NeuroToxicology 30S (2009) S1–S16

Comparative functional observational battery study of twelve commercialpyrethroid insecticides in male rats following acute oral exposure§

Myra L. Weiner a,1, Mark Nemec b, Larry Sheets c, Dana Sargent d,*, Charles Breckenridge e

a TOXpertise, LLC, Princeton, NJ 08540, United Statesb Wil Research Laboratories, LLC, Ashland, OH 44805, United Statesc Bayer CropScience, Stilwell, KS 66085, United Statesd Bayer CropScience, 2 TW Alexander Drive, P.O. Box 12014, Research Triangle Park, NC 27709, United Statese Syngenta Crop Protection Inc., Greensboro, NC 27419, United States

A R T I C L E I N F O

Article history:

Received 16 April 2009

Accepted 24 August 2009

Available online 11 September 2009

Keywords:

Functional observational battery

Neurotoxicity

Pyrethroids

Common mechanism of toxicity

FQPA

A B S T R A C T

Twelve commercial pyrethroid insecticides (technical-grade active ingredients) were evaluated

individually for acute neurobehavioral manifestations of toxicity under conditions suited to assist

with determining whether they act by a common mechanism of toxicity. The pyrethroids that were

tested reflect a diversity of structures, including six with an a-cyano phenoxybenzyl moiety (b-

cyfluthrin, l-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate and fenpropathrin) and six without

this moiety (bifenthrin, S-bioallethrin, permethrin, pyrethrins, resmethrin and tefluthrin).

These chemicals also present a variety of behavioral effects, including ones that are historically

classified as causing a T (tremor), CS (choreoathetosis with salivation) or intermediate syndrome of

intoxication, and others that have not previously been classified. Each pyrethroid that was tested

consisted of the complement of isomers that occur in commercial products—a key factor for relevance for

environmental and human exposure and for comparisons, since the biological activity of the individual

isomers can vary tremendously. Young-adult male Sprague–Dawley rats (10 per dose group) were

administered a single dose of pyrethroid by oral gavage, in corn oil, at a volume of 5 ml/kg. Each was

tested at a range of two or three dose levels, including a minimally toxic dose, to establish the more

sensitive manifestations of toxicity, and a more toxic dose, to establish a more complete spectrum of

neurobehavioral manifestations. Animals were evaluated using a functional observational battery (FOB)

that was designed to characterize and distinguish effects classically associated with T or CS syndromes of

intoxication. The FOB was performed when manifestations of toxicity were most apparent at the time of

peak effect (2, 4, or 8 h post-dosing) by observers who were blinded to dose group assignment, thus

avoiding possible bias. The results from this study indicate that some pyrethroids clearly exhibit the

historic classification symptoms of the T and CS syndromes while others do so less obviously. Use of the

statistical technique of Principal Component Analysis (PCA) further helped interpret the study findings,

as described in the accompanying paper (Breckenridge et al., 2009). These results establish

manifestations of neurotoxicity in vivo that can be used as weight of evidence to determine whether

pyrethroid insecticides act through a common mechanism of toxicity in mammals. Based on a review of

the FOB data, analyzed by PCA, and other published data, two common mechanism groups are proposed.

Group 1 would include pyrethrins, bifenthrin, resmethrin, permethrin, S-bioallethrin and tefluthrin.

Group 2 would include cypermethrin, deltamethrin, esfenvalerate, b-cyfluthrin and l-cyhalothrin.

Fenpropathrin exhibited features of both groups.

� 2009 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

NeuroToxicology

§ This work was presented in part at the 2006 Society of Toxicology meeting:

Sheets et al. (2006).

* Corresponding author. Tel.: +1 919 549 2223; fax: +1 919 549 2925.

E-mail address: dana.sargent@bayercropscience.com (D. Sargent).1 Formerly with FMC Corporation, Princeton, NJ, Unites States.

0161-813X/$ – see front matter � 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2009.08.014

1. Introduction

The Food Quality Protection Act (FQPA) of 1996, whichamended the Federal Insecticide, Fungicide and Rodenticide Act(FIFRA), and the Federal Food, Drug and Cosmetics Act, requires theUS EPA to consider the cumulative effects of exposure to pesticideshaving a ‘‘common mechanism of toxicity’’. FQPA mandates thatthe risk assessment consider collectively exposure to all chemicals

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16S2

that act by a common mechanism of toxicity in a cumulative riskassessment, as well as to consider exposure to each chemical viavarious routes (e.g., dietary, dermal and inhalation) and sources(e.g., residues in water and on various foods) in an aggregate riskassessment. In order to support the grouping of different chemicalstogether for purposes of cumulative risk assessment, there must besufficient evidence to support a common adverse effect that isassociated with a common mechanism of action in specific targettissues. To date, cumulative risk assessments have been applied tothe organophosphorus and carbamate insecticides, for which acommon mechanism of toxicity (i.e., inhibition of acetylcholines-terase activity in the brain and other target tissues) has beenassociated with adverse effects (cholinergic signs of intoxication).However, the criteria that are required to establish a commonmechanism of toxicity with a specific toxic effect have not yet beenachieved with sufficient clarity for the pyrethroid insecticides. Foradditional details, the reader is referred to reviews of the publishedand proprietary data that pertain to the mechanisms of toxic actionof pyrethroid insecticides in mammals, including the shortcomingsof the existing knowledge for the pyrethroids (Soderlund et al.,2002; Shafer and Meyer, 2004; Wolansky and Harrill, 2008).

A determination of common mechanism of toxicity inmammals is complicated by the number of potential biologicaltarget sites and effects expressed by various pyrethroid insecti-cides on these targets (Soderlund et al., 2002; Wolansky andHarrill, 2008). In mammals, as in insects, pyrethroids exert toxicityby interfering with the closure of voltage-sensitive neuronalsodium ion channels, which would tend to support a commonmechanism of toxicity. However, unlike insects, mammals havemany sodium channel isoforms expressed at different locationsand stages of development, which vary in their sensitivity tovarious pyrethroids (Soderlund et al., 2002; Choi and Soderlund,2006). Pyrethroids have also been shown to act in vitro and in vivo

on voltage-sensitive calcium channels (Clark and Symington,2007; Symington and Clark, 2005; De Ondarza et al., 2003;Symington et al., 2008; Shafer and Meyer, 2004) and chloridechannels (Forshaw et al., 2000; Burr and Ray, 2004). It is consideredlikely that these differences of action on neuronal ion channelsamong the pyrethroid insecticides contribute to the diversity ofneurologic and behavioral manifestations of acute toxicity that areevident in the whole animal, and thus, could form the basis fordetermination for common mechanism groups.

The nervous system is generally considered to be a commontarget site for all pyrethroid insecticides, since they all produceacute signs of neurotoxicity at some dose level (Casida et al., 1983;Wolansky and Harrill, 2008; Soderlund et al., 2002). By compar-ison, other organ systems are only adversely affected by individualpyrethroid insecticides, not all pyrethroids as a class. Therefore, thenervous system is considered to be a common site of toxic actionfor all pyrethroids (Wolansky and Harrill, 2008; Soderlund et al.,2002). Historically, many pyrethroids have been grouped into twogroups based on the specific complement or syndrome ofneurobehavioral responses observed after acute exposure. Earlywork suggested that pyrethroids lacking an a-cyano substituent atthe phenoxybenzyl moiety of the molecule produced clinical signsof fine tremor, hyperthermia, aggressive sparring, increasedsensitivity to stimuli, and prostration, named appropriately the‘‘T syndrome’’. Pyrethroids containing the a-cyano phenoxybenzylmoiety produced clinical signs of choreoathetosis (writhing),profuse salivation, hypothermia, coarse tremor, clonic seizures,pawing and burrowing, abnormal hindlimb locomotion, namedappropriately, the ‘‘CS syndrome’’ (Verschoyle and Barnes, 1972;Barnes and Verschoyle, 1974; Verschoyle and Aldridge, 1980).Pyrethroids that produce the T and CS syndromes of intoxicationhave also been called Type I and Type II pyrethroids, respectively(Lawrence and Casida, 1982, reviewed by Soderlund et al., 2002).

While the aforementioned groupings tend to support twogroups of pyrethroids for cumulative risk assessment under FQPA,the existing database is inadequate to make this determination.First, many newer commercial pyrethroids have not beenevaluated to determine their placement into either category: TypeI (also considered T syndrome) or Type II (also considered CSsyndrome). In addition, the early acute studies generally used pureisomers, rather than the commercial mix of isomers, withintravenous (Verschoyle and Aldridge, 1980) or intracerebral(Lawrence and Casida, 1982) administration; whereas, thecommercial products to which humans are exposed contain amix of isomers. For human health risk assessment, it is importantto evaluate the product to which humans are exposed, by the sameroute of potential exposure (oral). Furthermore, inconsistencies instudy designs used to evaluate the various pyrethroids forneurotoxicity in vivo limit meaningful comparisons among thepyrethroids (Soderlund et al., 2002). Other differences amongstudies include the route of exposure, method of dose adminis-tration (e.g., vehicle and dose volume), methods used to evaluatethe animals, animal strain and study design. For example, the levelof detail used to describe the neurobehavioral effects (e.g., fineversus coarse tremor and the nature of gait or posture alterations)in currently available studies has been shown to limit thecharacterization or comparison of findings.

In a more recent study (Wolansky et al., 2006), elevencommercial pyrethroids (the same pyrethroids tested in thepresent study with the exception of pyrethrins) were compared foreffects on motor activity in a single study in rats via oraladministration. Doses causing a 30% decrease in motor activity(ED30) and threshold doses were determined, which providedcomparative data of motor activity effects of the elevenpyrethroids and confirms their general motor-depressant action.However, this study also has limitations with regard to providingevidence for a common mechanism for the pyrethroids. Motoractivity can be altered by a variety of drugs and chemicals in aquantitative manner via a multitude of mechanisms of action(Crofton et al., 1991; MacPhail et al., 1989; Ross, 2001), and itsmeasurement has been arguably considered as too variable and toodifficult to interpret as a primary index of neurotoxicity (Ross,2001). Therefore, though these same chemicals or classes ofchemicals may alter motor activity, a decrease in motor activitydoes not, by itself, define the chemical or classes of chemicals by aspecific mechanism. Unlike the responses in a motor activityassessment, the clinical signs or responses based on observationcan be considered to be more rigorous in their ability to specificallydefine the class of chemicals, particularly, for pyrethroids(Verschoyle and Aldridge, 1980).

Therefore, this study was performed to address a criticalshortcoming of the existing database for the pyrethroid insecti-cides: namely, standard conditions of evaluation on the functionalobservational battery of commercial pyrethroid insecticides in asingle study (Soderlund et al., 2002). Although the effects of one(Righi and Palermo-Neto, 2003) or two pyrethroids (McDaniel andMoser, 1993) have been evaluated previously for neurobehavioralfunctional observations, the full evaluation of 12 pyrethroids inone study has not been conducted previously. Thus, this study isthe first to compare the pyrethroids for neurotoxicity on thefunctional observational battery in an objective way, withoutregard to structure or identity, under Good Laboratory PracticeGuidelines. In addition, these pyrethroids were tested understandardized conditions that are appropriate for human riskassessment (e.g., relevant isomer mixes, route of exposure, animalmodel and dose selection), and to complement recent work with in

vitro ion channel model systems that investigated the biologicalactivity of the same active isomers that constitute thesecommercial products (Choi and Soderlund, 2006; Symington

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16 S3

et al., 2008; Burr and Ray, 2004). The present study was presentedat the 2006 Society of Toxicology meeting (Sheets et al., 2006).

2. Materials and methods

2.1. Test substances, dosing solution and vehicle

The following test substances were evaluated in this study:bifenthrin, S-bioallethrin, b-cyfluthrin, l-cyhalothrin, cyperme-thrin, resmethrin, deltamethrin, esfenvalerate, fenpropathrin,permethrin, pyrethrins and tefluthrin. Purity and stability ver-ification for the test substances were confirmed by the respectivesuppliers by Certificates of Analysis or GLP analyses. Supplierswere Bayer CropSciences (S-bioallethrin, b-Cyfluthrin, deltame-thrin, pyrethrins, resmethrin); E.I. DuPont (esfenvalerate); FMCCorporation (bifenthrin, cypermethrin, permethrin); SyngentaCorporation (tefluthrin, l-cyhalothrin) and Valent USA Corpora-tion (fenpropathrin). The vehicle utilized in preparation of the testmixtures and for administration to control animals was Mazola1

corn oil (Bestfoods, NJ).The test substance formulations were prepared as weight/

volume solutions in corn oil to be administered at a constantvolume of 5 ml/kg body weight. Control groups received corn oilat a constant dosage volume of 5 ml/kg. Dose volumes were notadjusted for purity. All solutions were prepared fresh for eachuse and were stirred continuously throughout use. No analyseswere performed on dose solutions. Fenpropathrin was storedfrozen under liquid nitrogen. All other test substances werestored at room temperature. Resmethrin was protected fromlight.

2.2. Administration

The selected route of administration was oral gavage. Thevehicle and test substance formulations were administered orallyby gastric intubation via a 16-gauge stainless steel gavage cannulaas a single dose. The day of dose administration was termed studyday 0 for that animal. Individual dosages were based on the day 0body weight.

2.3. Animals and animal husbandry

The Crl:CD1(SD)IGS BR rat was chosen for this study because itis recognized as an appropriate test animal species and strain forneurotoxicity studies (Moser et al., 1991) and is a widely usedstrain for which robust historical control data are available.

Male Crl:CD1(SD)IGS BR rats in good health were receivedfrom Charles River Laboratories, Inc., Raleigh, NC, at 28 or 29 daysof age and acclimated for a period of at least 14 days. Animals were

Table 1Pyrethroids evaluated, dose levels and time-to-peak effect.

Pyrethroid CAS number Structural classa

Pyrethrins 8003-34-7 Non-cyano

Resmethrin 10453-86-8 Non-cyano

S-Bioallethrin 28434-00-6 Non-cyano

Permethrin 52645-53-1 Non-cyano

Bifenthrin 82657-04-3 Non-cyano

Tefluthrin 75938-32-2 Non-cyano

Cypermethrin 52315-07-8 Cyano

Esfenvalerate 66230-04-4 Cyano

b-Cyfluthrin 68359-37-5 Cyano

Deltamethrin 52918-63-5 Cyano

Fenpropathrin 39515-41-8 Cyano

l-Cyhalothrin 91465-08-5 Cyano

a Pyrethroids lacking the a-cyano group on the phenoxybenzyl alcohol moiety (non-

moiety (cyano).

individually identified and housed in accordance with the ‘‘Guidefor the Care and Use of Laboratory Animals’’ (National Researchand Council, 1996) in a facility accredited by the Association forAssessment and Accreditation of Laboratory Animal Care Inter-national (AAALAC International). All animals were housed three/cage by sex for the first three days of the 14-day acclimationperiod, and housed individually thereafter, in an environmentallycontrolled room at relatively constant temperature (718 � 38F)and humidity (50 � 20%) with a 12 h light (6 am to 6 pm)–darkphotoperiod.

The basal diet used in this study, PMI Nutrition International,Inc. Certified Rodent LabDiet1 5002, is a certified feed, withappropriate analyses performed by the manufacturer, and adocumented water supply. Basal diet and water were providedfor ad libitum consumption throughout the study.

2.4. Dosage selection and times-to-peak effect

The dose levels and the times-to-peak effect for each testsubstance were based on data from a range-finding study at thesame laboratory. Each test substance was evaluated in the range-finding study at two or more dose levels to determine a range ofdose responses such that the low dose would show limited or noclinical signs and the high-dose would show the full-range of non-lethal, neutoxic effects when evaluated at the time-to-peak effect.Following the completion of the tests with two doses, additionaldose levels were evaluated on the following test substances toobtain better dose-response data: b-cyfluthrin, cypermethrin,deltamethrin, esfenvalerate and resmethrin. All treated andconcurrent control groups consisted of 10 male rats each. Thetime-to-peak effect was determined in the range-finding study.The test substances, CAS numbers, structural feature regarding theabsence or presence of an a-cyano group on the phenoxybenzylmoiety, dose levels and times-to-peak effects are summarized inTable 1.

Due to the large number of treated groups, the study wasconducted using a temporally blocked design so that representa-tive groups comprised of control and treated animals at the sametime-to-peak effect were present in each block. Table 2 sum-marizes the block design for this study. There were a total of sevenblocks, each with its own control.

2.5. Assignment of animals to treatment groups

Animals judged suitable for assignment to the study based onphysical examination and good health were selected for use,using a computerized randomization procedure to ensurehomogeneity of group mean body weights and variances. Thenthe animals were assigned to dose groups (10/dose group)

Dose levels (mg/kg) Time (h)-to-peak effect

400, 800 4

350, 500, 750 4

150, 200, 300 2

200, 250 4

40, 55 8

10, 15 8

65, 100, 150 4

15, 25, 50 8

12.5, 25, 45 2

12.5, 25, 35 4

15, 30 2

10, 20 4

cyano) or pyrethroids containing the a-cyano group on the phenoxybenzyl alcohol

Table 2Block design used to test pyrethroids: dose levels and times-to-peak effect.

Block Treatmenta Dose levels (mg/kg)

2 h time to peak effect

A Vehicle 0

b-Cyfluthrin 25, 45

S-Bioallethrin 200, 300

Fenpropathrin 15, 30

4 h time to peak effect

B Vehicle 0

l-Cyhalothrin 10, 20

Cypermethrin 100, 150

Permethrin 200, 250

4 h time to peak effect

C Vehicle 0

Resmethrin 500, 750

Deltamethrin 25, 35

Pyrethrins 400, 800

8 h time to peak effect

D Vehicle 0

Tefluthrin 10, 15

Esfenvalerate 25, 50

Bifenthrin 40, 50

2 h time to peak effect

F Vehicle 0

b-Cyfluthrin 12.5

S-Bioallethrin 150

4 h time to peak effect

G Vehicle 0

Resmethrin 350

Deltamethrin 12.5

Cypermethrin 65

8 h time to peak effect

H Vehicle 0

Esfenvalerate 15

a All groups included ten males/dose group with the exception of the vehicle

control in Block G. The Block G control group included 20 males/group due to an

error in formulation of the cypermethrin group, which was replaced and dosing was

repeated with naı̈ve animals. An additional 10 animals were added to the vehicle

control group for comparative purposes.

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16S4

accordingly. Individual body weights at randomization werewithin �20% of the mean.

2.6. Parameters evaluated

2.6.1. Clinical observations and survival

All animals were observed twice daily (morning and afternoon)for mortality and moribundity. Clinical observations were per-formed daily, except on the day of the Functional ObservationalBattery.

2.6.2. Body weights

Animals were randomized to groups based on body weight 1week prior to dosing so that body weights were similar on the dayof dosing. Individual body weights were recorded at randomiza-tion and prior to dose administration on study day 0. Body weightswere also recorded during the FOB and prior to terminaleuthanasia.

2.6.3. Functional observational battery (FOB)

The FOB used in this study was based on a standardizedprocedure developed and used by the laboratory. It is based onprocedures published in the literature (Gad, 1982; Haggerty,1989; Irwin, 1968; Moser et al., 1991, 1988; O’Donoghue, 1989)and in the US EPA OPPTS Health Effects Test Guideline 870.6200(US EPA, 1996). The FOB was modified to include additional details(e.g., the coarseness of tremor) to distinguish findings particularlyassociated with pyrethroid intoxication. Tremors were scored on a

severity scale of 1–5: 1 indicates no tremors; 2 indicates slight(1.5 mm) tremors; 3 indicates moderately coarse (3 mm) tremorswith slight impairment; 4 indicates markedly coarse (4.5 mm)tremors with marked impairment of locomotion and 5 indicatesextremely coarse (6 mm) tremors and locomotion impossible. Foraerial righting and landing footsplay, the animals landed on awell-cushioned surface and the test was not performed if theanimal was judged unable to perform the test. Observations wererecorded for all animals at the time of peak effect after testsubstance administration. Study technicians were given specialtraining to distinguish classical symptoms of pyrethroid intox-ication. Testing was performed without the technician’s knowl-edge of dose group assignment and inter-observer reliability wasestablished to verify consistency among the technicians byverification of training in the laboratory with standard pyre-throids. The FOB was performed in a sound-attenuated roomequipped with a white noise generator set to operate at70 � 10 dB, while home cage observations were performed in theanimal room.

The FOB consisted of six types of observations: home cage,handling, open field, sensory, neuromuscular and physiologicalobservations. Table 3 summarizes the specific parameters eval-uated for each category of the FOB observations.

2.6.4. Macroscopic examination (unscheduled deaths)

Animals found dead during the study underwent a grossnecropsy examination. This included, but was not limited to,examination of the external surface, all orifices, and the cranial,thoracic, abdominal and pelvic cavities, including viscera.

2.6.5. Scheduled euthanasia

Following clinical observations on the day after treatment,surviving animals were euthanized by carbon dioxide inhalationand discarded without necropsy.

2.6.6. Statistical methods

Each mean was presented with the standard deviation (S.D.),and the number of animals (N) used to calculate the mean. Thenumeric data were subjected to statistical analyses by theDunnett’s Test, except for hindlimb resistance and extensorstrength in the neuromuscular parameters, which were subjectedto the Fisher’s Exact Test. In addition, Principal ComponentAnalysis and Factor Analysis were used for a more thoroughinterpretation of these data, as described in detail in Part II of thispaper in this journal (Breckenridge et al., 2009).

3. Results

3.1. Clinical observations, gross observations and survival

One male each in the high-dose groups of b-cyfluthrin (45 mg/kg), resmethrin (750 mg/kg) and bifenthrin (55 mg/kg) and twomales in the S-bioallethrin group (300 mg/kg) were found deadwithin 24 h after treatment. The deaths did not interrupt theevaluation of the neurotoxicity component of the study. Thesedeaths were attributed to the respective test substances and reflectthat the objective to test the highest dose possible to reveal the fullspectrum of acute neurobehavioral signs of toxicity was achievedor slightly exceeded for these products.

Signs of toxicity had resolved in all surviving animals by the dayafter treatment with the following exceptions: tremors for onemale in the 55 mg/kg bifenthrin group on the day after treatment;dried yellow matting in the ventral abdominal area for four and sixmales in the 25 and 35 mg/kg deltamethrin groups, respectively,and dried brown material at the base of the tail for five males eachin these same groups.

Table 3Functional observational battery evaluations.

Type of observations Parameters

Home cage Posture Biting

Convulsions/tremors Palpebral (eyelid) closure

Feces consistency

Handling Ease of removal from cage Ease of handling animal in hand

Lacrimation/chromodacryorrhea Salivation

Pilorection Fur appearance

Papebral closure Respiratory rate/character

Eye prominence Mucous membrane/eye/skin color

Red/crusty deposits Muscle tone

Open field Mobility Gait

Rearing Arousal

Convulsions/tremors Urination

Grooming Defecation

Bizarre/stereotypic behavior Gait score

Time to first step (s) Backing

Sensory Approach response Touch response

Startle response Tail Pinch response

Pupil response Eyeblink response

Forelimb response Hindlimb response

Air-righting response Olfactory response

Neuromuscular Hindlimb extensor strength Grip strength: hind and fore limb

Hindlimb foot splay Rotorod performance

Physiological Body temperature Body weight

Catalepsy

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16 S5

3.2. Body weight

Body weight for test substance-treated animals was generallysimilar to the concurrent control group values on the day of dosingand at the scheduled euthanasia. Treatment-related reductions inbody weight one day after treatment were found for three testmaterials: b-cyfluthrin (25 and 45 mg/kg), l-cyhalothrin (10 and20 mg/kg) and bifenthrin (55 mg/kg).

3.3. FOB

FOB findings at the times of peak effect for the Type I and Type IIpyrethroids are summarized in Tables 4 and 5, respectively. Thefindings are grouped into home cage, handling, open field, sensoryand neuromuscular observations and are listed from top to bottomin each table for each treatment group. The pyrethroids are listedfrom least potent to most potent, based on administered dose, fromleft to right across the top of each table. Detailed neuromuscularobservations (grip strength, rotarod performance and hindlimbfootsplay) and physiological observations are summarized sepa-rately in Tables 6–9 below.

3.4. Home cage observations

Home cage observations for the Type I pyrethroids consisted oftremors, ranging in intensity from slight to moderate (Table 4).Two animals exposed to the high-dose of S-bioallethrin alsoexhibited markedly coarse tremors. One animal each in the high-dose groups of S-bioallethrin and bifenthrin exhibited extremelycoarse tremors. All Type I pyrethroids also showed a dose-related,intensity-related incidence in clonic convulsions. The most severeconvulsions, whole body convulsions, were displayed by animalstreated with pyrethrins, resmethrin, S-bioallethrin, permethrinand bifenthrin, particularly at the higher doses. Other home cageobservations not associated with control animals includedflattened limbs, rearing and splayed hindlimbs, which occurredin only one or two animals/group.

Home cage observations for the Type II pyrethroids exhibitedfar fewer incidences of convulsions and no incidences of tremors

greater than slight tremors (Table 5). Slight tremors were noted inonly one or two animals among all animals treated with Type IIpyrethroids, and three animals exhibited slight tremors in thehigh-dose group of fenpropathrin. One or two animals per groupexhibited clonic convulsions when treated with one of several TypeII pyrethroids. Four animals at the low dose of b-cyfluthrinexhibited clonic convulsions with repetitive movement of themouth/jaw and three animals at the high-dose of deltamethrinexhibited whole body clonic convulsions. Splayed hindlimbs andflattened, extended limbs were exhibited by animals treated withdeltamethrin at the mid- and high-doses. Splayed hindlimbs werenoted among two animals each in the cypermethrin or l-cyhalothrin high-dose groups. There were isolated incidences ofbiting of self for cypermethrin, b-cyfluthrin and fenpropathrin andbiting of cage for b-cyfluthrin, deltamethrin and l-cyhalothrin.

3.5. Handling observations

Handling observations following treatment with the Type Ipyrethroids consisted primarily of single animals displaying ventralstaining, except for bifenthrin (where the incidence was higher) andS-bioallethrin (where ventral staining was not evident) (Table 4).Animals treated with bifenthrin also displayed ventral wetness,abdominogenital wetness, slightly soiled fur, pale mucous mem-branes, pale skin, and pulsating eyes (spontaneous nystagmus).Salivation with incidences greater than control animals was limitedto animals treated with bifenthrin (40 and 55 mg/kg doses) and withS-bioallethrin (200 and 200 mg/kg doses).

Handling observations following treatment with the Type IIpyrethroids showed a significant incidence of salivation for all testsubstances, particularly at the mid- and high-dose levels (Table 5).Lacrimation was observed in animals treated with l-cyhalothrin,b-cyfluthrin and deltamethrin (one animal in the mid-dose group).Ventral staining, ventral wetness, or abdominogenital wetness wasobserved in all animals treated with Type II pyrethroids at one ortwo dose levels, with the exception of fenpropathrin. Pulsatingeyes (spontaneous nystagmus) were reported in a single animalfrom each of the high-dose groups for esfenvalerate anddeltamethrin.

Table 4Summary of FOB findings for Type I pyrethroids.

Test substance

Pyrethrins Resmethrin S-Bioallethrin Permethrin Bifenthrin Tefluthrin

4a 4a 2a 4a 8a 8a

400b 800b 350b 500b 750b 150b 200b 300b 200b 250b 40b 55b 10b 15b

Home cage observations

Sitting, head held low 1 1 (4) 2 (4) 1

Flattened, limbs may be extended 1 1 1 1 1

Rearing 2 1 1 1 2 1

Splayed hindlimbs 1 1 2 1 1 (1) 2 (1) 1 (1) 2 (1)

Clonic convulsions (repetitive movement of mouth/jaw) 1 (1) 3 (1) 2 1 1 3

Clonic convulsions (back twitches) 1 3 5 5 2 1 3 5 2

Clonic convulsions (head/body twitches) myoclonus 1 1 4 6 3 6 2 2 1 2 1 1

Clonic convulsions (irregular jerking, limbs) 1 3 1 1 2 2 4 3 2 2

Clonic convulsions (whole body) 1 3 2 1 4 1 5 1 4

Slight tremors 1 2 3 6 7 3 4 2 4 8 6 6 2 3

Moderately coarse tremors 1 1 2 1 1 1 3

Markedly coarse tremors 2

Extremely coarse tremors 1 1

Biting of self 1 (1)

Handling observations

Salivation 1 (1) 1 (1) 2 1 1 (1) 4 (1) 2 (1)

Ventral staining 1 1 1 1 1 3 5 1 1

Ventral wetness 1 2

Abdominogenital wetness 1

Slightly soiled fur 2 1

Red deposits—nose 2 (1) 1 (1) 3 (1) 3 (1) 1 2 3 6 (1) 9 (1)

Pale mucous membranes 1

Pale skin 2

Pulsating eyes 4 1

Exophthalmus 1

Moderately difficult to remove from cage 1 1 1 1

High difficulty in handling 1

Open field observations

Ataxia, excessive sway, rocks, lurches 1 1 4 1 1 3 1 1

Slightly impaired mobility 1 2 2 1 1 1 1

Moderately impaired mobility 2 1

Walking on tiptoes 1 (1) 3 (1) 2 (1) 1 1 1 1 8 2 1

Body drags, body sways, abdomen contacts surface 1 2

Hindlimbs splayed or dragging 1 1 2 1 2

Hunched body 1 1 3 1 1 2 3 2 1 1 5 4 2 1

Gait impairment—slight 3 1 1

Gait impairment—considerable 3

Gait impairment—severe, cannot walk without falling 1

Clonic convulsions (back twitches) 1 3 1 2 2 1

Clonic convulsions (head/body twitches) 2 (1) 3 (1) 9 (1) 5 (1) 3 5 7 2 (1) 4 (1) 6 7 1 4

Clonic convulsions (irregular jerking, limbs) 2 1 1 1 7 7 3 2

Clonic convulsions (whole body) 2 1 3 3 1

Slight tremors 1 (1) 3 (1) 6 (1) 3 (1) 6 (1) 6 4 3 2 (1) 1 (1) 8 4 2 3

Moderately coarse tremors 3 3 3 1 1 2 5 1 4 1

Markedly coarse tremors 1 1 1 1 3 2

Extremely coarse tremors 2 1

Low arousal level 1 2

Stereotypic behavior (head flick)

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M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16 S7

3.6. Open Field Observations

Findings following treatments with all Type I pyrethroidsincluded tremors, ranging in severity from slight to extreme, andclonic convulsions, ranging in severity from back twitches to head/body twitches, to irregular jerking limbs, to whole body convul-sions (Table 4). All Type I pyrethroids exhibited hunched body,walking on tiptoes and some degree of clonic convulsions. Wholebody clonic convulsions were noted in groups treated withresmethrin, S-bioallethrin, bifenthrin and tefluthrin. Ataxia withexcessive sway, rocks and lurches was noted for resmethrin, S-bioallethrin, permethrin, bifenthrin and tefluthrin.

Following treatment with Type II pyrethroids, slight tremorswere observed for esfenvalerate, b-cyfluthrin and fenpropathrin inone or two animals each, except for two animals in the high-dosegroup of fenpropathrin with moderately coarse tremors (Table 5).Scattered, generally low incidences of clonic convulsions werenoted for all Type II compounds, except l-cyhalothrin. Gaitimpairment was noted, with severity ranging from ‘‘slight’’ to‘‘severe’’ for all Type II pyrethroids. Other notable findings includeddragging or splayed hindlimbs for all Type II pyrethroids, exceptfenpropathrin; walking on tiptoes for all compounds, except b-cyfluthrin and fenpropathrin; some degree of impaired mobilityfor all compounds, except fenpropathrin; low arousal level for allcompounds, except fenpropathrin, and stereotypic head flickbehavior for all compounds. Isolated incidences of stereotypicpawing/burrowing behavior were noted for esfenvalerate and b-cyfluthrin; as was stereotypic writhing behavior for deltamethrin.

3.7. Sensory observations

All animals treated with Type I pyrethroids exhibited exag-gerated hindlimb flexion. Animals treated with S-bioallethrin orpermethrin also exhibited no hindlimb extension. Other significantsensory observations, noted in groups treated with S-bioallethrinand permethrin only, included more energetic touch, approach andstartle responses and slightly uncoordinated air-righting reflex(Table 4).

Animals treated with Type II pyrethroids generally gave moreenergetic response to touch, except for those treated with b-cyfluthrin and fenpropathrin, and more energetic response tostartle response. However, the startle responses for groups treatedwith cypermethrin, deltamethrin and l-cyhalothrin should bedisregarded due to high incidence of this response amongrespective control groups. Bizarre reactions to tail pinch werenoted for animals treated with cypermethrin and esfenvalerate;bizarre reactions to the startle response were noted for animalstreated with esfenvalerate and deltamethrin, and no reaction toolfactory orientation was noted for animals treated with cyperme-thrin, b-cyfluthrin, deltamethrin or l-cyhalothrin. The air-rightingreflex was altered for a few animals treated with cypermethrin, b-cyfluthrin or l-cyhalothrin (Table 5).

3.8. Physiological observations

The physiological observations from the FOB for catalepsy, bodytemperature and body weight are listed vertically in Tables 6 and 7following animal treatments with the Type I and II pyrethroids,respectively. For each parameter, separate control groups for the2-, 4- and 8-h times-to-peak effect were included. Times-to-peakeffect are indicated horizontally for each pyrethroid. Numericalvalues can be compared to the appropriate control groups of thesame time-to-peak effect. All pyrethroids were evaluated in thesame blocks with separate control groups as indicated in Section 2.Numerical mean values for treatment groups were compared tothe appropriate controls using the Dunnett’s Test. The pyrethroids

Table 5Summary of FOB findings for Type II pyrethroids.

Test substance

Cypermethrin Esfenvalerate b-Cyfluthrin Deltamethrin Fenpropa-thrin

l-Cyhalo-thrin

4a 8a 2a 4a 2a 4a

65b 100b 150b 15b 25b 50b 12.5b 25b 45b 12.5b 25b 35b 15b 30b 10b 20b

Home cage observations

Sitting, head held low 1 2 2 1 4 (4) 7 (4) 2 3 4

Flattened, limbs may be extended 1 6 1

Rearing 1 2 (1) 1 2 (1) 1 (1)

Splayed hindlimbs 2 2 (1) 5 4 2

Prostration 2 2

Clonic convulsions (repetitive movement of mouth/jaw) 2 (1) 3 (1) 2 4 1 1 1 (1) (1) 1 1 (1) 2 (1)

Clonic convulsions (back twitches) 2

Clonic convulsions (head/body twitches) 1 2 1

Clonic convulsions (irregular jerking, limbs) 1 1 1 2 1

Clonic convulsions (whole body) 3

Slight tremors 1 1 2 1 1 1 2 3 1 1

Moderately coarse tremors

Markedly coarse tremors

Extremely coarse tremors

Biting of self 1 2 1

Biting of cage 1 1 3 1

Handling observations

Exaggerated hindlimbflexion 2 1 1

Salivation 1 (1) 2 (1) 4 (1) 1 (1) 4 (1) 5 (1) 4 10 1 (1) 5 (1) 10 (1) 2 2 4 (1) 10 (1)

Lacrimation 2 1 2 5

Ventral staining 2 7 5 1 5 1 4

Ventral wetness 1 4 3 2 5 4 10 3 10

Abdominogenital wetness 2 2 2 4 8 6

Slightly soiled fur 1 2 2 2 2 1

Red deposits—mouth 1 2 2

Red deposits—nose 1 (1) 2 (1) 2 (1)

Red and/or crusty deposits—eyes 1

Pale mucous membranes

Pale skin 1 1 1 3

Pulsating eyes 1 1

Exophthalmus

Moderately difficult removal from cage 1 2 1 1 1

High difficulty in handling 1 3

Open field observations

Ataxia, excessive sway, rocks, lurches 3 8 4 2 5 4 4 2 2 9

Slightly impaired mobility 3 2 1 2 2 2 1 5

Moderately impaired mobility 3 1 1 1 2 2 4

Totally impaired mobility 1 2 5 1

Walking on tiptoes 2 (1) 2 (1) 3 (1) 3 3 3 2 (1) 5 (1) 2 (1) 2 (1) 7 (1)

Body drags, body sways, abdomen contacts surface 1 2 5 1

Hindlimbs splayed or dragging 4 3 2 2 1 1 2 3 7 1

Hunched body 2 7 8 3 2 5 4 6 5 1 1 4 9

Gait impairment—slight 2 1 5 2 1 2 1

Gait impairment—considerable 3 2 2 1 1 1 2 1 5

Gait impairment—marked, falls often 3 2 1 1 4

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Gait impairment—severe, cannot walk without falling 1 2 6 1

Clonic convulsions (repetitive movement of mouth/jaw) 1 1 2 5

Clonic convulsions (back twitches)

Clonic convulsions (head/body twitches) 1 5 3 1 3

Clonic convulsions (irregular jerking, limbs) 1 6 3 1 1 2 3

Clonic convulsions (whole body) 1 1 2

Slght tremors 1 (1) 1 (1) 1 (1) 2 1 2 1 2

Moderately coarse tremors 2

Markedly coarse tremors

Extremely coarse tremors

Low arousal level 2 (1) 4 (1) 4 (1) 3 2 5 4 (1) 7 (1) 1 (1) 8 (1)

Somewhat high arousal level

Very high arousal level

Stereotypic behavior (head flick) 2 2 2 3 3 4 1 1 1 2 2 6 5

Stereotypic behavior (pawing, burrowing) 1 1

Stereotypic behavior (writhing) 2 3

Sensory observations

Approach reaction—no response 1 2 3 1 3

Approach reaction—more energetic response (more than slight) 1

Touch response—no reaction 1 1 2 3 5

Touch response—more energetic response (more than slight) 1 1 2 1 1 (5) 4 (5) 1

Startle response—no reaction 1 1 1 2 1 3

Startle response—more energetic response (more than slight) 1 (5) 3 (5) 1 2 1 1 1 1 (5) 1 (5) 2 1 (5)

Startle response—bizarre reaction 1 1 4

Tail pinch response—bizarre reaction 1 1 1

No pupil response

Olfactory orientation—no reaction 2 3 1 2 3 1 2 7

Air-righting reflex-lands on back 1

Air-righting reflex-lands on side 1 3 1 1 2

No hindlimb extension 2 4 3 1 1 1 6

No forelimb extension

Neuromuscular observations

Reduced hindlimb resistance 1 1 2 2 2 7 4

Note: Numericals represent the numbers of animals withfindings. Occurrences of findings in the controlgroup are indicatedinparenthesis next to the group finding. If the finding wasnot observedinthe control, nonumberhasbeen included.a Time-to-peak effect (h).b Dose levels (mg/kg).

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Table 6Physiological observations for Type I pyrethroids.

Controlsa Test substance

Pyrethrins Resmethrin S-Bioallethrin Permethrin Bifenthrin Tefluthrin

4b 4b 2b 4b 8b 8b

400c 800c 350c 500c 750c 150c 200c 300c 200c 250c 40c 55c 10c 15c

Catalepsy (s):

control, 2 h

0.2�0.05 0.2�0.00

0.3�0.09

0.3�0.09

0.2�0.00

Catalepsy (s):

control, 4 h

0.2�0.07 0.2�0.03

0.2�0.05

0.2�0.00

0.2�0.07

0.2�0.07

0.2�0.07

0.3�0.160.2�0.04

0.2�0.00

Catalepsy (s):

control, 8 h

0.2�0.04 0.2�0.04

0.3**�0.10

0.3�0.07

0.2�0.05

Body temperature

(8C): control, 2 h

39.0�0.39 39.0�0.85 39.5�0.46

38.8�0.44

Body temperature

(8C): control,

4 h

39.2�0.42 39.0�0.32

39.1�0.54

38.5�0.26

39.4�0.26

39.3�0.31

38.9�0.40

39.2�0.42 39.3�0.28

38.8�0.36

38.5�0.21

Body temperature

(8C): control, 8 h

38.7�0.30 39.4**�0.49

39.7**�0.33

38.8�0.37

39.0�0.46

Body weight (g):

control, 2 h

204.7�10.39 208.8�18.47

201.5�16.57

203.4�13.18207.6�13.11

Body weight (g):

control, 4 h

206.8�18.28 229.4�18.59

225.2�21.15

185.1�16.50

223.1�19.30

226.0�14.83

205.9�20.12

201.8�17.21231.2�13.59

187.9�10.25

Body weight (g):

control, 8 h

219.5�11.48 208.3�13.12

205.9�18.53

214.6�11.19

211.7�17.80

Values express mean and standard deviation (S.D.) of ten animals in each group.a A concurrent control group was assigned to each time to peak effect and/or testing group; all of the mean (�S.D.) control values for the Type I pyrethroids are represented in this table. Groupings of treatment groups with same time-

to-peak controls are explained in Section 2 and Table 2.b Time-to-peak effect (h).c Dose levels (mg/kg).** Significantly different from the respective control groupsa at the 0.01 level using Dunnett’s Test.

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Table 7Physiological observations for Type II pyrethroids.

Controlsa Test substance

Cypermethrin Esfenvalerate b-Cyfluthrin Deltamethrin Fenpropathrin l-Cyhalothrin

4b 8b 2b 4b 2b 4b

65c 100c 150c 15c 25c 50c 12.5c 25c 45c 12.5c 25c 35c 15c 30c 10c 20c

Catalepsy (s): control, 2 h 0.2�0.05 0.2�0.00

0.2�0.00

0.2�0.07

0.2�0.00

0.3�0.11 0.3�0.05

0.2�0.07

0.3�0.050.2�0.00

Catalepsy (s): control, 4 h 0.2�0.07 0.3�0.17

0.4�0.36

0.2�0.03

0.3�0.07

0.7**�0.56 0.2�0.07

0.4�0.240.2�0.04

0.2�0.00

Catalepsy (s): control, 8 h 0.2�0.04 0.2�0.00

0.2�0.07

Body temperature (8C):

control, 2 h

39.0�0.39 38.6�0.33

37.8�1.12**

37.4�0.49**

39.0�0.27

38.8�0.4238.8�0.44

Body temperature (8C):

control, 4 h

39.2�0.42 38.2�1.18

37.6�0.92**

38.5�0.25

36.4�1.77**

36.1�1.26**

38.7�0.81

36.1**�1.0638.8�0.36

38.5�0.21

Body Temperature (8C):

control, 8 h

38.7�0.30 38.7�0.33

38.9�0.36

38.6�0.3838.5�0.23

Body weight (g):

control, 2 h

204.7�10.39 207.2�16.79

203.1�15.97

201.6�13.56

203.5�15.71

201.7�12.85207.6�13.11

Body weight (g):

control, 4 h

206.8�18.28 203.5�9.91

204.3�17.53

185.1�11.12

224.5�18.56

218.5�17.98

201.8�23.57

199.3�9.62231.2�13.59

187.9�10.25

Body weight (g):

control, 8 h

219.5�11.48 196.8�10.79

209.8�15.67

209.3�16.81198.5�12.85

Values express mean and standard deviation (S.D.) of ten animals in each group.a A concurrent control group was assigned to each time to peak effect and/or testing group; all of the mean (�S.D.) control values for the Type II pyrethroids are represented in this table. Groupings of treatment groups with same time-

to-peak controls are explained in Section 2 and Table 2.b Time-to-peak effect (h).c Dose levels (mg/kg).** Significantly different from the respective control groupsa at the 0.01 level using Dunnett’s Test.

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Table 8Neuromuscular observations for Type I pyrethroids.

Controlsa Test substance

Pyrethrins Resmethrin S-Bioallethrin Permethrin Bifenthrin Tefluthrin

4b 4b 2b 4b 8b 8b

400c 800c 350c 500c 750c 150c 200c 300c 200c 250c 40c 55c 10c 15c

Grip stength (g): mean� S.D.

Forelimb,

2-h control

448.2�71.65 436.7�88.84

406.6�81.27

330.8*�70.29490.1�86.73

Forelimb,

4-h control

510.9�72.28 543.5�103.75

439.1�99.20

406.7�74.40

410.1�113.58

517.4�102.30

423.2�136.26

426.0�86.69518.4�102.52

483.4�74.73

Forelimb,

8-h control

536.6�118.13 300.8**�102.10

311.6**�71.25

450.7�136.26

417.4�151.71

Hindlimb,

2-h control

175.1�41.97 270.8�55.47

162.6�61.81

133.4�31.27264.1�36.39

Hindlimb,

4-h control

235.1�40.56 260.0�48.46

177.0*�43.80

244.2�31.15

199.1�40.73

202.4�68.83

204.3�52.20

180.7�45.38240.0�60.47

265.7�38.61

Hindlimb,

8-h control

238.2�47.48 170.1�61.20

177.6�58.86

221.6�69.58

188.3�66.19

Rotarod performance (s): mean� S.D.

2-h control 50.4 �47.76 48.4�42.46

78.6�36.60

48.0�49.5087.1�46.69

4-h control 95.3�43.54 93.7�40.9

109.7�25.22

82.3�45.49

55.7�39.06

63.3�41.04

94.6�33.84

108.7�35.7385.9�42.45

106.2�30.16

8-h control 69.8�50.43 77.3�50.29

50.2�52.27

109.0�34.88

83.5�50.75

Hindlimb footsplay (mm): mean� S.D.

2-h control 65.2�12.15 64.7�9.62

64.4�13.53

49.1�20.8761.9�10.75

4-h control 71.1�8.97 79.0�12.62

66.0�15.74

70.2�14.40

69.9�12.86

64.0�15.03

76.5�13.18

66.8�12.1672.6�12.38

72.0�10.65

8-h control 66.6�13.91 53.6�11.58

46.9�12.04

59.9�17.25

65.1�12.69

Values express mean and standard deviation of ten animals in each group.a A concurrent control group was assigned to each time of peak effect and/or testing group; all of the mean (�S.D.) control values for the Type I pyrethroids are represented in this table. Groupings of treatment groups with same time-

to-peak controls are explained in Section 2 and Table 2.b Time-to-peak effect.c Dose levels (mg/kg).* Significantly different from the control at the 0.05 level using the Dunnett’s Test.** Significantly different from the controls at the 0.01 level using Dunnett’s Test. The Fisher’s Exact Test was performed on the hindlimb resistance and extensor strength only. All other values used the Dunnett’s Test.

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Table 9Neuromuscular observations for Type II pyrethroids.

Controlsa Test substance

Cypermethrin Esfenvalerate b-Cyfluthrin Deltamethrin Fenpropathrin l-Cyhalothrin

4b 8b 2b 4b 2b 4b

65c 100c 150c 15c 25c 50c 12.5c 25c 45c 12.5c 25c 35c 15c 30c 10c 20c

Grip strength (g): mean� S.D.

Forelimb, 2-h control 448.2�71.65 478.3�111.93

327.5*�73.62

335.1*�120.73

378.3�109.09

418.3�86.78490.1�86.73

Forelimb, 4-h control 510.9�72.28 505.8�50.62

463.4�80.29

374.9�107.41

430.9�96.20

325.1**�166.21

175.8**�130.48

513.3�156.62

341.8*�154.44518.4�102.52

483.4�74.73

492.4�91.62

Forelimb, 8-h control 536.6�118.13 385.1�79.42

410.9�115.24

379.*�79.71456.6�95.80

Hindlimb, 2-h control 175.1�41.97 252.5�30.40

146.6�24.92

149.3�33.88

156.6�36.51

141.8�40.50264.1�36.39

Hindlimb, 4-h control 235.1�40.56 285.0�63.34

189.2�46.82

156.6**�56.78

214.2*�55.73

148.2**�57.40

85.7**�34.63

195.9�51.85

153.2**�50.70240.0�60.47

265.7�38.61

279.1�53.55

Hindlimb, 8-h control 238.2�47.48 236.0�42.15

214.0�82.86

184.4�34.82264.3�50.40

Rotarod performance (s): mean� S.D.

2-h control 50.4�47.76 99.9�33.66

52.0�49.58

94.1�45.69

86.3�48.77

93.0�39.7187.1�46.69

106.12�30.16

4-h control 95.3�43.54 82.6�39.25

79.9�51.61

38.5*�43.81

76.7�55.98

73.3�52.54

30.3*�49.97

109.8�29.84

53.2�45.3485.9�42.45

106.2�30.16

86.8�43.93

8-h control 69.8�50.43 88.3�38.62

108.5�35.36

64.0�44.06112.8�15.83

Hindlimb footsplay (mm): mean� S.D.

2-h control 65.2�12.15 75.5*�13.53

55.1�18.73

60.5�21.52

60.2�16.30

64.1�19.48

4-h control 71.1�8.97 69.0�5.75

78.2�11.68

66.4�18.45

57.3*�14.19

59.2�21.83

45.9**�22.68

67.4�14.00

51.0*�21.8572.6�12.38

72.0�10.65

65.3�13.63

8-h control 66.6�13.91 59.8�19.54

53.8�19.93

54.8�22.6569.3�13.34

Values express mean and standard deviation of ten animals in each group.a A concurrent control group was assigned to each time of peak effect and/or testing group; all of the mean (�S.D.) control values for the Type I pyrethroids are represented in this table. Groupings of treatment groups with same time-

to-peak controls are explained in Section 2 and Table 2.b Time-to-peak effect.c Dose levels (mg/kg).* Significantly different from the control at the 0.05 level using the Dunnett’s Test.** Significantly different from the controls at the 0.01 level using Dunnett’s Test. The Fisher’s Exact Test was performed on the hindlimb resistance and extensor strength only. All other values used the Dunnett’s Test.

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M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16S14

are listed from left to right across the top of each table from leastpotent to most potent, based on administered dose.

There were only a few statistically significant physiologicalfindings among animals treated with either Type I or Type IIpyrethroids. Only bifenthrin treatment resulted in significantlylonger catalepsy response at the high-dose and increased bodytemperature at both doses (Table 6). There were no othersignificant findings for Type I compounds.

As noted in Table 7, body temperature was significantly reducedby treatment with either cypermethrin, deltamethrin, l-cyhalo-thrin, or b-cyfluthrin. Deltamethrin caused a statistically sig-nificant longer catalepsy response among animals given the high-dose level. No significant effects on the physiological parameterswere caused by either esfenvalerate or fenpropathrin.

3.9. Neuromuscular observations

There were no significant general neuromuscular findings forthe Type I pyrethroids (Table 4). General neuromuscular finding forthe Type II pyrethroids included reduced hindlimb resistance formid- or high-dose animals treated with cypermethrin, esfenvale-rate, b-cyfluthrin, deltamethrin or l-cyhalothrin (Table 5).

Detailed quantitative neuromuscular observations for gripstrength (forelimb and hindlimb), rotorod performance andhindlimb footsplay are summarized for Type I and Type IIpyrethroids in Tables 8 and 9, respectively. Data represent themean and standard deviations for each group for each measure-ment. Treated groups are compared to their respective 2-, 4- or 8-htime-to-peak effect control groups using the Dunnett’s Test.

Statistically significant findings for the Type I pyrethroidsincluded reduced hindlimb grip strength for the high-dose grouptreated with pyrethrins; reduced forelimb grip strength for thehigh-dose group treated with S-bioallethrin, and for both dosegroups treated with bifenthrin (Table 8).

Statistically significant neuromuscular findings for Type IIpyrethroids included reduced forelimb grip strength for esfenva-lerate, b-cyfluthrin, deltamethrin and l-cyhalothrin; reducedhindlimb grip strength for cypermethrin, deltamethrin and l-cyhalothrin; slower rotorod performance for cypermethrin anddeltamethrin; and reduced hindlimb footsplay for deltamethrinand l-cyhalothrin (Table 9). The statistically elevated hindlimbfootsplay for the low dose group given b-cyfluthrin is notconsidered biologically significant.

4. Discussion

Pyrethroids act on the nervous system as a primary target organ(Wolansky et al., 2006; Wolansky and Harrill, 2008; Soderlundet al., 2002). The most common and shared effect of pyrethroids isthe induction of neurotoxicity following acute exposure. This studywas designed to characterize and compare the neurobehavioralmanifestations of toxicity of 12 commercial pyrethroid insecticidesin rats under standardized conditions following acute oralexposure, at minimally toxic and near-lethal doses. It is the firstmajor GLP study to compare all twelve compounds simultaneouslyfor effects on the functional observational battery. Thus, it providesdata to assist with determining whether there is evidence of one ormore common mode(s) or mechanism(s) of toxicity withcommercial pyrethroid insecticides as well as for possible futureclassification of new pyrethroids (Communication: US EPAScientific Advisory Panel Meeting, June 16–17, 2009).

Regulatory guideline acute neurotoxicity studies in rats havebeen conducted on some commercial pyrethroids via oralexposure, and an extensive review by Soderlund et al. (2002)found inconsistencies between the results of these studies and theclassical T and CS syndrome classifications. In addition, the

regulatory studies themselves cannot be readily compared forevidence of common effect and/or common mechanism becausethey used different test conditions, rat strains, vehicles, dosevolumes, criteria to characterize the effect, etc. Neurobehavioralassessment of the pyrethroids is known to be particularlyinfluenced by methodological changes in the route, vehicle, dosingvolume, species and strain (Crofton et al., 1995; Soderlund et al.,2002; Wolansky and Harrill, 2008).

Wolansky et al. (2007) found that dose volume itself influencesthe potency of bifenthrin on neurobehavioral endpoints. Doses ofbifenthrin of 1, 6, 12 and 20 mg/kg in 1 ml corn oil/kg werecompared to doses of bifenthrin of 6, 12, 20 and 26 mg/kg in 5 mlcorn oil/kg for potency on motor activity and functional observa-tional battery tests in male Long Evans rats. Bifenthrin effects wereapproximately two-fold more potent at dose volumes of 1 ml/kgbody weight, compared to 5 ml/kg body weight on both measures(Wolansky et al., 2007). The present study dosed bifenthrin at 40and 55 mg/kg in 5 ml corn oil/kg to male Sprague–Dawley rats. Thetime-to-peak effect was later (8 h) compared to bifenthrin dosed in1 ml/kg (3.5–4 h). The test substances are considered comparablein composition; therefore, it can be concluded that dose volumemay have a considerable impact on potency and time-to-peakeffect, but not necessarily on the types of responses. Thedifferences in potency and time-to-peak effect noted for bifenthrindue to dose volume are most likely related to toxicokineticdifferences.

Evidence for an effect of dose volume on potency can also befound by comparing the data on cypermethrin and permethrindosed in a dose volume of 1 ml corn oil/kg in the study by McDanieland Moser (1993) and these same pyrethroids dosed in a dosevolume of 5 ml corn oil/kg in the present study. McDaniel andMoser (1993) compared the effects on motor activity andfunctional observational battery in rats dosed with 20, 60 and120 mg/kg cypermethrin and 25, 75 and 150 mg/kg permethrin in1 ml corn oil/kg body weight. Each pyrethroid produced a distinctsyndrome of neurobehavioral responses which was different fromthat of the other pyrethroid (cypermethrin and permethrin). Theauthors confirm the sensitivity of the FOB to distinguish Type I andII pyrethroids at low doses. In contrast, higher doses of bothpyrethroids were needed at a dose volume of 5 ml corn oil/kg toproduce comparable toxicity in the present study: 65, 100, 150 mg/kg cypermethrin and 200 and 250 mg/kg permethrin. It should benoted that the test materials in the study by McDaniel and Moser(1993) may have had a different isomer composition and puritythat would be expected to contribute to toxicity and potencydifferences. Thus, the two studies are not directly comparable.Nevertheless, this comparison underscores the importance of dosevolume in comparing different studies from different laboratories.As with many other studies evaluated in the Soderlund et al. (2002)review, a study of the neurobehavioral effects of cyhalothrin dosedin distilled water in Wistar rats cannot be compared to the presentstudy because animals were dosed daily for 7 consecutive days, adifferent vehicle (water) was used and an undefined commercialformulation was used (92.7% purity of active ingredient, otheringredients not described) (Righi and Palermo-Neto, 2003).

Therefore, the present study provides comparative data ontwelve commercial pyrethroids under identical test conditions. Inthis way, the rather unique clinical signs of neurotoxicity exhibitedby the commercial pyrethroids could be evaluated with respect tocommon mechanism(s) in an accurate side-by-side comparison.The FOB is considered an appropriate measure to evaluate andcompare pyrethroid neurotoxicity in order to show distinctionsand commonality among both non-cyano and a-cyano-containingpyrethroids (McDaniel and Moser, 1993).

The complexity of the current dataset provides a challenge toidentify associations among the set of tested pyrethroids using a

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16 S15

visual or manual approach. The approach used in the earlyclassification studies did not include full dose-response andincidence data on the pyrethroid isomers tested. For example,Verschoyle and Aldridge (1980) simply labeled pyrethroid isomersas ‘‘T’’ or ‘‘CS’’ based on single doses at near lethal or lethal levelswithout regard to incidence, severity or dose-response. Soderlundet al. (2002) compared the different regulatory acute oralneurotoxicity studies for the presence (+) or absence (�) of keyclinical T and CS signs without consideration of dose-responserelationships (see their Tables 3 and 4). For the purposes ofevaluation of the large dataset in the present study, two statisticalapproaches were employed to provide a better interpretation ofthe findings: Principal Component Analysis (PCA) and FactorAnalysis (FA). Both techniques are powerful statistical tools ideallysuitable for the present dataset. The PCA approach is able tosimultaneously analyze 56 separate neurobehavioral findingsacross the 38 control and treatment groups that comprise thisstudy in an objective manner. PCA also avoids the limitations ofvisual or subjective examination. PCA can demonstrate associa-tions or correlations empirically, based on mathematical models.Such associations provide a deeper and more rigorous under-standing of possible mechanisms of pyrethroid toxicity. Thecomplete results of the PCA and FA evaluations of the present FOBdata are included in Part II of this paper (Breckenridge et al., 2009).

Breckenridge et al. (2009) found that the different behavioralresponse patterns of pyrethroids could be defined by four principalcomponents, which demonstrate a dose-response relationshipwith one or more pyrethroids. Based on this analysis, individualchemicals were arrayed by a group of highly correlated (p � 0.5)behaviors, which constituted principle component factors. Thenon-cyano pyrethroids tended to array themselves in a dose-response manner primarily along two of the principal componentsthat were described by the following behavioral effects: tremors,myoclonus, elevated temperature, exaggerated response to sen-sory stimuli, difficulty handling, head flicking, jerking movements,pulsating eyes (spontaneous nystagmus). The a-cyano pyrethroidsarrayed themselves in a dose-related manner, primarily along twodifferent principal components described by these behavioraleffects: excessive salivation, impaired mobility, lower tempera-ture, non-reactivity to sensory stimuli, lacrimation, writhing,neuromuscular weakness, abnormal posture. For ease of compar-ison, while not strictly consistent with historical characterizations,the principle components that best described the Type Ipyrethroids were designated Ta and Tb, while those for Type IIpyrethroids were labeled CSa and CSb.

The results from the present study confirm the early reports ofdifferences in behavioral response patterns associated with Type Iand Type II pyrethroids, which were described by Verschoyle andAldridge (1980), Casida et al. (1983) and Soderlund et al. (2002).The magnitude and pattern of responses varied with individualpyrethroids, with some showing weaker responses (fenpropathrin,pyrethrin) and others showing stronger responses (l-cyhalothrin,deltamethrin, resmethrin, bifenthrin). Two cyano (Type II)pyrethroids (deltamethrin, esfenvalerate) showed some degreeof activity typical of non-cyano (Type I) pyrethroids. Fenpropathrinshowed a weak association with both T and CS signs in the FOBusing PCA (Breckenridge et al., 2009).

PCA and FA techniques were further used to evaluate the resultsfrom in vitro ion channel studies on the effects of the pyrethroidson the sodium, calcium and chloride channels (Breckenridge et al.,2009). Additional studies include (1) the effects of pure pyrethroidisomers on mammalian sodium ion channel isoforms expressed inXenopus oocytes (Choi and Soderlund, 2006); (2) modulation ofcalcium ion flux, membrane depolarization and glutamate releasefrom rat brain synaptosomes (Symington and Clark, 2005;Symington et al., 2008); and (3) change in open chloride ion

channel probability in mouse neuroblastoma cells (Burr and Ray,2004; Forshaw et al., 2000). Addition of the ion channel results tothose of the FOB study in a full PCA analysis provides compellingsupport for the common mechanism of toxicity of the pyrethroidsto be separated into at least two major classifications, the Type Iand Type II (Breckenridge et al., 2009). The results of the PCA/FAindicate that the responses of the ion channels to pyrethroidsgenerally support the responses in the rat FOB; namely, theexistence of two separate groups of pyrethroids, each showingsimilar characteristics in these diverse systems. SupplementalTable S1, Evidence for Common Mechanism Groups for Pyre-throids, summarizes the evidence for two possible commonmechanism groups for the pyrethroids, based on the US EPAfive-step process for identifying common mechanism(s) of toxicity(US EPA, 1999).

Although there is no known common toxophore that mediatesacute toxicity of pyrethroids, the presence of the a-cyanosubstituent in the 3-phenoxybenzyl alcohol moiety of the moleculeconfers greater potency by an estimated order of magnitude in acutelethality studies in rodents (see reviews by Soderlund et al., 2002;Wolansky and Harrill, 2008). In addition, the manifestations of theparticular toxic effect of neurotoxicity can also be related to thepresence or absence of the a-cyano substituent, as noted in the earlydistinctions of the two structural classes of the early pyrethroids(Verschoyle and Aldridge, 1980; Lawrence and Casida, 1982) and bythe present study using the FOB. In the present study the potency ofthe a-cyano-containing pyrethroids was generally higher than thenon-cyano pyrethroids. The lowest dose tested ranged from10 to 65 mg/kg for the a-cyano pyrethroids with l-cyhalothrin(10 mg/kg)> deltamethrin and b-cyfluthrin (12.5 mg/kg) >fenpropathrin and esfenvalerate (15 mg/kg)> cypermethrin(65 mg/kg). The potency of the non-cyano pyrethroids was generallylower than the a-cyano pyrethroids based on the lowest dose tested:tefluthrin (10 mg/kg) > bifenthrin (40 mg/kg)> S-bioallethrin(150 mg/kg)> permethrin (200 mg/kg) > resmethrin (350 mg/kg)> pyrethrins (400 mg/kg).

5. Summary

It is proposed that the evidence to date supports one commonmechanism group that contains pyrethrins, bifenthrin, resmethrin,permethrin, S-bioallethrin and tefluthrin, with a second commonmechanism group that contains cypermethrin, deltamethrin,esfenvalerate, b-cyfluthrin l-cyhalothrin. Fenpropathrin exhibitsresponses in several systems that are similar to both groups. Furtheranalysis of these and other data will be required to make a refinedgrouping of the pyrethroids for cumulative risk assessment.

Conflict of interest

Myra L. Weiner: Former employee of FMC Corporation, apyrethroid manufacturer and member of the Pyrethroid WorkingGroup (sponsor of the data generated) during the conduct of thestudy. Larry Sheets: My employer, Bayer CropScience, manufac-tures and sells some of the pyrethroids that were evaluated in thisstudy. Dana Sargent: Employee of Bayer CropScience, a pyrethroidmanufacturer and member of the Pyrethroid Working Group(sponsor of the data generated). Charles Breckenridge: Employedby Syngenta, a basic manufacturer of b-cyfluthrin, cypermethrin,permethrin and resmethrin. These chemicals were evaluated inthis study. Mark Nemec: No competing interest.

Acknowledgments

This research was sponsored by the Pyrethroid Working Group,a trade association of manufacturers of commercial pyrethroid

M.L. Weiner et al. / NeuroToxicology 30S (2009) S1–S16S16

pesticidess registered in the United States. Member companiesinclude Amvac Chemical Corporation, Bayer CropScience, Chemi-nova, Dupont Crop Protection, FMC Corporation, Syngenta CropProtection and Valent USA Corporation.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.neuro.2009.08.014.

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