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Evidence for Dose-Additive Effects of Pyrethroids on Motor Activity in Rats
Marcelo J. Wolansky,1 Chris Gennings,2 Michael J. DeVito,3 and Kevin M. Crofton4
1Departamento de Química Biológica (Área Toxicología), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Buenos Aires, Argentina; 2Solveritas, LLC, Richmond, Virginia, USA; 3Division of Experimental Toxicology, and 4Division of Neurotoxicology, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA are associated with two high-dose neuro logic Background: Pyrethroids are neurotoxic insecticides used in a variety of indoor and outdoor
syndromes, T syndrome and the CS syndrome applications. Previous research characterized the acute dose–effect functions for 11 pyrethroids
(Aldridge 1990; Barnes and Verschoyle 1974; administered orally in corn oil (1 mL/kg) based on assessment of motor activity.
Lawrence and Casida 1982; Ray and Forshaw oBjectives: We used a mixture of these 11 pyrethroids and the same testing paradigm used in
2000; Ray and Fry 2006; Verschoyle and single-compound assays to test the hypothesis that cumulative neurotoxic effects of pyrethroid mix-
Aldridge 1980), and dose-dependent changes tures can be predicted using the default dose–addition theory.
in motor and sensory behaviors at lower doses Methods: Mixing ratios of the 11 pyrethroids in the tested mixture were based on the ED30 (effec-
(Chanh et al. 1984; Crofton and Reiter 1984 tive dose that produces a 30% decrease in response) of the individual chemical (i.e., the mixture
1988; Hornychova et al. 1995; McDaniel and comprised equi potent amounts of each pyrethroid). The highest concentration of each individual
Moser 1993; Nishimura et al. 1984; Wolansky chemical in the mixture was less than the threshold for inducing behavioral effects. Adult male rats
et al. 2006; Wolansky and Harrill 2008).
received acute oral exposure to corn oil (control) or dilutions of the stock mixture solution. The
Currently, the link between alterations mixture of 11 pyrethroids was administered either simultaneously (2 hr before testing) or after a
in neuronal firing and downstream neuro- sequence based on times of peak effect for the individual chemicals (4, 2, and 1 hr before testing). A
threshold additivity model was fit to the single-chemical data to predict the theoretical dose–effect
behavioral syndromes is correlative and not relationship for the mixture under the assumption of dose additivity.
causative. Mechanistic pathways linking sites of action (e.g., sodium channels) to neurologic results: When subthreshold doses of individual chemicals were combined in the mixtures, we
found significant dose-related decreases in motor activity. Further, we found no departure from the
outcomes have not been fully elucidated (Gray predicted dose-additive curve regardless of the mixture dosing protocol used.
1985; Ray and Fry 2006; Shafer et al. 2005; Soderlund et al. 2002). This has resulted in conclusion: In this article we present the first in vivo evidence on pyrethroid cumulative effects
supporting the default assumption of dose addition.
uncertainty about whether a common mecha- nism of toxicity exists for all pyrethroids key words: additivity, cumulative, mixtures, neurotoxicity, pyrethroids. Environ Health Perspect
(Soderlund et al. 2002). Pyrethroid actions 117:1563–1570 (2009). doi:10.1289/ehp.0900667 available via http://dx.doi.org/ [Online 8 June
on many other neuronal target sites have been reported and include calcium (Ca++), potassium (K+), and chloride (Cl–) channels (Burr and Pyrethroids are synthetic insecticides derived cumulative risk of chemicals having a common Ray 2004; Lawrence and Casida 1982; Ray and from pyrethrins (Casida 1980; Elliott 1978). "mechanism of toxicity." For chemicals consid- Fry 2006; Shafer and Meyer 2004). Although Pyrethroids are increasingly used in a wide ered to have a common mechanism of toxicity those mechanisms of action are not as well array of pesticide applications, including (commonality of target tissue, target site, and established (Shafer and Meyer 2004) as actions veteri nary, agriculture, and home pest control primary toxicologic effects for the members of on Na+ channels, alterations in these ion chan- (Amweg et al. 2005). Recent reports indicate a chemical class), dose additivity is the default nels will also disrupt neuronal firing rates.
low-level exposure to multiple pyrethroids hypothe sis for assessing the hazard of mixtures No published data exist to determine in humans (Becker et al. 2006; Fortin et al. (U.S. EPA 1999, 2002a). The overall assump- whether dose addition predicts the effects 2008; Heudorf et al. 2004; Lu et al. 2006, tion under dose additivity is that the toxicity of the combined exposure to pyrethroids 2009; Morgan et al. 2007; Riederer et al. of each component of the mixture behaves 2008; Tulve et al. 2006). Pyrethroids have as a known dilution of a reference chemical Address correspondence to K.M. Crofton, been classified as type I or type II based on selected as the index compound (U.S. EPA Neurotoxicology Division, MD-B105-04, NHEERL, acute high-dose biological effects and chemical 2000). This approach was used for the cumula- U.S. EPA, Research Triangle Park, NC 27711 USA. structure (Gammon et al. 1981; Gray 1985; tive risk assessment of cholinesterase-inhibiting Telephone: (919) 541-2672. Fax: (919) 541-4849. Lawrence and Casida 1982; Verschoyle and organo phosphate (U.S. EPA 2002b) and car- Aldridge 1972, 1980). Type I compounds lack bamate (U.S. EPA 2007) pesticides. The pres- Supplemental Material is available online an α-cyano group on the phenoxy benzyl moi- ent study was part of a larger research effort to (doi:10.1289/ehp.0900667.S1 via http://dx.doi.org/). Raw data used in this study are available for alter- ety and produce toxic signs charac terized by determine whether or not pyrethroid pesticides native analyses from the corresponding author.
aggressive sparring and tremors (T syndrome). share a common mechanism and may thus be We thank D. Sargent, P. Parsons, L. Sheets, Type II compounds contain an α-cyano group subject to cumulative risk assessment.
P. Devine, J. Sharp, and M. Weiner for pyrethroid on the phenoxybenzyl moiety, and acute expo- Pyrethroids act primarily on the nervous samples. We gratefully acknowledge A. Lowit, sures produce a syndrome characterized by system (Narahashi 2000; Soderlund et al. T. Shafer, L. Teuschler, and L. Sheets for comments choreoathetosis and salivation (CS syndrome). 2002). A proposed mechanism of action for on an earlier version of the manuscript. This article has been reviewed by the National A limited number of pyrethroids elicit both all pyrethroids is the prolongation of the open Health and Environmental Effects Research tremors and salivation (Gammon et al. 1981; state of neuronal voltage-dependent sodium Laboratory, U.S. Environmental Protection Agency, Lawrence and Casida 1982; Verschoyle and channels (Narahashi 1971; Vijverberg and and approved for publication. Mention of trade Aldridge 1980) and have been classified as van den Bercken 1990). This action results in names or commercial products does not constitute type I/II or TS syndrome compounds.
altered neuronal excitability characterized by endorsement or recommendation for use.
The Food Quality Protection Act (FQPA in vitro and in vivo changes in neuronal fir- C.G. is a founder and principal in Solveritas, LLC. The other authors declare they have no competing 1996) requires the U.S. Environmental ing rates (e.g., repetitive firing or depolarizing financial interests.
Protection Agency (EPA) to consider the block of the neuron) (Narahashi 2000) that Received 6 February 2009; accepted 8 June 2009.
Environmental Health Perspectives • volume 117 number 10 October 2009 Wolansky et al.
in mammals. Some in vitro evidence on et al. 1981; Righi and Palermo-Neto 2003; on a 12:12-hr photo period (0600:1800 hours). the action of pyrethroid mixtures has been Wolansky et al. 2006). In addition, recent Food (Purina 5001 Lab Chow; Ralston-Purina, reported. Whole-cell and patch-clamp assays research has characterized extensive dose–effect St. Louis, MO, USA) and tap water were in cultured neurons from rat suggest that functions for 11 pyrethroids on motor activ- provided ad libitum. Tap water (city water; type I and type II pyrethroids interact with ity. Data from these 11 compounds were used Durham, NC, USA) was filtered through Na+ channel binding sites by either com- to compute ED30 values (dose that decreases sand and activated charcoal filters and then petitive or allo steric actions (Motomura and activity by 30%) and relative potencies rechlorinated to 4–5 ppm Cl– before use. Narahashi 2001; Song and Narahashi 1996). (Wolansky et al. 2006). In the present study, Colony rooms were maintained at 22 ± 2°C In addition, structure-dependent inter action we used a mixture of these 11 pyrethroids to and relative humidity at 55 ± 20%. The facil- among pyrethroids has been proposed to test the hypothesis of dose addition.
ity is approved by the American Association occur in Cl– channels: pretreatment with cis- We also tested the hypothesis that for Accreditation of Laboratory Animal Care. resmethrin (type I) antagonizes the effects of kinetic differences between the 11 pyre- All animals were treated humanely and with fenpropathrin, a mixed type I/II pyrethroid throids would result in less than dose addi- regard to alleviation of suffering. All experi- (Burr and Ray 2004). Unfortunately, these tion if all compounds were administered at mental protocols were approved in advance studies were not designed to test the hypoth- once. Toxicokinetic differences for individual by the National Health and Environmental esis of dose addition (Burr and Ray 2004). pyrethroids result in variation in the time Effects Research Laboratory's Animal Care and Soderlund et al. (2002) highlighted the need of peak effects of more than 4 hr for the 11 Use Committee.
for empirical data to test the hypothesis of pyrethroids tested (Soderlund et al. 2002; Chemicals. Technical grade samples of
additivity for pyrethroids using robust statisti- Wolansky et al. 2006; Wolansky and Harril pyrethroids were kindly supplied by their cal models (e.g., Casey et al. 2004; Feron and 2008). To test this hypothesis, we used two manu facturers: permethrin, bifenthrin, Groten 2002; Olmstead and LeBlanc 2005).
alternative oral dosing protocols: simultaneous and cypermethrin (FMC Corporation, In the present study, we tested the hypoth- (SLT) adminis tration of all chemicals with Philadelphia, PA, USA); esfenvalerate esis that pyrethroids act in a dose-additive a 2-hr dose-to-test interval, and a sequential (Dupont Crop Protection, Wilmington, DE, manner. The hypothesis was tested using the (SQT) protocol where the 11 compounds USA); deltamethrin and β-cyfluthrin (Bayer "single-chemical-required" (SCR) method were administered either 1, 2, or 4 hr before CropScience LP, Research Triangle Park, NC, (Casey et al. 2004) that compares a thresh- testing, depending on each chemical's time of USA); tefluthrin and λ-cyhalothrin (Syngenta old additivity model (Gennings et al. 2004), peak effect. In addition, we conducted a time Crop Protection, Greensboro, NC, USA); and fit to single-compound data, with a similarly course study to determine the time of peak fenpropathrin, resmethrin, and S-bioallethrin parameterized model for data obtained from effect for the mixture when administered using (Valent USA Corp., Walnut Creek, CA, an experimentally tested mixture. We used the SLT protocol. The empirical mixture data USA). Information on the chemical purity and motor activity as the dependent variable in for both the SLT and SQT exposure protocols isomer composition was reported previously this study for two reasons. First, motor activity were fit to a threshold dose– response curve (Wolansky et al. 2006). Doses were calcu- is a valid test method routinely used in acute and compared with the theoretical outcome lated based on percent active ingredient in the and sub chronic regulatory neuro toxicity stud- predicted by the additivity model using the technical product. Mixture stock and dosing ies [Organization for European Cooperation SCR method of analysis [modeling procedures solutions were prepared daily by codissolving and Development (OECD) 1997, 2007; described by Casey et al. (2004)].
pyrethroids in corn oil (Sigma Chemical Co., Soderlund 2001; Soderlund et al. 2002; U.S. St. Louis, MO, USA) according to the dos- EPA 1998a, 1998b). Second, motor activity Materials and Methods
ing protocol described below. Solutions were has been extensively evaluated for a number of Subjects. Male Long-Evans rats (Charles River stirred and gently heated (40–50°C) before
pyrethroids in rodents. Seventeen pyrethroid Laboratories Inc., Wilmington, MA, USA) dosing to assure full solubility and then used preparations, assessed under a variety of dosing were obtained at 55–57 days of age and housed at room temperature.
and testing conditions across laboratories, pro- two per cage in standard polycarbonate hang- Mixture composition. The fixed ratio
duced decreased activity (Crofton et al. 1995; ing cages (45 cm × 24 cm × 20 cm) contain- of individual pyrethroids in the stock solu- Crofton and Reiter 1984, 1988; De Souza ing heat-sterilized pine shavings (Beta Chips; tion (i.e., ai proportion; for each chemical i, Spinosa et al. 1999; Hornychova et al. 1995; Northeastern Products, Inc., Warrensburg, ai = dosei/dosemixture) was based on the indi- Hoy et al. 2000; Mandhane and Chopde NY, USA). All animals were given a 5- to vidual relative potency factors (RPFs) obtained 1997; McDaniel and Moser 1993; Reiter 9-day acclimation period and were maintained from single-compound assays (Wolansky et al. 2006). Each RPF was calculated as the Table 1. Summary of pyrethroid type, threshold dose, ED30, percentage of total mixture dose mass, and
the absolute dose for each chemical.
ratio of the ED30 for the index compound Pyrethroid dose (mg/kg) 30 for delta methrin = 2.50 mg/kg) ED a (mg/kg) divided by the ED 30 for each chemical. The 30 was chosen as a biologically signifi- cant effect on motor activity (Crofton et al. 1991). The absolute doses of each chemical in the stock solution (i.e., the highest mixture dose examined) were equal to 33% of the ED30 for the chemical [see also Supplemental Material, Table 1 (doi:10.1289/ehp.0900667.
S1 via http://dx.doi.org/)]. This dose is approximately 20% lower than the thresh- old dose previously calculated using the SCR approach on each individual dose–effect data The fixed mixing ratio was based on the ratio of the ED30 for the each chemical compared with an index compound (i.e., ED30 for deltamethrin = 2.50 mg/kg). set (Wolansky et al. 2006). Table 1 lists the aData from Wolansky et al. (2006).
chemical names, potency information (derived volume 117 number 10 October 2009 • Environmental Health Perspectives Pyrethroids and mixtures from Wolansky et al. 2006), mixing ratios, three mixture stocks and dilutions were 1 hr xi represents the concentration/dose of the ith and stock mixture solution composition. Two for stock B1, 2 hr for stock B2, and 4 hr for component in combination with the c agents sets of stock solutions were made. Stock A stock B3. Seven mixture dosages, from 1% to that yield the same response. According to this contained all 11 chemicals in the ratio and 100% of the concentration of the stock solu- definition, if the substances interact in an addi- amounts described above. The second set, tion, were tested in each dose–response experi- tive fashion, then used for the sequential dosing, consisted of ment as follows: SLT protocol, 1%, 4%, 10%, three separate stocks: Stock B1 contained 33%, 50%, 66%, and 100%; SQT protocol, S-bioallethrin only; stock B2 contained per- 1%, 4%, 20%, 30%, 40%, 50%, and 100%.
methrin, cypermethrin, delta methrin, esfenva- Motor activity testing. The same end
If the left-hand side of Equation 1, termed lerate, β-cyfluthrin, fenpro pathrin, tefluthrin, point and testing procedures used in single- the "interaction index," is < 1, then a greater and λ-cyhalothrin; and stock B3 contained compound assessments (Wolansky et al. 2006) than additive inter action (e.g., synergism) can bifenthrin and resmethrin [see Supplemental were used to examine mixture dose–effect be claimed at the combination of interest. If Material, Table 2 (doi:10.1289/ehp.0900667. relationships. Rats were transferred from the the left-hand side of Equation 1 is > 1, then a S1)]. The overall ratio and composition of holding room to the adjacent testing room in less than additive interaction (e.g., antagonism) stocks B1, B2, and B3 were equivalent to individual polycarbonate transfer cages and can be claimed with the combination. This stock A. All dosing solutions were made daily. were allowed to acclimate for 5 min before test- definition of additivity is a general form for Exposure. Before dosing, animals were ing. Motor activity was then measured for 1 hr dose addition. It should be pointed out that
moved from the colony room to an iso- using 16 figure-eight mazes, each consisting of use of the toxic equivalence factor approach lated dosing room within the testing labora- a series of inter connected alleys (10 × 10 cm) (Safe 1998) assumes common dose–response tory. After a 1-hr acclimation, animals were converging on a central arena and covered slopes across the chemicals under study; the removed from home cages, dosed, and then with trans parent acrylic plastic (Norton et al. general dose-addition definition of Equation 1 returned to the home cages until the next 1975; Reiter et al. 1975). Total activity was does not require such an assumption.
dosing time or testing. All rats were ran- calculated as the sum of horizontal and vertical We combined the 11 chemicals according domly assigned to treatment groups. Body activity photo cell counts. Photobeam calibra- to the specified mixing ratio (Table 1) and weights were counter balanced across groups. tion was checked daily before testing. Maze evaluated them experimentally. The mixing Experimentally naive groups of rats were used assignments, order of testing, and time of day ratio is denoted in terms of the proportion, ai, for each experiment. Dose selection for the were counterbalanced across treatment groups. of each chemical in the mixture (Table 1) such dose–response studies was based on pilot work All testing was conducted between 0900 and that the summation of ai for the 11 chemicals (data not shown), with the goal to identify at 1700 hours.
equals 1, and the dose xi of each chemical in least two no-effect levels.
Statistical analysis. We analyzed motor the mixture is
We used vehicle control and two dose activity data (i.e., total photocell counts for levels for the mixture time-course study: the 1-hr test session) by two-way analysis of x i = ait (i.e., total mixture dose = t = 0 (corn oil only), 76, and 152 mg pyrethroid/ variance (ANOVA) using SAS software ver- kg. The two doses were chosen to produce sion 9.1 (SAS Institute Inc., Cary, NC, USA), The SCR approach (Casey et al. 2004) mild and moderate clinical signs of pyrethroid with mixture dose and time as independent allows for different slope parameters for each exposure. The high dose produced mild tremor variables. For the SQT and SLT mixture-dose chemical and fixed-ratio mixture. The single- in most of the animals that lasted from 1 to studies, a two-way ANOVA was used, with chemical data were modeled (termed the addi- 4 hr, whereas the lower dose produced a small block and mixture-dose treatment as inde- tivity model) using a nonlinear exponential percentage of animals exhibiting transient bur- pendent variables and total activity counts as threshold model for the mean motor activity rowing and pawing behaviors but no tremor. the dependent variable. We performed mean (percent of control) of the form Independent groups of animals were exposed contrast testing using Duncan's new mul- for 1, 2, 4, 8, 24, or 48 hr before testing (n = 8 tiple range test (SAS). Data from one test- or 12 per group, except at 48 hr, n = 4 for the ing run in the time-course study (n = 8 rats) µ = 76 mg/kg/group).
were excluded from formal analysis because i xi ≤ db We used two experimental designs for of excessive noise from building construction. the dose response that differed only in the In addition, one rat was excluded because where α + γ = 100, α is the maximum effect dosing protocol used to administer all the of excessive toxicity (8 hr, high dose). In the parameter, xi is the absolute dose of the ith chemicals. The SLT protocol used mixture SLT experiment, data from one rat (1.5 mg/ chemical, βi are the slope parameters for the stock A, whereby all 11 pyrethroids were kg), and in the SQT experiment data from administered at the same time in one mixture three rats (one each from the 1.5, 30.5, and Table 2. Individual times to peak effect and the
2 hr before testing (three replicate test blocks 76.2 mg/kg groups), were excluded because of mixture dosing protocols for each chemical. with four rats per mixture dose per block). aspiration of the gavage fluid into the lungs.
Mixture dosing protocol The 2-hr time point was based on the time of Determination of departure from additivity time to peak (hours before testing) peak effect of the mixture determined in the for the motor activity data from two mixture Compound SLT dosing SQT dosing time-course study. The SQT protocol used dose–response studies (SLT and SQT proto- mixture stocks B1, B2, and B3 (two replicate cols) used the SCR method. The definition Permethrin test blocks with six rats per mixture dose per of additivity given by Berenbaum (1985) can Cypermethrin block). Because of known differences in the be related to the isobologram for a combina- kinetics and time course of effects of the differ- tion of chemicals (Loewe 1953; Loewe and β-Cyfluthrin ent pyrethroids, this protocol allowed sequen- Muischnek 1926) through the inter action Fenpropathrin tial dosing where the 11 pyrethroids were index. That is, in a combination of c (here, Tefluthrin adminis tered according to their previously c = 11) chemicals, E i represents the concen- determined time of peak effects (Table 2). tration or dose of the ith component alone Bifenthrin The time between dosing and testing for the that yields a fixed response (i.e., ED Environmental Health Perspectives • volume 117 number 10 October 2009 Wolansky et al.
individual chemicals (i = 1, . . , 11), and which is associated with the threshold additiv- signs of pyrethroid toxicity. Mild whole-body δ is the threshold parameter such that the ity model given in Equation 2a, or tremors were present in most animals at the dose threshold for each individual chemical highest mixture dose (152.4 mg/kg) from 1 to µ = α + γ exp(θadd t), i = δ/βi, i = 1, . . , 11. The γ 4 hr post dosing. Signs of high-dose pyrethroid parameter was constrained to be γ = 100 – α, toxicity such as excessive salivation, whole- so that the mean response for the vehicle- which is associated with the nonlinear additivity body tremors, and choreo athetotic movements control groups is 100%. It is important to model given in Equation 2b.
(Aldridge 1990; McDaniel and Moser 1993; note that the form of the additivity model Thus, the slope parameter associated with Soderlund et al. 2002) were not observed in does not include information about schedul- mixture along the specified fixed-ratio ray any animals, with one exception: One rat in ing of dosing, because single-chemical data under additivity is the time-course experiment exhibited clini- were available only with the SLT protocol. cal signs of excessive pyrethroid exposure and Therefore, for the present study we assumed iadd = / biai .
raspy breath sounds, possibly due to partial that the model represented by Equation 2a aspiration of the gavage solution into the lungs. is an additivity model for the case where the By replacing the unknown parameters Data from this animal were not used.
timing of the dosing has a negligible effect.
in Equations 3a and 3b with parameter esti- The time-course study revealed a rapid When the dose thresholds for all single mates, a plot of the dose–response curve decline in motor activity with a peak decrease chemicals are estimated outside of the experi- under additivity for a specified fixed ratio of at 1–2 hr post dosing (Figure 1). Activity recov- mental region, the model in Equation 2a is the chemicals was produced. The predicted ered to control levels at 24–48 hr post dosing. over parameterized. The corresponding non- mixture dose–response relationship was The time course of effects was similar for both linear smooth additivity model is given by estimated using only single-chemical dose– mixture-dose groups. These conclusions were response data (Equation 2) and then predict- ing along the mixture ray with the constraint add = a + c exp f / i [2b] of additivity given in Equation 1.
Following Gennings et al. (2002) and We estimated unknown parameters Casey et al. (2004), the mixture data along the using the maximum quasi-likelihood method fixed mixture ray is fit to a similarly param- (McCullagh and Nelder 1989). The esti- eterized mixture model of the form mated additivity model was used to predict the mean response along the fixed mixing imixt > d ratio of the 11 chemicals in terms of total Total activity (counts/hr)
mix t - dmix h mixt # dmix 152 mg mixture/kg dose. Threshold estimates, ED values (i.e., the response associated with a for a threshold model. The hypothesis of addi- Time (hr)
10% motor activity decrease), and the cor- tivity along the specified ratio of the chemicals responding large-sample 95% confidence is a hypothesis of coincidence (i.e., the rela- Figure 1. Time course of cumulative effects of
11 pyrethroids on figure-eight maze activity (mean intervals (CIs) for each single chemical were tionships are the same) between the additivity ± SE). The arrow indicates the time of peak effects computed, as well as dose thresholds, ED10 model in Equation 2 and the mixture model for the tested mixture. values, and ED30 values for the mixture. In given in Equation 4 (i.e., for the threshold this work, all of these parameter estimates additivity models), Table 3. Estimated model parameters from the
were computed using empirical data from threshold additivity model (Equation 2a) and the single compounds and mixtures. However, H mixture model (Equation 3a) fit simultaneously.
0: {imix = iadd and dmix = dadd} versus for single chemicals, dose thresholds, ED H1: {imix ≠ iadd or dmix ≠ dadd}. values, and RPFs for motor function had Single-chemical slope parameters been previously obtained using the same SCR To determine whether there was a statisti- model parameterized using only experimen- cally significant deviation from additivity, we β 1 (β-cyfluthrin) tal data from single compounds (Wolansky used a quasi-likelihood ratio test to compare β 2 (bifenthrin) et al. 2006). All 11 chemicals in this study the empirical mixture model with the restricted β 3 (S-bioallethrin) are associated with significant decreases in additivity model based on an F-distribution β 4 (cypermethrin) β 5 (deltamethrin) motor activity as their doses increase, as (e.g., Casey et al. 2004). The restricted addi- β 6 (esfenvalerate) evidenced by negative and significant slope tivity model (Casey et al. 2004) included only β 7 (fenpropathrin) parameters. The significance of the thresholds the single-chemical dose–response model β 8 (λ-cyhalothrin) can be described by the significance of the parameters but used both the single- chemical β 9 (permethrin) threshold parameter (δadd) in the additiv- and mixture data. We used this restricted β 10 (resmethrin) ity model (p < 0.001) and the 95% CIs on model to predict the mean responses for the β 11 (tefluthrin) the dose thresholds that did not include zero mixture data using the constraint of additiv- Mixture slope parameters (Wolansky et al. 2006). We estimated the ity given in Equation 1. Finally, we compared θ1 (SLT) curve for the mixture dose–response relation- the predicted responses from the mixture data θ2 (SQT) Threshold parameters ship using the model expressed by under the hypothesis of additivity (Casey et al. δ 2004; Gennings et al. 2002) with the observed sample means using an F-test.
/ ibait > d Data are the estimated slopes for single chemicals (β parameters) and the mixture (θ parameters) adminis- f / ibait - dp / ibait ≤ db tered using two alternative dosing protocols (i.e., SLT and We observed no mortality in the experiments SQT protocols), and the estimated thresholds (δ parame- in this study. The two higher mixture dosages add t > d ters) for the additivity model (δ add) and the two dosing add t ≤ d , (i.e., 76.2 and 152.4 mg/kg) evoked few clinical protocols (δmix_1 and δmix_2). volume 117 number 10 October 2009 • Environmental Health Perspectives Pyrethroids and mixtures supported by a significant mixture-dose × time (θ values) were negative and significant, model fit simultaneously. The simultaneous interaction [F(10,147) = 5.18, p < 0.0001] indicating that as the dose of each individ- fit of the additivity model and the mixture and significant main effects of mixture dose ual chemical or the total dose of the mixture model accommodates a common maximum [F(2,147) = 62.48, p < 0.0001] and testing increases, the mean motor activity decreases.
effect parameter (α), which was estimated time [F(4,147) = 15.5, p < 0.0001]. The activ- Figure 2 shows the plots of individual to be 26.5 (i.e., 26.5% of control; Table 1). ity of all mixture-dose groups was significantly data points for each chemical and the fit dose The γ parameter was constrained to be decreased compared with controls at 1, 2, 4, response using the additivity model. These γ = 100 – α, so that the mean response for and 8 hr (p < 0.05).
plots illustrate the wide potency range of the the control groups is 100%. Tables 4 and 5 Parameter estimates and correspond- individual chemicals, from 10 to 900 mg/kg, list the model estimates for the threshold ing p-values from the additivity model in and the dose-related decrease in activity dose and ED30 dose. Note that the individual Equation 2a, from the simultaneously fit for all 11 chemi cals. These plots also illus- chemical estimates in Tables 3–5 are in some single-chemical data and mixture data, are trate the estimated thresholds for each of cases marginally different than previously provided in Table 3. The slope parameters the dose–response functions. Table 3 lists published estimates (Wolansky et al. 2006) associated with each of the 11 single chemi- the estimated model parameters from the because of the inclusion of the mixtures data cals (β values) and for the fixed-ratio mixtures threshold additivity model and the mixture in the SCR model in the present study: The Total activity (% control)
Total activity (% control)
Total activity (% control)
Dose (mg/kg)
Dose (mg/kg)
Dose (mg/kg)
Total activity (% control) 20
Total activity (% control) 20
Total activity (% control) 20
10 20 30 40 50 60 70 80 90 100 110 120 Dose (mg/kg)
Dose (mg/kg)
Dose (mg/kg)
Total activity (% control)
Total activity (% control)
Total activity (% control)
Dose (mg/kg)
Dose (mg/kg)
Dose (mg/kg)
Total activity (% control)
Total activity (% control)
Dose (mg/kg)
Dose (mg/kg)
Figure 2. Observed data (individual data points) and the model-predicted dose–response curve from the additivity threshold model given in Equation 2a for each
of the 11 pyrethroids. Environmental Health Perspectives • volume 117 number 10 October 2009 Wolansky et al.
empirical mixtures data were not available for lack of interaction between replicate testing rats. The present results provide the first inclusion at the time the previous estimates blocks and mixture dose, and the significant in vivo evidence on cumulative actions of were published.
main effect of replicate block, we conducted all pyrethroid mixtures in mammals. These data The mixture dose–response studies demon- additional analyses on the motor activity counts suggest that dose-additive approaches should strated dose-related decreases in activity regard- expressed as percentage of block control values.
be used when assessing the risk of exposures to less of the dosing protocol (Figure 3). For the Results of the SCR method demonstrated chemical mixtures that contain pyrethroids.
SQT group, the pyrethroid mixture decreased no significant difference between the predicted The time course of effect of the pyrethroid activity by approximately 58% in the two response and the empirical data for both the mixture was consistent with patterns reported highest mixture-dose groups, with significant SLT and SQT exposure protocols. Table 3 lists for a number of single-compound assessments. decreases in all mixture doses ≥ 30.4 mg/kg the slope estimates for individual chemicals The time course for the mixture showed a (p < 0.05). There was no interaction between and the dosing protocols, as well as the thresh- maximum decrease in activity at 1–2 hr and testing block and mixture dose [F(14,92) = 0.66, old estimates for both protocols. The empirical recovery within 24–48 hr. Previous reports p < 0.8030], but there were significant main fit for the mixture administered using the SLT from studies using similar dosing protocols effects of dose [F(7,92) = 12.84, p < 0.0001] protocol was not different from that predicted in rats demon strated maximal decreases in and block [F(2,92) = 6.15, p < 0.0035]. The assuming additivity, and the null hypothe- figure-eight maze activity at 1–4 hr post dosing main effect of block was due to slight differ- sis was not rejected [F(2,1037) = 0.015, (Crofton et al. 1995; Crofton and Reiter ences in the overall baseline for activity counts p = 0.985; Figure 3A]. The small shift to 1984, 1988; McDaniel and Moser 1993; over the different test days (data not shown). the left in the dose–response relationship Wolansky et al. 2006). In addition, allethrin, For the SLT protocol, the pyrethroid mixture between the empirical and predicted curves S-bioallethrin, permethrin, fenvalerate, delta- decreased activity by 60% in the highest mix- for the SQT protocol (Figure 3B) was not methrin, and cypermethrin evoke altera- ture-dose group, with significant decreases in all significant, and the null hypothesis was not tions in motor-related end points in small mixture doses ≥ 50.2 mg/kg (p < 0.05). There rejected [F(2,1037) = 2.65, p = 0.071]. The rodents evident as early as 0.5–1.5 hr after was no interaction between testing block and two thresholds were not statistically different; systemic exposure (De Souza Spinosa et al. mixture dose [F(14,94) = 1.24, p < 0.2643], however, the SQT protocol threshold value 1999; Hoy et al. 2000; McDaniel and Moser but there were significant main effects of (5.38 mg/kg) was 3.7-fold lower than that 1993; Nishimura et al. 1984; Wolansky et al. dose [F(7,94) = 20.45, p < 0.0001] and block using the SLT protocol (19.82 mg/kg). This 2006). Likewise, the extended effect on activ- [F(2,94) = 2.85, p < 0.0414]. Because of the difference was 1.5-fold when we compared ity through 8 hr is consistent with the pro- mixture ED30 values (SQT, 29.27 mg/kg, longed syndromes evoked by resmethrin and Table 4. Estimated dose thresholds for each of the
vs. SLT, 49.81 mg/kg). The CIs were wide bifenthrin (Crofton and Reiter 1984; Holton 11 chemicals with 95% large sample CIs.
and included zero, and although the thresh- et al. 1997; Soderlund et al. 2002; White old for the mixture administered using the et al. 1976; Wolansky et al. 2006, 2007). SQT dosing protocol was numerically lower Reports of longer times to onset after acute than that for the mixture where the chemi- exposures (e.g., Soderlund et al. 2002) have cals were administered together at once been attributed to larger dosing volumes that (see Figure 3A,B), a test of coincidence delay absorption (Kim et al. 2007; Wolansky in the two mixture curves was not rejected et al. 2007). The present data are also consis- [F(2,1037) = 0.90, p = 0.407; Table 3].
tent with the fact that most of the 11 chemi- cals have individual times to peak effects of 1.5–2.5 hr post exposure (Table 2). These data In the present study we tested the hypothe sis support the hypothesis that the toxico kinetics that the combined action of 11 pyrethroid of pyrethroids may not be altered in mixtures insecticides on motor function is predicted composed of low-levels of the individual insec- by dose addition. We designed the mixture ticides. Toxicokinetic studies of pyrethroid so that the highest mixture dose contained mixtures are needed to test this hypothesis.
aThreshold values for the individual chemicals vary
doses of each pyrethroid that were below their The mixture of 11 pyrethroids produced slightly from those in Table 1 because of inclusion of the single-chemical and mixture data in the computation of individual thresholds for effect. The results dose-dependent decreases in motor activ- the estimates (see "Materials and Methods" for details). demon strated that the additivity model pre- ity. This is consistent with previous reports dicted the measured effects on behavior in of dose-related decreases in activity in the Table 5. Estimated ED30 values for each of the
11 chemicals with 95% large-sample CIs.
Predicted effect addition Predicted dose addition Total activity (% control)
Total activity (% control) 20
Mixture dose (mg/kg)
Mixture dose (mg/kg)
Figure 3. Dose–response relationships for the cumulative effects of 11 pyrethroids on figure-eight maze
activity (mean ± SD). (A) SLT group. (B) SQT group. The departure of the experimental data from the pre- dictive curve modeled assuming dose addition was not significant. volume 117 number 10 October 2009 • Environmental Health Perspectives Pyrethroids and mixtures figure-eight maze in rats (Crofton et al. 1995; type II pyrethroids may compete for binding much lower compared with the rat acute oral Crofton and Reiter 1984, 1988; Gilbert et al. to a Cl– channel target site. Furthermore, these exposures used in the present study. Acute 1990; McDaniel and Moser 1993; Reiter et al. authors reported that binary mixtures did not oral bolus doses may result in higher peak tis- 1981) and in other assessments with motor lead to effect-additive outcomes. Song and sue concentrations, compared with dietary end points carried out in mice (Chanh et al. Narahashi (1996) concluded that tetramethrin and dermal human exposures (Conolly et al. 1984; Mandhane and Chopde 1997). Thus, may displace fenvalerate or interact allosteri- 1999). Urinary levels of pyrethroid metabo- decreased locomotor behavior appears as a cally with sodium-channel protein binding sites lites range from non detectable to as high as common finding of acute pyrethroid exposure in an ex vivo rat dorsal root ganglion prepara- 50 µg/L, with median levels between 1 and to both individual compounds and mixtures.
tion. Although these reports suggest that pyre- 5 µg/L (Lu et al. 2009; Morgan et al. 2007). An important finding of the present study throids do not act in an effect-additive manner, Comparable data are not available in rats. is that low doses of the individual chemi cals, the experimental designs used preclude any Toxicokinetic models are needed that will when combined in a mixture, decreased motor definitive conclusions concerning additivity allow comparison between effective doses in activity. The threshold for decreased activ- from these reports. First, the electro physiologic rats and aggregate human exposures.
ity was 5.4 mg total pyrethroids/kg when the work (Ray et al. 2006) used dose levels that SQT protocol was used (Figure 3). The abso- exceeded known lethal doses. In addition, all Conclusions
lute amount of each individual pyrethroid at of these previous reports lacked, either by study In summary, the present data demonstrate this mixture dose is approximately 3% of the design or by statistical approach, the ability that sub threshold doses of individual pyre- threshold dose for altering motor activity when to test the hypothesis of additivity. The use of throids, when combined in a mixture, pro- given alone in the single-compound assays rigorous statistical models is critical for testing duce measurable neurotoxicity in rats. These (Table 1). These data clearly demonstrate two hypothe ses of effect addition or dose addition findings provide the first in vivo evidence of key findings. First, that effect addition, defined and determining whether antagonism or syner- cumulative actions of pyrethroid mixtures as a simple summation of the effects (i.e., sum gism exists (Feron and Groten 2002; Gennings in mammals and suggest that dose-additive of decreases in motor activity) of all chemicals et al. 2004; Hertzberg and Teuschler 2002; approaches should be used for considering the in a mixture, under estimates the potency of the LeBlanc and Olmstead 2004; Teuschler 2007). combined toxicity of pyrethroid insecticides.
tested mixture by a wide margin. Second, these The finding of dose addition for both the data demon strate that low doses of individual SLT and SQT protocols suggests a lack of pyrethroids, when acutely administered as a toxico kinetic or enzymatic interactions at low mixture, produce measurable effects on motor doses, which has been shown for other mix- Aldridge WN. 1990. An assessment of the toxicological proper- behavior in the rat.
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volume 117 number 10 October 2009 • Environmental Health Perspectives

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