resistant; TTX-S, sensitive to TTX.

(a) Data in the first four columns are based on information
presented by Goldin et al. (2000) and Novakovic et al. (2001).
(b) Felts et al. (1997). (c) Sarao et al. (1991). (d) Smith and
Soderlund (1998). (e) Gustafson et al. (1993). (f) Kallen et al.
(1990). (g) Nang et al. (2001). (h) Donahue et al. (2000).
(i) Plummer et al. (1997). (j) Benn et al. (2001).
(k) Smith and Soderlund (2001).

Table 2. Summary of peer-reviewed developmental neurotoxicity
studies with pyrethroids. (a)

Species/compound        Dose/route/vehicle              Dosing period

Rat (Wistar)
  Cyhalothrin         0.02% in drinking water,        PND0-21
    (type II)         0.4% sucrose
                      + "cyhalothrin vehicle"

                      0.018%, 1 mL dermal,            "Entire
                      daily, "cyhalothrin             pregnancy"
                      vehicle"

  Fenvalerate         10 mg/kg, i.p.;                 GD18 and
    (type II)         saline                          PND2-5
  Deltamethrin        0.08 mg/kg, p.o.                GD6-15,
    (type II)         "deltamethrin vehicle"          once daily

Mouse
  Bioallethrin        0.72 and 72 mg/kg               PND10-16,
    (type I)          20% fat emulsion (egg           once daily
                      lecithin/peanut oil)
  Deltamethrin        0.71 and 1.2 mg/kg
    (type II)         20% fat emulsion (egg
                      lecithin/peanut oil)
  Bioallethrin        0.7 mg/kg, p.o.;
    (type I)          20% fat emulsion (egg
                      lecithin/peanut oil)
  Deltamethrin        0.7 mg/kg, p.o.,
    (type II)         20% fat emulsion
                      (egg lecithin/peanut oil)
  Bioallethrin        0.42, 0.70, 42 mg/kg,
                      p.o., 20% fat emulsion
                      (egg lecithin/peanut
                      oil)
  Bioallethrin        0.7 mg/kg, p.o., 20% fat        PIN D10-16, once
                      emulsion (egg lecithin/         daily; again at
                      peanut oil)                     5 months for
                      4 treatment groups:             7 days, once
                      vehicle as pup and              daily
                      5 months, VB, vehicle as
                      pup, bioallethrin at
                      5 months, BV, bioallethrin
                      as pup, vehicle at
                      5 months, BB, bioallethrin
                      as pup and 5 months
Rat (Wistar)
  Deltamethrin        0.7 mg/kg, i.p.;                PND9-13
    (type II)         propylene glycol
Rat (Druckrey)
  Cypermethrin        Experiment 2: 5 mg/kg,          PND10-13,
                      p.o. (corn oil vehicle)         17, or 30

                      Experiment 3: 2.5 mg/kg,        PND10-17
                      p.o. (corn oil vehicle),
                      (1/100 L[D.sub.50])

Allethrin             18 hr/day inhalation of         PND2-19
                      vapors, unknown
                      commercial product
                      containing 3.6%
                      Allethrin, 96% kerosine,
                      0.3% stabilizer
Deltamethrin          1.0 mg/kg, p.o.,                GD14-20
                      deltamethrin
                      formulation in corn
                      oil
Rat (Wistar)
  Deltamethrin        7 mg/kg, p.o.                   GD5-21
                      2.8% EC formulation,
                      peanut oil
  Fenvalerate         10 mg/kg, p.o., 20% EC
                      formulation, peanut oil
  Cypermethrin        15 mg/kg, p.o., 25% EC
                      formulation, peanut oil
  Deltamethrin
                      7 mg/kg; 2.8%                   PND22-37
                      EC formulation,
                      corn oil
Rat (Charles
Wistar)
  Fenvalerate         10 mg/kg, p.o.;                 GD5-21
                      corn oil                        (gestational
                                                      exposure)
                                                      or PND1-15
                                                      (lactational
                                                      exposurel
                                                      Biochemical
                                                      outcomes measured
                                                      at 3 weeks of age
  Cypermethrin        15 mg/kg, p.o.,                 GD5-21
                      corn oil                        (gestational
                                                      exposure)
                                                      or PND1-15
                                                      (lactational
                                                      exposure)
                                                      Biochemical
                                                      outcomes measured
                                                      at 3 weeks of age

Rat (Wistar)
  d-Allethrin         0.43-74.2 mg/[m.sup.3]          PND10-16, 6hr/day
                      Inhalation; unknown
                      vehicle
Mouse (ICR)
  Permethrin          Experiment 1: 0.33 to           PND0-21
    (cis or trans)    33 [micro]g/ml cis-permethrin
                      or 33 [micro]g/mL trans-
                      permethrin in drinking water,
                      0.33 [micro]g/mL DMSO vehicle

                      Experiment 2: 1 mg/day          PND0-35
                      cis-permethrin, p.o.
                      corn oil
Mouse (NMRI)
  Deltamethrin        0.7 mg/ml p.o., 20% fat         PND10-16
                      emulsion (egg
                      lecithin/peanut oil)
                      Hypothermic, normothermic,
                      and hyperthermic groups
   S-bioallethrin      0.7 mg/mL p.o., 20% fat         PND10-16
                      emulsion (egg
                      lecithin/peanut oil)
                      Hypothermic, normothermic
                      and hyperthermic groups

Species/compound                       Effects

Rat (Wistar)
  Cyhalothrin         [down arrow] learning avoidance latencies
    (type II)           at PND90, 0 effect on motor
                        activity in pup
                      Delayed development of fur,
                        ear/eye opening, and testes
                        descent. PND90: [down arrow] hole-board
                        head dips; 0 effect avoidance,
                        and locomotion in open field
                      0 change in sexual behaviors in
                        males or females
  Fenvalerate         0 effect: testis descent, weight,
    (type II)           monoamine levels, stereotyped
                        behavior, locomotion, rearing
                        [down arrow] pup weight on PND21,
                        [down arrow] ductus deferens and seminal
                        vesicle weight, female sexual
                        behavior at PND120
  Deltamethrin        PND21: [up arrow] rearing in males, 0
    (type II)           effect on locomotion frequency
                        in males or females
                      PND60 males: [down arrow] immobility
                        time in forced swim test
                        [up arrow] DOPAC, DOPAC/DA, NA
                        0 effect on 5HT, 5HIAA,
                        HVA/DA; 0 effect in
                        PND60 females
Mouse
  Bioallethrin        PND17: [up arrow] mAChR density and
    (type I)            altered ratio of high- and low-
                        affinity QNB binding sites in
                        CTX but not HP with
                        deltamethrin and bioallethrin
                        at low (0.7 mg/kg) but not
                        high doses
  Deltamethrin        0 change in nAChR density
    (type II)
  Bioallethrin        4 months: T motor activity
    (type I)          with lack of habituation,
                      [up arrow] mAChR density in CTX;
                      0 change in mAChR in HP, STR
  Deltamethrin        4 months: [up arrow] motor activity
    (type II)         with lack of habituation;
                      0 change in mAChR in CTX,
                      HP, STR
  Bioallethrin        PND17: [up arrow] mAChR density in
                      CTX; [up arrow] low-affinity QNB
                      (mAChR) binding
                      4 months: [up arrow] motor activity
                      with lack of habituation,
                      [down arrow] mAChR density in CTX
  Bioallethrin        5 months: [up arrow] motor activity
                        with lack of habituation in BB
                        and BV groups
                      Performance in [H.sub.2]O maze:
                        [down arrow] reversal in BB groups; 0 effect
                        in BV, VB groups
                      mAChR density in CTX: [up arrow] in
                        BB treatment group, 0 effect in
                        BV, VS groups
Rat (Wistar)
  Deltamethrin        Examined on PNDs 12, 15, 21,
    (type II)           and 30: delayed cerebellar
                        cytogenesis and morphogenesis
                        of interneurons, vascular
                        damage with focal
                        degeneration; [down arrow] brain and
                        body weight
Rat (Druckrey)
  Cypermethrin        [up arrow] BBB permeability at
                        PNDs 13, 17, and 30 by 71, 61,
                        and 80%; effect recovered by
                        PND60 following withdrawal on
                        PND18
                      [up arrow] BBB permeability by 28%
Allethrin             [up arrow] body (23%) and brain (17%)
                        weights, [up arrow] BBB permeability, LH
                        levels on PND10 but not PND18:
                        [up arrow] (small) in conjugated dienes
                        (measure of lipid peroxidation)
                        on PND10; [down arrow] GSH 17% on PND10;
                        [up arrow] GSH by 28% on PND18
Deltamethrin          Delayed surface righting reflex
                        6 and 12 weeks postnatal:
                        [up arrow] AChE activity, [up arrow] GAP-43
                        immunochistochemistry (both %
                        area and total number of positive
                        cells), [down arrow] QNB [B.sub.max];
                        [down arrow] relearning in Y-maze task
Rat (Wistar)
  Deltamethrin        [down arrow] weight of unspecified brain
                        regions at PND22(?);
                        [[up arrow]] resorptions and neonatal death,
                        delayed surface righting, eye
                        opening, fur development, incisor
                        eruption, and pinna detachment,
                        [down arrow] grip strength, [down arrow] motor
                        activity at PNDs 21 and 42, altered regional
                        polyamine levels
  Fenvalerate         Delayed surface righting, eye
                        opening, fur development, incisor
                        eruption, and pinna detachment,
                        [down arrow] grip strength, 0 effect on motor
                        activity, altered regional
                        polyamine levels
  Cypermethrin        Delayed surface righting, eye
                        opening, fur development, incisor,
                        eruption and pinna detachment,
                        0 effect on motor activity; altered
                        regional polyamine levels
  Deltamethrin
                      [down arrow] hippocampal weight without
                        effect on other brain regions,
                      [up arrow] mitochondrial monamine
                      oxidase and microsomal AChE
                        without effect on Na/K
                        ATPase; [up arrow] spontaneous
                        locomotor activity,
                        [down arrow] conditioned avoidance
                        response; altered regional
                        polyamine levels
Rat (Charles
Wistar)
  Fenvalerate         0 effect on dam weight, food/
                        water intake, gestation length,
                        no. of offspring, sex ratio
                      Gestational exposure: [down arrow] MAO,
                        Na/K-ATPase activity, spiro-
                        peridol binding; [up arrow] AChE activity
                      Lactational exposure:
                        [down arrow] MAO, AChE activity,
                        [up arrow] spiroperidol, QNB binding
  Cypermethrin        0 effect on dam weight, food/
                        water intake, gestation length,
                        no. of offspring, sex ratio
                      Gestational exposure: 0 effect
                      on MAO, Na/K-ATPase, AChE
                      activity; spiroperidol binding
                      [down arrow] QNB binding
                      Lactational exposure:
                      [down arrow] Na/K-ATPase, AChE activity,
                      [up arrow]spiroperidol, QNB binding
Rat (Wistar)
  d-Allethrin         0 Effects on weight gain, motor
                        activity, mAChR density when
                        assessed on PND17 and
                        4 months
                      0 effect in Morris water
                        maze at 11 months
Mouse (ICR)
  Permethrin          0 effect on weight in
    (cis or trans)      dam, pups; concentration-
                        dependent decrease in c-fos
                        mRNA in cerebellum at PND21;
                        trend toward decrease in BDNF
                        mRNA at PND21; 0 effect on
                        [beta]-actin mRNA
                      [down arrow] c-fos mRNA at PND21
                        only; 0 effect on [beta]-actin mRNA
                        at any time
Mouse (NMRI)
  Deltamethrin        Pup mortality in hypothermic
                        groups (control and S-bioallethrin),
                        including cannibalism; hypo-
                        thermic pups displayed reduced
                        motility, body weight gain
                        PND10-17 was affected by
                        conditions of hypothermia,
                        hyperthermia; rectal temperature
                        was affected by environmental
                        temperature, differences in
                        temperature between control
                        and deltamethrin-treated animals
                        were present in hypothermic but
                        not hyperthermic animals,
                        environmental temperature altered
                        brain weight, with effects of
                        S-bioallethrin and deltamethrin
                        observed only in hypothermic
                        animals, both deltamethrin and
                        S-bioallethrin decreased
                        brain/body weight ratios in
                        hypothermic animals; QNB
                        binding: on PND17, mAChR
                        density was increased in both
                        sexes by S-bioallethrin in
  S-bioallethrin        hypothermic and normothermic
                        groups, no differences were
                        observed in the hyperthermic
                        group or in the deltamethrin-
                        treated groups

Species/compound         Reference

Rat (Wistar)
  Cyhalothrin         Moniz et al. 1990
    (type II)
                      Gomes et al.
                        1991a
                      Gomes et al.
                        1991b
  Fenvalerate         Moniz et al. 1999
    (type II)
  Deltamethrin        Lazarini et al.
    (type II)           2001
Mouse
  Bioallethrin        Eriksson and
    (type I)          Nordberg 1990
  Deltamethrin
    (type II)
  Bioallethrin        Eriksson and
    (type I)            Fredriksson 1991
  Deltamethrin
    (type II)
  Bioallethrin        Ahlbom et al. 1994
  Bioallethrin        Talts et al. 1998
Rat (Wistar)
  Deltamethrin        Patro et al. 1997
    (type II)
Rat (Druckrey)
  Cypermethrin        Gupta et al.
                      1999a
  Allethrin           Gupta et al.
                      1999b
  Deltamethrin        Aziz et al. 2001
Rat (Wistar)
  Deltamethrin        Husain et al. 1992
  Fenvalerate
  Cypermethrin
  Deltamethrin
                     Husain et al. 1994
Rat (Charles
Wistar)
  Fenvalerate         Malaviya et al.
                      1993
  Cypermethrin        Malaviya et al.
                      1993
Rat (Wistar)
  d-Allethrin         Tsuji et al. 2002
Mouse (ICR)
  Permethrin          Imamura et al.
    (cis or trans)    2002
Mouse (NMRI)
  Deltamethrin        Pauluhn and
                      Schmuck 2003
  S-bioallethrin

Species/compound                       Comments

Rat (Wistar)
  Cyhalothrin         Strengths: maternal behavior examined
    (type II)           in Moniz et al., 1990 (no effect); culling
                        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 GDQ-PNDQ
                        (Gomes et al. 1991a, 1991b),
                        inappropriate statistical models,
                        minimal description of results; not clear
                        that litter is statistical unit (numbers
                        of replicates in figure legends do not
                        always agree with number of treatment
                        groups)
  Fenvalerate         Strengths: litter as statistical unit; more
    (type II)           complete and appropriate statistical
                        analysis, but still some incorrect uses
                        of t-test (Moniz et al. 1999), maternal
                        weight examined/reported; Lazarini
                        et al. (2001) considered sex differences,
                        only papers examining reproductive
  Deltamethrin          behavior, culling, male/female ratios
    (type II)           described and even. Housing as adults
                        described
                      Limitations: deltamethrin commercial
                        product, unknown ("deltamethrin
                        vehicle") vehicle composition, purity of
                        fenvalerate not known, discrepancies
                        between text and figures in Moniz et al.
                        (1999 their Figure 3), differences in
                        control testes descent day in Moniz
                        et al. (1999) vs. Gomes et al. (1991,
                        1991 b)(19 vs. 23 days)
Mouse
  Bioallethrin        Strengths: consistent demonstration of
    (type I)            increased motor activity and lack of
                        habituation with bioallethrin and
                        deltamethrin; dosing occurs over
                        a critical period of brain development;
                        dose response demonstrated for
                        bioallethrin for behavior and bio-
                        chemistry effects present 3.5-4 months
  Deltamethrin          postdosing, behavior, biochemistry
    (type II)           measured in same animals; changes in
                        mAChR binding in CTX ~10% at
                        4 months, but changes not observed
  Bioallethrin          after 5 months (bioallethrin); consistent
    (type I)            effects over several different studies;
                        history of publications with motor
                        activity and QNB binding
                      Limitations: statistical analysis of
  Deltamethrin          biochemical data increases the
    (type II)           possibility of type I error; unclear that
                        litter is unit of treatment; in some cases,
                        changes as small as 1-3% reported
  Bioallethrin          as significant (biochemistry); sex
                        differences not considered/included;
                        toxicity observed at high dose of
                        deltamethin and bioallethrin by Eriksson
                        and Nordberg (1990), with tolerance
                        developing by the fourth day of dosing
  Bioallethrin

Rat (Wistar)
  Deltamethrin        Strengths: only study examining
    (type II)           morphology, culled litters to equal
                        numbers, time course examined, within-
                        litter dosing design
                      Limitations: effects may be due to
                        decreased growth, not direct neuro-
                        toxicity; inappropriate statistical models,
                        no control for "maternal" neglect
                        effects in control vs. treated pups
Rat (Druckrey)
  Cypermethrin        Strengths: control data demonstrate
                        maturation of BBB; within-paper
                        replication of effect; technical grade
                        (94.5% purity) cypermethrin (b)
                      Limitation: litter was not the
                        statistical unit
  Allethrin           Strengths: replication of fluorescence
                        levels on PND10 compared with Gupta
                        et al. (1999a); litters culled to
                        8 pups/dam (size of litter is known)
                      Limitations: unknown formulation;
                        exposure to kerosine > > allethrin, no
                        kerosine control
  Deltamethrin        Strengths: examined two time points,
                        behavioral and biochemical changes
                      Limitations: unknown formulation, corn
                        oil used as "control"; unclear that litter
                        is statistical unit, maze learning
                        procedure is poorly described, and
                        "relearning" is poorly defined
Rat (Wistar)
  Deltamethrin        Strengths: work uniquely covers effects
                        of pyrethroids on different periods of
                        perinatal development from shortly
                        after conception to post-weaning, and
                        suggests that effects may depend on
                        the exposure period (includes Malaviya
                        et al. 1993). However, different
                        compounds were utilized; effects on
                        maternal parameters, general toxicity
  Fenvalerate           recorded; litter size adjusted to an
                        average of 8 pups/litter
                      Limitations: formulated products used,
                        lack of relevant vehicle controls; general
                        or less specific toxicity may be indicated
                        by changes in fur development, pinna
  Cypermethrin          detachment, statistical models are often
                        inappropriate, descriptions of
                        comparisons (data sets) used for
                        statistical tests are sometimes unclear
                        or confusing; not clear that litter is the
                        statistical unit
  Deltamethrin
                      Strengths: work uniquely covers effects
                        of pyrethroids on different periods of
                        perinatal development from shortly
                        after conception to post-weaning, and
                        suggests that effects may depend on
                        the exposure period (includes Malaviya
                        et al. 1993). However, different
                        compounds were utilized; effects on
                        maternal parameters, general toxicity
                        recorded, litter size adjusted to an
Rat (Charles            average of 8 pups/litter
Wistar)               Limitations: formulated products used;
  Fenvalerate           lack of relevant vehicle controls, general
                        or less specific toxicity may be indicated
                        by changes in fur development, pinna
                        detachment; statistical models are often
                        inappropriate, descriptions of
                        comparisons (data sets) used for
                        statistical tests are sometimes unclear
                        or confusing, not clear that litter is the
                        statistical unit
  Cypermethrin

Rat (Wistar)
  d-Allethrin         Strengths: measured air levels of
                        allethrin during exposure, provides
                       additional exposure information;
                        multiple dose levels, litter controlled (c)
                      Limitations: absence of positive controls,
                        this would demonstrate that lack of
                        effect is true negative
Mouse (ICR)
  Permethrin          Strengths: water consumption (ingested
    (cis or trans)      dose) measured, replication of c-fos
                        decrease by different routes of
                        exposure, similar findings following
                        in vitro exposure to cerebellar granule
                        cells (Imamura et al. 2000)
                      Limitations: did not use litter as
                        statistical unit. 3-4 samples/litter,
                        BDNF data variable
Mouse (NMRI)
  Deltamethrin        Strengths: technical compound of known
                        purity used (99.8% for deltamethrin and
                        95.7% for S-bioallethrin); statistical
                        analysis using ANOVAs, randomized
                        selection of pups and dams for treatment
                        groups from a pool.
                      Limitations: pup mortality observed in
                        control, S-bioallethrin groups with no
                        information provided regarding number
                        of pups lost/cannibalized; replacement
                        pups came from a pool of pups that had
                        been housed under "normal conditions,"
                        which likely differed in temperature from
                        group that lost pups (hypothermic pups),
                        sample size for various end points is
                        difficult to determine, examined only
                        PND17 animals, not known if temperature
                        differences could contribute to long-term
                        changes in mAChR expression,
                        randomized assignment of pups to dams
                        does not control for maternal effects,
                        did not demonstrate that typical p.o.
                        dosing causes hypothermia; because of
                        design of study (incomplete block design),
                        comparisons between vehicle and
  S-bioallethrin        pyrethroid treatments cannot be made;
                        study design was to compare effects of
                        different temperature conditions within
                        these treatments

Abbreviations: 5HIAA, 5-hydroxyindoleacetic acid; 5HT, serotonin; ACNE,
acetylcholinesterase, ANOVA, analysis of variance; BBB, blood-brain
barrier; BDNF, brain-derived neurotropic factor; [B.sub.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; L[D.sub.50], dose lethal to 50%; LH, luteinizing
hormone; MAO, monoamine oxidase; NA, noradrenaline; nAChR, nicotinic
acetylcholine receptor; p.o., per us, 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. 1999al; data from A.K.
Agarwal (personal communication). Not reported in original publication
(Tsuji et al. 2002); data from R. Tsuji (personal communication).

Table 3. Summary of developmental neurotoxicity studies with pyrethroid
compounds in NMRI mice dosed once daily on PND10-16 (Muhammad and Ray,
unpublished data).

Compound                       Dose/route/vehicle

d-Allethrin, 93% purity    0.7 mg/kg
  (cis/trans)                egg lecithin/peanut oil
  Experiment 13              (1:10) 40% fat emulsion
S-Bioallethrin             0.7, 3.5 mg/kg
  (traps)                    egg lecithin/peanut oil
  Experiment 17a             (1:10) 40% fat emulsion
S-Bioallethrin             Attempt to replicate
  (trans)                    experiment 17a
  Experiment 19a
S-Bioallethrin             0.7 mg/kg, corn oil
  (trans)
  Experiment 25a
S-Bioallethrin             0.7 mg/kg, corn oil
  (trans)
  Experiment 26a
Deltamethrin               0.7 mg/kg, egg
  Experiment 12              lecithin/peanut oil (1:10)
                             40% fat emulsion
Deltamethrin               0.7 mg/kg, corn oil
  Experiment 23
Deltamethrin               0.7 mg/kg, corn oil
  Experiment 25
Deltamethrin               0.7 mg/kg, corn oil
  Experiment 26

Compound                                     Effects

d-Allethrin, 93% purity    4 months: 0 effect on motor activity, 0
  (cis/trans)                effect on mAChR (QNB) binding in CTX
  Experiment 13
S-Bioallethrin             4 months: [up arroq] motor activity,
  (traps)                    habituation (slow mobile counts, 0 effect
  Experiment 17a             on mAChR in CTX
S-Bioallethrin             4 months: [up arrow] mAChR in CTX, CB
  (trans)                    (3.5 mg/kg); [up arrow] mAChR brainstem
  Experiment 19a             (0.7 and 3.5 mg/kg); [down arrow]
                             habituation (slow mobile counts) by 0.7
                             mg/kg dose, [up arrow] DOPAC, HVA in
                             striatum; [up arrow] saxitoxin binding in
                             CB and MB, [down arrow] in CTX
S-Bioallethrin             PND17: 0 effect on mAChR in CTX
  (trans)                  4 months: no data provided, despite mention
  Experiment 25a             that motor activity and mAChR were
                             assessed
S-Bioallethrin             4 months: significant delay in habituation
  (trans)                    of slow rearing, fast rearing, total
  Experiment 26a             rearing, and rearing time, 0 effect on
                             mobile activity and time, 0 effect on
                             mAChR
Deltamethrin               4 months: [up arrow] rearing time fast and
  Experiment 12              total mobile counts slow, fast, and total
                             rearing; delayed habituation of counts,
                             slow mobile counts, and mobile time
                             mAChR not examined
Deltamethrin               4 months: [up arrow] mAChR in CTX; no effect
  Experiment 23              on any measure of motor activity
Deltamethrin               PND17: [up arrow] mAChR; motor activity not
  Experiment 25              examined
Deltamethrin               4 months: significant delay in habituation
  Experiment 26              of slow mobile counts, mobile and rearing
                             time, 0 change in mAChR (increased but not
                             significant)

Compound                                     Comments

d-Allethrin, 93% purity    Strengths: each chemical was examined in
  (cis/trans)                several cohorts in this study, closely
  Experiment 13              replicates methodology of  Eriksson and
                             co-workers (see Table 2) for motor
                             activity measurements, examined vehicle
                             differences; technical compounds of known
                             purity (100% for deltamethrin and 95.2%
                             for S-bioallethrin)
S-Bioallethrin             Limitations: not published, peer-reviewed or
  (traps)                    submitted to any regulatory agency; litter
  Experiment 17a             was not used as statistical unit;
S-Bioallethrin               statistical models not well described,
  (trans)                    t-tests used for biochemical measures,
  Experiment 19a             date of study unknown, circa mid-1990s
S-Bioallethrin
  (trans)
  Experiment 25a
S-Bioallethrin
  (trans)
  Experiment 26a
Deltamethrin
  Experiment 12
Deltamethrin
  Experiment 23
Deltamethrin
  Experiment 25
Deltamethrin
  Experiment 26

Abbreviations: CB, cerebellum; CTX, cortex; HVA, homovanillic acid;
MB, midbrain.

Table 4. Summary of data from studies in NMRI mice (dosed once
daily on PND10-16) submitted to the U.S. EPA.

Compound      Dose/route/vehicle                     Effects

d-Allethrin   0.15, 4, or 100 mg/         PND17: motor activity:
              [m.sup.3] 6 hr/day,           increased habituation in
              inhalation,                   0.15 mg/[m.sup.3] females
              polyethylene glycol           when compared to control,
                                            effects not dose-related,
                                            mAChR: 25% [up arrow] in
                                            ONE in cortex, smaller
                                            changes in hippocampus and
                                            striatum; nAChR: 40-60%
                                            [down arrow] in cortex,
                                            hippocampus, and striatum
                                            in both sexes, AChE:
                                            [up arrow] by 70-80% in
                                            striatum but not
                                            significant due to large
                                            variability, ChAT: 0 effect
                                          4 months: motor activity: no
                                            significant effects, mAChR:
                                            0 effect, nAChR: large
                                            sporadic changes but no
                                            clear sex- or dose-related
                                            trends, AChE: 0 effect;
                                            ChAT: 0 effect
Cyfluthrin    6, 15 or 50 mg/[m.sup.3],   All pups died in 50 mg/
              6 hr/day, inhalation;         [m.sup.3] dose group; 15
              polyethylene glycol           mg/[m.sup.3] pups had
                                            clinical signs including
                                            "clonic seizures"
                                            (probably tremors and/or
                                            choreoathetosis),
                                            [down arrow] pup
                                            weight in 15 mg/[m.sup.3]
                                            and in 5 mg/[m.sup.3]
                                            females
                                          PND17: no measurements
                                          4 months motor activity: 15
                                            mg/[m.sup.3] females were
                                            hyperactive and had
                                            decreased habituation in
                                            horizontal and vertical v
                                            activity; mAChR:
                                            [down arrow] QNB binding
                                            (not statistically
                                            significant) of ~22% in
                                            15 mg/[m.sup.3] males

Compound             References                     Comments

d-Allethrin   Ivens et al.,               Strengths: technical
              unpublished                   compound, 95% purity; group
              data                          sizes of 10; litter was
                                            statistical unit; good
                                            statistical analysis, males
                                            and females considered
                                            separately; second control
                                            group was included, closely
                                            replicates methodology of
                                            Eriksson and co-workers
                                            (see Table 2) for motor
                                            activity measurements
                                          Limitations: not peer-
                                            reviewed or published, some
                                            biochemical measurements
                                            were variable and not
                                            dose-related
Cyfluthrin    Jekat et al.,               Strengths: technical
              unpublished                   compound, 96.8% purity,
              data                          group sizes of 10, litter
                                            was statistical unit, good
                                            within-lab replicability
                                            for motor activity
                                            [comparison of data with
                                            Ivens et al. (unpublished
                                            data)]; closely replicates
                                            methodology of Eriksson and
                                            co-workers (see Table 2)
                                            for motor activity
                                            measurements
                                          Limitations: not peer-
                                            reviewed or published; only
                                            examined adults; general
                                            toxicity observed, QNB data
                                            variable, no dose-related
                                            effects, difficult to
                                            compare with other studies
                                            because presented either
                                            as dpm or percent of
                                            control

Abbreviations: AChE, acetylcholinesterase; ChAT, choline
acetyltransferase; nAChR, nicotinic acetylcholine receptor.

Table 5. Summary of effects on mAChR and motor activity after
developmental exposure to pyrethroids.

                               MAChR expression (a)

Compound (b)             Preweaning                     Adult

d-Allethrin      [up arrow] CTX               0 CTX
d-Allethrin      0                            0
Bioallethrin     0 CTX                        [up arrow] CTX (c)
Bioallethrin     ND                           0 CTX
Bioallethrin/
  bioallethrin   ND                           [up arrow] CTX
Bioallethrin     ND                           [up arrow] CTX; 0 HP, STR
Bioallethrin     [up arrow] CTX               [up arrow] CTX
Bioallethrin     [up arrow] CTX               ND
S-Bioallethrin   [up arrow] CTX               ND
Cyfluthrin       ND                           0 CTX
Cypermethin      [down arrow] STR             ND
                   (gestation experiment),
                   [up arrow] STR
                   (lactation experiment)
Deltamethrin     ND                           [down arrow] HP
Deltamethrin     [up arrow] CTX               [up arrow] CTX
Deltamethrin     ND                           0 CTX, HP, STR
Deltamethrin     [down arrow] HP              ND
Deltamethrin     0 CTX                        ND
Fenvalerate      0 STR (gestation             ND
                   experiment), [up arrow]
                   STR lactation experiment

                          Motor activity

Compound (b)     Preweaning                     Adult

d-Allethrin      [up arrow] HB    0
d-Allethrin      ND               0
Bioallethrin     ND               [up arrow] MA, [down arrow] HB
Bioallethrin     ND               [up arrow] MA, [down arrow] HB
Bioallethrin/
  bioallethrin   ND               [up arrow] MA, [down arrow] HB
Bioallethrin     0 MA, 0 HB       [up arrow] MA, [down arrow] HB
Bioallethrin     ND               [up arrow] MA, [down arrow] HB
Bioallethrin     ND               ND
S-Bioallethrin   ND               ND
Cyfluthrin       ND               In females, [up arrow] MA,
                                    [down arrow] HB
Cypermethin      ND               ND

Deltamethrin     ND               ND
Deltamethrin     ND               [down arrow] HB
Deltamethrin     0 MA, 0 HB       [up arrow] MA, [down arrow] HB
Deltamethrin     ND               ND
Deltamethrin     ND               ND
Fenvalerate      ND               ND

Compound (b)             Reference

d-Allethrin      Ivens et al., unpublished data
d-Allethrin      Tsuji et al. 2002
Bioallethrin     Muhammad and Ray, unpublished data
Bioallethrin     Talts et al. 1998
Bioallethrin/
  bioallethrin
Bioallethrin     Eriksson and Fredriksson 1991
Bioallethrin     Ahlbom et al. 1994
Bioallethrin     Eriksson and Nordberg 1990
S-Bioallethrin   Pauluhn and Schmuck 2003
Cyfluthrin       Jekat et al., unpublished data
Cypermethin      Malaviya et al. 1993

Deltamethrin     Aziz et al. 2001
Deltamethrin     Muhammad and Ray, unpublished data
Deltamethrin     Friksson and Fredriksson 1991
Deltamethrin     Eriksson and Nordberg 1990
Deltamethrin     Pauluhn and Schmuck 2003
Fenvalerate      Malaviya et al. 1993

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 for complete details.