Relative Potencies for Acute Effects of Pyrethroids on Motor Function
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《毒物学科学杂志》
National Research Council, Research Triangle Park, North Carolina 27711
Solveritas, LLC, Virginia Bio-Technology Research Park, Richmond VA 23219
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
The prevalence of pyrethroids in insecticide formulations has increased in the last decade. A common mode-of-action has been proposed for pyrethroids based on in vitro studies, which includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of membrane polarization, and abnormal discharge in targeted neurons. The objective of this work was to characterize individual dose-response curves for in vivo motor function and calculate relative potencies for eleven commonly used pyrethroids. Acute oral dose-response functions were determined in adult male Long Evans rats for five Type I (bifenthrin, S-bioallethrin, permethrin, resmethrin, tefluthrin), five Type II (-cyfluthrin, -cyhalothrin, cypermethrin, deltamethrin, esfenvalerate) and one mixed Type I/II (fenpropathrin) pyrethroids (n = 8–18 per dose; 6–11 dose levels per chemical, vehicle = corn oil, at 1 ml/kg). Motor function was measured using figure-8 mazes. Animals were tested for 1 h during the period of peak effects. All pyrethroids, regardless of structural class, produced dose-dependent decreases in motor activity. Relative potencies were calculated based on the computed ED30s. Deltamethrin, with an ED30 of 2.51 mg/kg, was chosen as the index chemical. Relative potency ratios ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). Additional work with environmentally-based mixtures is needed to test the hypothesis of dose-additivity of pyrethroids.
Key Words: pyrethroids; motor function, relative potency.
INTRODUCTION
Pyrethrins are derived from the flowers of Chrysanthemum cinerariaefolium that have been used as insecticides for more than a century (LaForge and Markwood, 1938). Pyrethroids are structural derivatives of pyrethrins that have greater potency and environmental stability (Casida, 1980; Elliott, 1978). Pyrethroids and pyrethrins are used in a wide array of indoor and outdoor applications, including medicinal, veterinary, and agricultural usages (ATSDR, 2003). Pyrethroid usage has been estimated at 23% of the worldwide insecticide market (Casida and Quistad, 1998). Agricultural and home use of pyrethroids is increasing, partly due to phase-outs of older insecticides (Amweg et al., 2005).
Pyrethroids are classified as Type I or Type II according to both chemical structure and biological effects of high-dose acute exposures (Gammon et al., 1981; Gray, 1985; Lawrence and Casida, 1982; Verschoyle and Aldridge, 1972, 1980). Compounds lacking an -cyano group on the phenoxybenzyl moiety produce toxic signs characterized by aggressive sparring and tremors (Type I, or T-syndrome). The presence of an -cyano group on the phenoxybenzyl moiety leads to a syndrome characterized by choreoathetosis and salivation (Type II, or CS-syndrome). There are a few compounds with mixed signs, including both tremors and salivation (Gammon et al., 1981; Lawrence and Casida, 1982; Verschoyle and Aldridge, 1980). Accordingly, these compounds have been labeled Type I/II or TS.
Pyrethroids act primarily on the nervous system. The commonly accepted mechanism-of-action of pyrethroids is the prolongation of the open state of voltage-dependent sodium channels in nervous tissue (Narahashi, 2000; Soderlund et al., 2002; Vijverberg and van den Bercken, 1990). These altered sodium channels result in repetitive firing or depolarizing block of the neuron, depending on how long the channel open state is prolonged (Soderlund et al., 2002; Narahashi, 2000). Other channel and receptor systems in neuronal tissues have been proposed to play a role in the generation of compound-specific clinical symptoms in mammals, including calcium channels and GABAA receptors (Crofton and Reiter, 1987; Hildebrand et al., 2004; Lawrence and Casida, 1983; Soderlund et al., 2002). However, the role of these other channels or receptors in the action of pyrethroids is not well established (Ogata et al., 1988; Shafer and Meyer, 2004; Soderlund et al., 2002).
The Food Quality Protection Act (FQPA), promulgated in 1996, requires the U.S. EPA to consider the cumulative toxicity of pesticides having a common mode-of-action. Risk-assessment approaches to additivity assume, where data are lacking, that chemicals with similar modes-of-action act in a dose-additive fashion (U.S. EPA, 1986, 2000). There are currently no published data on the additivity, or lack thereof, for pyrethroids. In addition, there is debate about whether a common mode-of-action exists for all pyrethroids (Soderlund et al., 2002). Research needs to reduce uncertainty in pyrethroid cumulative risk assessments include: (1) dose-response functions for individual pyrethroids, (2) calculation of relative potencies, and (3) studies that test additivity of relevant mixtures of pyrethroids. This paper addressed the first two items. We characterized dose-responses for the acute effects of 11 pyrethroids on motor function. A simple assessment of motor activity was used, as this behavior has been extensively characterized for a number of pyrethroids (Crofton et al., 1995; Crofton and Reiter, 1984, 1988; Hornychova et al., 1995; Hoy et al., 2000; McDaniel and Moser, 1993). These motor activity data were analyzed using a nonlinear exponential threshold model (Casey et al., 2004) to estimate ED30s (effective dose that produces a 30% decrease in activity) and threshold doses (the highest dose that has no effect on activity). The ED30s were used to calculate relative potencies (Safe, 1998; U.S. EPA, 2000; Villeneuve et al., 2000; Wilkinson et al., 2000). These data will be utilized in future work testing the assumption of dose-additivity for environmentally relevant mixtures of pyrethroids.
MATERIALS AND METHODS
Subjects.
Male Long Evans rats (CRL, Wilmington, MA) were obtained at 55–57 days of age, and were housed two per cage in standard polycarbonate hanging cages (45 cm x 24 cm x 20 cm) containing heat-sterilized pine shavings (Beta Chips, Northeastern Products, Inc., Warrensburg, NY). All animals were given a 5–9 day acclimation period and were maintained on a 12:12 h photoperiod (0600:1800). Food (Purina 5001 Lab Chow) and tap water were provided ad libitum. Colony rooms were maintained at 22.0 ± 2.0°C and relative humidity at 55 ± 20%. The facility is approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All experimental protocols were approved in advance by the National Health and Environmental Effects Research Laboratory's Animal Care and Use Committee.
Chemicals.
Eleven pyrethroids were tested. Table 1 lists the common names, chemical formulas, isomer composition, purity, molecular weights, LD50s, and the number of doses and dose ranges for each pyrethroid. Doses were calculated based on percent active ingredient in the technical product (purity is listed in Table 1). Pesticides were kindly supplied by their manufacturers: permethrin, bifenthrin, and cypermethrin (FMC Corporation, Philadelphia, PA); esfenvalerate (Dupont Crop Protection, Wilmington, DE); deltamethrin and -cyfluthrin (Bayer Cropscience, Research Triangle Park, NC); tefluthrin and -cyhalothrin (Syngenta Crop Protection, Greensboro, NC); and fenpropathrin, resmethrin, and S-bioallethrin (Valent USA Corporation, Walnut Creek, CA). Note that these pyrethroids were from the same lot # (or an equivalent lot having similar purity and isomer composition) as those used in the manufacturer-sponsored studies summarized in Soderlund et al. (2002). Pyrethroid dosing solutions were prepared daily by dissolving in corn oil (Sigma, Co., USA). An exception was -cyfluthrin, which was first dissolved in a small volume of acetone; then a measured volume of corn oil was added to obtain a stock solution based on final volume, with serial dilutions used to prepare final dose concentrations. Acetone was allowed to vaporize overnight within a fume hood in the dark before the solution was used the following morning. High concentrations of resmethrin (400 mg/ml) gradually precipitated over the course of a few hours; therefore these solutions were intermittently stirred and gently heated (40–50°C) to maintain solubility. Dosing solutions were used at room temperature.
Animal treatment.
Pyrethroids were administered by gavage in 1 ml/kg corn oil. Six to eleven doses were examined per compound, with dose groups balanced for time-of-day and test chamber. Dose selection was based on pilot studies, with the goal to have at least three no-effect levels. Prior to dosing, animals were moved from the colony room to an isolated dosing room within the testing laboratory. After a minimum 1 h acclimation, animals were removed from home cages, dosed, and then returned to the home cages until testing. Dose levels inducing excessive toxicity (i.e., leading to prolonged Type I or II clinical signs, or mortality) were not included in final experiments. This was done to ensure estimations of alterations in general motor function and not decreases due to excessive toxicity. For most cases, 8–18 animals per group were tested. Each experiment was divided into at least two blocks. Control animals (vehicle only) were included in each block. Nonintubated animals were included in some of the initial experiments to ensure a lack of effect of the vehicle and intubation procedures. All rats were randomly assigned to treatment groups and to individual mazes. Independent groups of rats were used for each experiment.
Motor activity.
Rats were placed into individual plastic cages with pine shavings and allowed to acclimate to the test room, which was maintained at the same environmental conditions as the animal colony and dosing room, for 5 min before being tested. Motor activity was measured for 1 h using 16 figure-eight mazes, each consisting of a series of interconnected alleys (10 x 10 cm) converging on a central arena and covered with transparent acrylic plastic (Norton et al., 1975; Reiter et al., 1975). Horizontal and vertical activity were detected by photo-transistor/photodiode pairs, eight equally spaced around the mazes at 0.5 in. above the floor (horizontal), and four pairs located 3 in. above the floor in the central arena. Photodetectors were sampled at a 1-kHz rate, and each time a photobeam was interrupted, an activity count was registered. Total activity was calculated as the sum of horizontal and vertical activity counts. Photobeam calibration was checked daily prior to testing. Maze assignments, order of testing, and time of day were counterbalanced across treatment groups. All testing was conducted between 0900 and 1700 h.
Testing was conducted at the time of peak effects. The time of peak effect for some pyrethroids was selected from previous work done under similar dosing and motor activity testing conditions (Crofton and Reiter, 1984, 1988; McDaniel and Moser, 1993). For other compounds, the time of peak effects was obtained from pilot time-course studies using motor activity testing or behavioral observations. Time from dosing to testing was as follows: 1 h, S-bioallethrin; 1.5 h, permethrin, cypermethrin; 2 h, -cyfluthrin, esfenvalerate, deltamethrin, tefluthrin and fenpropathrin; 2.5 h, -cyhalothrin; and 4 h, bifenthrin, resmethrin. All animals were observed before and after motor activity testing for signs of excessive toxicity.
Statistical analysis.
Activity data were analyzed using a nonlinear exponential threshold additivity model (Casey et al., 2004). The model is algebraically equivalent to the definition of additivity (i.e., zero interaction) given by Berenbaum (1985) and can be related to the isobologram for a combination of chemicals (Loewe, 1953) through the interaction index. This is the model that will be used to analyze data from future mixtures research. The method of maximum quasi-likelihood was used to estimate model parameters. The adequacy of the fit of the additivity model to the single chemical data was assessed graphically and through goodness-of-fit statistics. This additivity model was used to determine the dose associated with a 30% decrease in motor activity (ED30) for each pyrethroid. Approximate 95% confidence intervals were computed for the ED30s by applying the delta method. The nonlinear exponential threshold additivity model was also used to obtain the threshold dose and its 95% confidence intervals for each single compound. This threshold dose represents an estimate of the highest no-effect dose level at which treated rats would not display any decrease in motor activity. Relative potencies were calculated from ED30s using deltamethrin as the index chemical. Deltamethrin was chosen due to its extensive toxicological database and the reliability of effects on motor activity within this laboratory (Crofton et al., 1995; Crofton and Reiter, 1984, 1987; Gilbert et al., 1990).
RESULTS
Pilot studies provided dose ranges, excluding excessive toxicity, for use in the formal dose-response experiments. In the formal studies, no signs of excessive toxicity for most of the pyrethroids were observed with cage-side observations conducted before and after motor activity testing. Only in the case of tefluthrin (12 mg/kg), fenpropathrin (24 mg/kg), and permethrin (200 mg/kg) were excessive signs (i.e., prolonged (>4 h) clinical signs, or clear and complete Type I or Type II syndromes) seen at the highest doses and only with a small percentage of animals. These doses were not used in any data analyses.
All pyrethroids induced dose-dependent decreases in motor activity (Fig. 1). ED30s and threshold doses are listed in Table 2. ED30s varied by more than two orders of magnitude (Table 2) from the least (resmethrin) to the most potent (esfenvalerate) compound. Threshold doses for the eleven pyrethroids were approximately 39% of the ED30s (Table 2). The predictability of the model for each of the single chemical data showed significant Spearman's rho coefficients in all cases (mean = 0.68 range: 0.5–0.81). Table 3 lists estimated model parameters. All estimated parameters were significant. Relative potencies, calculated as ratio of the ED30 for the index chemical, deltamethrin (ED30 = 2.51 mg/kg), over each chemical's ED30, are seen in Table 4.
DISCUSSION
Motor activity was used as an endpoint to determine dose-response relationships after acute exposure to each of eleven pyrethroid insecticides. All pyrethroids tested produced dose-dependent decreases in motor activity. Relative potencies of the eleven pyrethroids spanned more than two orders of magnitude. The present findings confirm the generality of the motor-depressant effect of pyrethroids, expand these to include other pyrethroids, and more importantly, provide extensive dose-response data for each chemical.
Dose-dependent decreases in motor activity are consistent with a wide variety of previous reports on the acute effects of pyrethroids. Past work from this laboratory and others (Crofton et al., 1995; Crofton and Reiter, 1984, 1987, 1988; Gilbert et al., 1990; McDaniel and Moser, 1993) demonstrated decreased motor activity for five of the currently tested compounds using the same test method (i.e., figure-eight mazes). Decreases in motor activity have been demonstrated in other testing devices after acute or short-term exposure, including permethrin (Hoy et al., 2000), fenvalerate (De Souza Spinosa et al., 1999), cyhalothrin (Righi and Palermo-Neto, 2003), -cyhalothrin (Hornychova et al., 1995; Ratnasooriya et al., 2002), and cypermethrin (Hornychova et al., 1995). The acute motor depressant effect has been also observed in mice after oral exposure to fenvalerate (Mandhane and Chopde, 1997) and deltamethrin (Chanh et al., 1984).
There are a few reports of acute pyrethroid exposures resulting in increased motor activity. Increased motor activity was reported in mice acutely exposed to commercial formulations of fenvalerate and permethrin (Mitchell et al., 1988). Husain et al. (1996) found an increase in motor activity 1 day after a 15-day exposure to a deltamethrin formulation. These reports of increased activity are difficult to interpret and may have resulted from unknowns in the commercial formulations used. Mitchell et al. (1988) used commercial formulations where the pyrethroids were 30% or less of the administered product. Husain et al. (1996) used Decis, an emulsifiable formulation containing only 2.8% deltamethrin. Another report found no effect on motor activity in rats monitored using an automated open field following acute deltamethrin exposure (10 mg/kg) using a commercial formulation (Dayal et al., 2003). This negative finding may be due to use of a commercial product containing only 2.8% deltamethrin (Dayal et al., 2003), making the dose of deltamethrin used only 0.28 mg/kg. This dose falls well below the threshold dose (0.99 mg/kg) in the current study. The importance of precise reporting of the exact nature of the tested agent, as well as the potential confounds that result from other components in commercial formulations, has been previously noted (Shafer and Meyer, 2004; Shafer et al., 2005).
Potency estimates (ED30s) for the eleven pyrethroids ranged from 1.2 mg/kg for esfenvalerate to 292.8 mg/kg for resmethrin. Using deltamethrin as an index chemical, relative potencies ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). The model applied to fit the dose-response datasets also computed a threshold dose for each pyrethroid. Threshold doses for the eleven pyrethroids followed the same potency relationships calculated for the ED30s. This wide range of relative potencies is likely caused by a number of toxicokinetic and toxicodynamic factors. The presence of an -cyano group on the alcohol moiety of the pyrethroid confers increased potency in both insects and mammals (Soderlund et al., 2002; Valentine, 1990). This is evident comparing permethrin and cypermethrin (Tables 2 and 4). Another important factor in potency is the enrichment of active isomers in the technical product used (Glickman and Casida, 1982; Verschoyle and Barnes, 1972). Pyrethroid structures include chiral carbons (usually two to three). The activity of these analogs is highly dependent on the stereoisomeric configuration of the molecule and the rate of degradation by metabolizing enzymes (Glickman and Casida, 1982). Pyrethroids in the cis- configurations (i.e., deltamethrin, -cyhalothrin, tefluthrin, and bifenthrin) are more potent than those in the trans- configuration (Verschoyle and Aldridge, 1980). Thus, the applicability of relative potencies will be dependent on the isomeric composition of the test material.
The use of the relative potencies reported here to calculate cumulative risks should be tempered by uncertainties. The first uncertainty is that the relative potencies for motor activity may not predict all behavioral effects of pyrethroids. With only a few exceptions, high doses (i.e., lethal) of pyrethroids are well known to produce two very different syndromes of toxicity (Verschoyle and Aldridge, 1980). In addition, the acoustic startle response, a simple sensory-evoked motor reflex (Davis et al., 1982), is differentially affected by some pyrethroids. Depending on structure, pyrethroids may increase or decrease this reflex behavior (Crofton and Reiter, 1984, 1988; Hijzen et al., 1988; Hijzen and Slangen, 1988). Changes in motor activity have long been used in risk assessment and critically evaluated concerning specificity and reliability (Crofton et al., 1991; Gerber and O'Shaughnessy, 1986; Kulig et al., 1996; MacPhail et al., 1989; Reiter and MacPhail, 1982; Stanton, 1994). Motor activity, like many behavioral functions, can be altered in both humans and laboratory animals by a wide variety of drugs and toxicants (Crofton et al., 1991; MacPhail et al., 1989; Tyron, 1985). Another uncertainty in the use of the present data in cumulative risk derives from the lack of data on the combined action of pyrethroid insecticides on any aspects of nervous system function. Predicting the effects of mixtures based on data from individual chemicals is difficult (Borgert et al., 2004; Teuschler et al., 2002; Wilkinson et al., 2000). Concurrent exposures may interfere with the metabolism and kinetics of each individual chemical and/or its metabolites (Aldridge, 1990; Wilkinson et al., 2000). Pyrethroid metabolic pathways have both common and compound-specific steps (Roberts and Hutson, 1998), and the toxicity of pyrethroid metabolites remains poorly evaluated (Beres et al., 2000; NRCC, 1986). In addition, the severity of the effects of pyrethroids is influenced by route of exposure, vehicle, and dosing volume (Crofton et al., 1995; Nishimura et al., 1984; Soderlund et al., 2002; Verschoyle and Aldridge, 1980). Furthermore, these acute data may or may not predict effects of longer-term exposures. Extrapolation of acute neurotoxicity findings can be difficult on both the quantitative and qualitative level (Bass et al., 1985). Thus, the assumption of dose-additivity should be empirically tested before ruling out antagonistic or synergistic effects (Borgert et al., 2004). Future work should include assessing other behavioral endpoints, and testing the hypothesis of additivity for mixtures of pyrethroids.
NOTES
The information in this document has been funded wholly (or in part) by the U.S. Environmental Protection Agency. This manuscript has been reviewed following the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
The authors thank the following for help obtaining pyrethroid samples: Drs. Dana Sargent, Paul Parsons, Larry Sheets, Patricia Devine, Janice Sharp, and Myra Weiner. Drs. William Sette and Tim Shafer are gratefully acknowledged for helpful comments on an earlier version of this manuscript. Thanks to Josh Harrill for help with pyrethroid structural chemistry.
Note: All raw data used in this manuscript are freely available for alternative analyses. Please direct requests to the corresponding author.
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Solveritas, LLC, Virginia Bio-Technology Research Park, Richmond VA 23219
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
The prevalence of pyrethroids in insecticide formulations has increased in the last decade. A common mode-of-action has been proposed for pyrethroids based on in vitro studies, which includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of membrane polarization, and abnormal discharge in targeted neurons. The objective of this work was to characterize individual dose-response curves for in vivo motor function and calculate relative potencies for eleven commonly used pyrethroids. Acute oral dose-response functions were determined in adult male Long Evans rats for five Type I (bifenthrin, S-bioallethrin, permethrin, resmethrin, tefluthrin), five Type II (-cyfluthrin, -cyhalothrin, cypermethrin, deltamethrin, esfenvalerate) and one mixed Type I/II (fenpropathrin) pyrethroids (n = 8–18 per dose; 6–11 dose levels per chemical, vehicle = corn oil, at 1 ml/kg). Motor function was measured using figure-8 mazes. Animals were tested for 1 h during the period of peak effects. All pyrethroids, regardless of structural class, produced dose-dependent decreases in motor activity. Relative potencies were calculated based on the computed ED30s. Deltamethrin, with an ED30 of 2.51 mg/kg, was chosen as the index chemical. Relative potency ratios ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). Additional work with environmentally-based mixtures is needed to test the hypothesis of dose-additivity of pyrethroids.
Key Words: pyrethroids; motor function, relative potency.
INTRODUCTION
Pyrethrins are derived from the flowers of Chrysanthemum cinerariaefolium that have been used as insecticides for more than a century (LaForge and Markwood, 1938). Pyrethroids are structural derivatives of pyrethrins that have greater potency and environmental stability (Casida, 1980; Elliott, 1978). Pyrethroids and pyrethrins are used in a wide array of indoor and outdoor applications, including medicinal, veterinary, and agricultural usages (ATSDR, 2003). Pyrethroid usage has been estimated at 23% of the worldwide insecticide market (Casida and Quistad, 1998). Agricultural and home use of pyrethroids is increasing, partly due to phase-outs of older insecticides (Amweg et al., 2005).
Pyrethroids are classified as Type I or Type II according to both chemical structure and biological effects of high-dose acute exposures (Gammon et al., 1981; Gray, 1985; Lawrence and Casida, 1982; Verschoyle and Aldridge, 1972, 1980). Compounds lacking an -cyano group on the phenoxybenzyl moiety produce toxic signs characterized by aggressive sparring and tremors (Type I, or T-syndrome). The presence of an -cyano group on the phenoxybenzyl moiety leads to a syndrome characterized by choreoathetosis and salivation (Type II, or CS-syndrome). There are a few compounds with mixed signs, including both tremors and salivation (Gammon et al., 1981; Lawrence and Casida, 1982; Verschoyle and Aldridge, 1980). Accordingly, these compounds have been labeled Type I/II or TS.
Pyrethroids act primarily on the nervous system. The commonly accepted mechanism-of-action of pyrethroids is the prolongation of the open state of voltage-dependent sodium channels in nervous tissue (Narahashi, 2000; Soderlund et al., 2002; Vijverberg and van den Bercken, 1990). These altered sodium channels result in repetitive firing or depolarizing block of the neuron, depending on how long the channel open state is prolonged (Soderlund et al., 2002; Narahashi, 2000). Other channel and receptor systems in neuronal tissues have been proposed to play a role in the generation of compound-specific clinical symptoms in mammals, including calcium channels and GABAA receptors (Crofton and Reiter, 1987; Hildebrand et al., 2004; Lawrence and Casida, 1983; Soderlund et al., 2002). However, the role of these other channels or receptors in the action of pyrethroids is not well established (Ogata et al., 1988; Shafer and Meyer, 2004; Soderlund et al., 2002).
The Food Quality Protection Act (FQPA), promulgated in 1996, requires the U.S. EPA to consider the cumulative toxicity of pesticides having a common mode-of-action. Risk-assessment approaches to additivity assume, where data are lacking, that chemicals with similar modes-of-action act in a dose-additive fashion (U.S. EPA, 1986, 2000). There are currently no published data on the additivity, or lack thereof, for pyrethroids. In addition, there is debate about whether a common mode-of-action exists for all pyrethroids (Soderlund et al., 2002). Research needs to reduce uncertainty in pyrethroid cumulative risk assessments include: (1) dose-response functions for individual pyrethroids, (2) calculation of relative potencies, and (3) studies that test additivity of relevant mixtures of pyrethroids. This paper addressed the first two items. We characterized dose-responses for the acute effects of 11 pyrethroids on motor function. A simple assessment of motor activity was used, as this behavior has been extensively characterized for a number of pyrethroids (Crofton et al., 1995; Crofton and Reiter, 1984, 1988; Hornychova et al., 1995; Hoy et al., 2000; McDaniel and Moser, 1993). These motor activity data were analyzed using a nonlinear exponential threshold model (Casey et al., 2004) to estimate ED30s (effective dose that produces a 30% decrease in activity) and threshold doses (the highest dose that has no effect on activity). The ED30s were used to calculate relative potencies (Safe, 1998; U.S. EPA, 2000; Villeneuve et al., 2000; Wilkinson et al., 2000). These data will be utilized in future work testing the assumption of dose-additivity for environmentally relevant mixtures of pyrethroids.
MATERIALS AND METHODS
Subjects.
Male Long Evans rats (CRL, Wilmington, MA) were obtained at 55–57 days of age, and were housed two per cage in standard polycarbonate hanging cages (45 cm x 24 cm x 20 cm) containing heat-sterilized pine shavings (Beta Chips, Northeastern Products, Inc., Warrensburg, NY). All animals were given a 5–9 day acclimation period and were maintained on a 12:12 h photoperiod (0600:1800). Food (Purina 5001 Lab Chow) and tap water were provided ad libitum. Colony rooms were maintained at 22.0 ± 2.0°C and relative humidity at 55 ± 20%. The facility is approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All experimental protocols were approved in advance by the National Health and Environmental Effects Research Laboratory's Animal Care and Use Committee.
Chemicals.
Eleven pyrethroids were tested. Table 1 lists the common names, chemical formulas, isomer composition, purity, molecular weights, LD50s, and the number of doses and dose ranges for each pyrethroid. Doses were calculated based on percent active ingredient in the technical product (purity is listed in Table 1). Pesticides were kindly supplied by their manufacturers: permethrin, bifenthrin, and cypermethrin (FMC Corporation, Philadelphia, PA); esfenvalerate (Dupont Crop Protection, Wilmington, DE); deltamethrin and -cyfluthrin (Bayer Cropscience, Research Triangle Park, NC); tefluthrin and -cyhalothrin (Syngenta Crop Protection, Greensboro, NC); and fenpropathrin, resmethrin, and S-bioallethrin (Valent USA Corporation, Walnut Creek, CA). Note that these pyrethroids were from the same lot # (or an equivalent lot having similar purity and isomer composition) as those used in the manufacturer-sponsored studies summarized in Soderlund et al. (2002). Pyrethroid dosing solutions were prepared daily by dissolving in corn oil (Sigma, Co., USA). An exception was -cyfluthrin, which was first dissolved in a small volume of acetone; then a measured volume of corn oil was added to obtain a stock solution based on final volume, with serial dilutions used to prepare final dose concentrations. Acetone was allowed to vaporize overnight within a fume hood in the dark before the solution was used the following morning. High concentrations of resmethrin (400 mg/ml) gradually precipitated over the course of a few hours; therefore these solutions were intermittently stirred and gently heated (40–50°C) to maintain solubility. Dosing solutions were used at room temperature.
Animal treatment.
Pyrethroids were administered by gavage in 1 ml/kg corn oil. Six to eleven doses were examined per compound, with dose groups balanced for time-of-day and test chamber. Dose selection was based on pilot studies, with the goal to have at least three no-effect levels. Prior to dosing, animals were moved from the colony room to an isolated dosing room within the testing laboratory. After a minimum 1 h acclimation, animals were removed from home cages, dosed, and then returned to the home cages until testing. Dose levels inducing excessive toxicity (i.e., leading to prolonged Type I or II clinical signs, or mortality) were not included in final experiments. This was done to ensure estimations of alterations in general motor function and not decreases due to excessive toxicity. For most cases, 8–18 animals per group were tested. Each experiment was divided into at least two blocks. Control animals (vehicle only) were included in each block. Nonintubated animals were included in some of the initial experiments to ensure a lack of effect of the vehicle and intubation procedures. All rats were randomly assigned to treatment groups and to individual mazes. Independent groups of rats were used for each experiment.
Motor activity.
Rats were placed into individual plastic cages with pine shavings and allowed to acclimate to the test room, which was maintained at the same environmental conditions as the animal colony and dosing room, for 5 min before being tested. Motor activity was measured for 1 h using 16 figure-eight mazes, each consisting of a series of interconnected alleys (10 x 10 cm) converging on a central arena and covered with transparent acrylic plastic (Norton et al., 1975; Reiter et al., 1975). Horizontal and vertical activity were detected by photo-transistor/photodiode pairs, eight equally spaced around the mazes at 0.5 in. above the floor (horizontal), and four pairs located 3 in. above the floor in the central arena. Photodetectors were sampled at a 1-kHz rate, and each time a photobeam was interrupted, an activity count was registered. Total activity was calculated as the sum of horizontal and vertical activity counts. Photobeam calibration was checked daily prior to testing. Maze assignments, order of testing, and time of day were counterbalanced across treatment groups. All testing was conducted between 0900 and 1700 h.
Testing was conducted at the time of peak effects. The time of peak effect for some pyrethroids was selected from previous work done under similar dosing and motor activity testing conditions (Crofton and Reiter, 1984, 1988; McDaniel and Moser, 1993). For other compounds, the time of peak effects was obtained from pilot time-course studies using motor activity testing or behavioral observations. Time from dosing to testing was as follows: 1 h, S-bioallethrin; 1.5 h, permethrin, cypermethrin; 2 h, -cyfluthrin, esfenvalerate, deltamethrin, tefluthrin and fenpropathrin; 2.5 h, -cyhalothrin; and 4 h, bifenthrin, resmethrin. All animals were observed before and after motor activity testing for signs of excessive toxicity.
Statistical analysis.
Activity data were analyzed using a nonlinear exponential threshold additivity model (Casey et al., 2004). The model is algebraically equivalent to the definition of additivity (i.e., zero interaction) given by Berenbaum (1985) and can be related to the isobologram for a combination of chemicals (Loewe, 1953) through the interaction index. This is the model that will be used to analyze data from future mixtures research. The method of maximum quasi-likelihood was used to estimate model parameters. The adequacy of the fit of the additivity model to the single chemical data was assessed graphically and through goodness-of-fit statistics. This additivity model was used to determine the dose associated with a 30% decrease in motor activity (ED30) for each pyrethroid. Approximate 95% confidence intervals were computed for the ED30s by applying the delta method. The nonlinear exponential threshold additivity model was also used to obtain the threshold dose and its 95% confidence intervals for each single compound. This threshold dose represents an estimate of the highest no-effect dose level at which treated rats would not display any decrease in motor activity. Relative potencies were calculated from ED30s using deltamethrin as the index chemical. Deltamethrin was chosen due to its extensive toxicological database and the reliability of effects on motor activity within this laboratory (Crofton et al., 1995; Crofton and Reiter, 1984, 1987; Gilbert et al., 1990).
RESULTS
Pilot studies provided dose ranges, excluding excessive toxicity, for use in the formal dose-response experiments. In the formal studies, no signs of excessive toxicity for most of the pyrethroids were observed with cage-side observations conducted before and after motor activity testing. Only in the case of tefluthrin (12 mg/kg), fenpropathrin (24 mg/kg), and permethrin (200 mg/kg) were excessive signs (i.e., prolonged (>4 h) clinical signs, or clear and complete Type I or Type II syndromes) seen at the highest doses and only with a small percentage of animals. These doses were not used in any data analyses.
All pyrethroids induced dose-dependent decreases in motor activity (Fig. 1). ED30s and threshold doses are listed in Table 2. ED30s varied by more than two orders of magnitude (Table 2) from the least (resmethrin) to the most potent (esfenvalerate) compound. Threshold doses for the eleven pyrethroids were approximately 39% of the ED30s (Table 2). The predictability of the model for each of the single chemical data showed significant Spearman's rho coefficients in all cases (mean = 0.68 range: 0.5–0.81). Table 3 lists estimated model parameters. All estimated parameters were significant. Relative potencies, calculated as ratio of the ED30 for the index chemical, deltamethrin (ED30 = 2.51 mg/kg), over each chemical's ED30, are seen in Table 4.
DISCUSSION
Motor activity was used as an endpoint to determine dose-response relationships after acute exposure to each of eleven pyrethroid insecticides. All pyrethroids tested produced dose-dependent decreases in motor activity. Relative potencies of the eleven pyrethroids spanned more than two orders of magnitude. The present findings confirm the generality of the motor-depressant effect of pyrethroids, expand these to include other pyrethroids, and more importantly, provide extensive dose-response data for each chemical.
Dose-dependent decreases in motor activity are consistent with a wide variety of previous reports on the acute effects of pyrethroids. Past work from this laboratory and others (Crofton et al., 1995; Crofton and Reiter, 1984, 1987, 1988; Gilbert et al., 1990; McDaniel and Moser, 1993) demonstrated decreased motor activity for five of the currently tested compounds using the same test method (i.e., figure-eight mazes). Decreases in motor activity have been demonstrated in other testing devices after acute or short-term exposure, including permethrin (Hoy et al., 2000), fenvalerate (De Souza Spinosa et al., 1999), cyhalothrin (Righi and Palermo-Neto, 2003), -cyhalothrin (Hornychova et al., 1995; Ratnasooriya et al., 2002), and cypermethrin (Hornychova et al., 1995). The acute motor depressant effect has been also observed in mice after oral exposure to fenvalerate (Mandhane and Chopde, 1997) and deltamethrin (Chanh et al., 1984).
There are a few reports of acute pyrethroid exposures resulting in increased motor activity. Increased motor activity was reported in mice acutely exposed to commercial formulations of fenvalerate and permethrin (Mitchell et al., 1988). Husain et al. (1996) found an increase in motor activity 1 day after a 15-day exposure to a deltamethrin formulation. These reports of increased activity are difficult to interpret and may have resulted from unknowns in the commercial formulations used. Mitchell et al. (1988) used commercial formulations where the pyrethroids were 30% or less of the administered product. Husain et al. (1996) used Decis, an emulsifiable formulation containing only 2.8% deltamethrin. Another report found no effect on motor activity in rats monitored using an automated open field following acute deltamethrin exposure (10 mg/kg) using a commercial formulation (Dayal et al., 2003). This negative finding may be due to use of a commercial product containing only 2.8% deltamethrin (Dayal et al., 2003), making the dose of deltamethrin used only 0.28 mg/kg. This dose falls well below the threshold dose (0.99 mg/kg) in the current study. The importance of precise reporting of the exact nature of the tested agent, as well as the potential confounds that result from other components in commercial formulations, has been previously noted (Shafer and Meyer, 2004; Shafer et al., 2005).
Potency estimates (ED30s) for the eleven pyrethroids ranged from 1.2 mg/kg for esfenvalerate to 292.8 mg/kg for resmethrin. Using deltamethrin as an index chemical, relative potencies ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). The model applied to fit the dose-response datasets also computed a threshold dose for each pyrethroid. Threshold doses for the eleven pyrethroids followed the same potency relationships calculated for the ED30s. This wide range of relative potencies is likely caused by a number of toxicokinetic and toxicodynamic factors. The presence of an -cyano group on the alcohol moiety of the pyrethroid confers increased potency in both insects and mammals (Soderlund et al., 2002; Valentine, 1990). This is evident comparing permethrin and cypermethrin (Tables 2 and 4). Another important factor in potency is the enrichment of active isomers in the technical product used (Glickman and Casida, 1982; Verschoyle and Barnes, 1972). Pyrethroid structures include chiral carbons (usually two to three). The activity of these analogs is highly dependent on the stereoisomeric configuration of the molecule and the rate of degradation by metabolizing enzymes (Glickman and Casida, 1982). Pyrethroids in the cis- configurations (i.e., deltamethrin, -cyhalothrin, tefluthrin, and bifenthrin) are more potent than those in the trans- configuration (Verschoyle and Aldridge, 1980). Thus, the applicability of relative potencies will be dependent on the isomeric composition of the test material.
The use of the relative potencies reported here to calculate cumulative risks should be tempered by uncertainties. The first uncertainty is that the relative potencies for motor activity may not predict all behavioral effects of pyrethroids. With only a few exceptions, high doses (i.e., lethal) of pyrethroids are well known to produce two very different syndromes of toxicity (Verschoyle and Aldridge, 1980). In addition, the acoustic startle response, a simple sensory-evoked motor reflex (Davis et al., 1982), is differentially affected by some pyrethroids. Depending on structure, pyrethroids may increase or decrease this reflex behavior (Crofton and Reiter, 1984, 1988; Hijzen et al., 1988; Hijzen and Slangen, 1988). Changes in motor activity have long been used in risk assessment and critically evaluated concerning specificity and reliability (Crofton et al., 1991; Gerber and O'Shaughnessy, 1986; Kulig et al., 1996; MacPhail et al., 1989; Reiter and MacPhail, 1982; Stanton, 1994). Motor activity, like many behavioral functions, can be altered in both humans and laboratory animals by a wide variety of drugs and toxicants (Crofton et al., 1991; MacPhail et al., 1989; Tyron, 1985). Another uncertainty in the use of the present data in cumulative risk derives from the lack of data on the combined action of pyrethroid insecticides on any aspects of nervous system function. Predicting the effects of mixtures based on data from individual chemicals is difficult (Borgert et al., 2004; Teuschler et al., 2002; Wilkinson et al., 2000). Concurrent exposures may interfere with the metabolism and kinetics of each individual chemical and/or its metabolites (Aldridge, 1990; Wilkinson et al., 2000). Pyrethroid metabolic pathways have both common and compound-specific steps (Roberts and Hutson, 1998), and the toxicity of pyrethroid metabolites remains poorly evaluated (Beres et al., 2000; NRCC, 1986). In addition, the severity of the effects of pyrethroids is influenced by route of exposure, vehicle, and dosing volume (Crofton et al., 1995; Nishimura et al., 1984; Soderlund et al., 2002; Verschoyle and Aldridge, 1980). Furthermore, these acute data may or may not predict effects of longer-term exposures. Extrapolation of acute neurotoxicity findings can be difficult on both the quantitative and qualitative level (Bass et al., 1985). Thus, the assumption of dose-additivity should be empirically tested before ruling out antagonistic or synergistic effects (Borgert et al., 2004). Future work should include assessing other behavioral endpoints, and testing the hypothesis of additivity for mixtures of pyrethroids.
NOTES
The information in this document has been funded wholly (or in part) by the U.S. Environmental Protection Agency. This manuscript has been reviewed following the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
ACKNOWLEDGMENTS
The authors thank the following for help obtaining pyrethroid samples: Drs. Dana Sargent, Paul Parsons, Larry Sheets, Patricia Devine, Janice Sharp, and Myra Weiner. Drs. William Sette and Tim Shafer are gratefully acknowledged for helpful comments on an earlier version of this manuscript. Thanks to Josh Harrill for help with pyrethroid structural chemistry.
Note: All raw data used in this manuscript are freely available for alternative analyses. Please direct requests to the corresponding author.
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