Differential lymphatic versus portal vein uptake of the synthetic pyrethroids deltamethrin and cis-permethrin in rats.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.

Toxicokinetics of cis- and trans-Permethrin: Influence of Isomer, Maturation, and Sex.

Permethrin exposure of children and adults is widespread in many populations, but knowledge of its relative toxicokinetics (TK) and health risks in immature age groups is lacking. Studies were conducted in rats to determine the influence of immaturity and sex (on plasma and target organ dosimetry of each of the insecticide's 2 isomers, cis- and trans-permethrin [CIS and TRANS]). Postnatal day 15, 21, and 90 (adult), Sprague Dawley rats were orally administered a graduated series of doses of CIS and TRANS in corn oil. Serial sacrifices were conducted over 24 h to obtain plasma, brain, liver, skeletal muscle, and fat profiles of CIS and TRANS. Levels of TRANS decreased relatively rapidly, despite administration of relatively high doses. Concentrations of each isomer in plasma, brain, and other tissues monitored were inversely proportional to the animals' age. The youngest pups exhibited 4-fold higher plasma and brain area under the curves than did adults. Little difference was observed in the TK of CIS or TRANS between adult male and female rats, other than higher initial plasma and liver CIS levels in females. Elevated exposure of the immature brain appears to be instrumental in increased susceptibility to the acute neurotoxicity of high-dose permethrin (Cantalamessa [1993]), but it remains to be established whether age-dependent TK is relevant to long-term, low-level risks.

Longitudinal investigation of haematological alterations among permethrin-exposed pesticide applicators in the Biomarkers of Exposure and Effect in Agriculture study.

OBJECTIVES: Permethrin use has been associated with an increased risk of multiple myeloma (MM) among pesticide applicators. However, the biological plausibility and mechanisms underlying this association are not fully understood. The aim of this study was to assess whether exposure to permethrin is related to haematological alterations among occupationally exposed pesticide applicators. METHODS: We conducted a longitudinal study among 33 pesticide applicators in the Biomarkers of Exposure and Effect in Agriculture study comparing haematological parameters in the offseason with the day after permethrin exposure and, for 27 participants, approximately 3 weeks postexposure. Complete blood counts with white blood cell differential and lymphocyte subsets were measured at each visit. Multivariate linear mixed effects models were used to assess the relationship between natural log-transformed haematological parameters and exposure to permethrin. RESULTS: The adjusted geometric mean immature granulocyte count was elevated among pesticide applicators following permethrin exposure compared with their offseason levels (37% increase, 95% CI 6% to 76%). Modest but statistically significant (p<0.05) alterations in red blood cell (RBC) parameters (eg, decreased RBC count and haemoglobin and increased mean corpuscular volume and RBC distribution width-SD) were also observed the day after permethrin use compared with offseason levels; decreases in RBC count and haemoglobin and increases in RBC distribution width-SD persisted approximately 3 weeks after permethrin use. CONCLUSIONS: Altered haematological parameters could be indicative of disrupted haematopoiesis, providing insights into the biological plausibility of the observed association between permethrin use and MM risk among pesticide applicators.

Effects of Acute Exposure of Permethrin in Adult and Developing Sprague-Dawley Rats on Acoustic Startle Response and Brain and Plasma Concentrations.

Permethrin is a type I (noncyano) pyrethroid that induces tremors at high concentrations and increases acoustic startle responses (ASRs) in adult rodents, however its effects in young rats have been investigated to a limited extent. ASR and tremor were assessed in adult and postnatal day (P)15 Sprague-Dawley rats at oral doses of 60, 90, or 120 mg/kg over an 8 h period. Permethrin increased ASR in adults, regardless of dose, and 20% of the high-dose rats showed tremor at later time points. For the P15 rats all doses induced tremor at all time points, and ASR was increased at 2 h in the 90 and 120 mg/kg groups with a trend in the 60 mg/kg group compared with controls. The 60 mg/kg group showed increased ASR at 4 and 6 h, whereas the 90 mg/kg group showed no differences from the controls at these times. The 120 mg/kg group showed decreased ASR from 4- to 8-h posttreatment. P15 and adult rats both showed plasma and brain cis- and trans-permethrin increases after dosing. After the same dose of permethrin, P15 rats had greater cis- and trans-permethrin in brain and plasma compared with adults. P15 rats had an increased tremor response compared with adults even at comparable brain permethrin concentrations. For ASR, P15 rats responded sooner and showed a biphasic pattern ranging from increased to decreased response as a function of dose and time, unlike adults that only showed increases. Overall, young rats showed greater effects from permethrin compared with adults.

Dose reconstruction in workers exposed to two major pyrethroid pesticides and determination of biological reference values using a toxicokinetic model.

A toxicokinetic model has been optimized to describe the time profiles of common biomarkers of exposure to permethrin and cypermethrin: trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids (trans-DCCA) and 3-phenoxybenzoic acid (3-PBA). The model then served to reproduce urinary time courses in exposed agricultural workers and predict corresponding absorbed doses. It allowed for the prediction of the main routes of exposure in workers during the study period. Modeling showed that simulating exposure mostly by the oral route, during the 3-day biomonitoring period, provided best-fits to the urinary time courses of most workers. This is compatible with an inadvertent oral exposure during work. According to best-fit scenarios, absorbed doses in workers reconstructed with the model reached a maximum of 2.4 µg/kg bw/day and were below the absorbed dose limits associated with an exposure to the reference dose values established by the U.S. Environmental Protection Agency (0.06 and 0.25 mg/kg bw/day for cypermethrin and permethrin, respectively) and the Acceptable Operator Exposure Level set by the European Commission (0.06 mg/kg bw/day for cypermethrin). Modeling was further used to derive biological reference values for cypermethrin and permethrin exposure. Respective values of 7 and 29 nmol/kg bw/day of trans-DCCA, and 3 and 13 nmol/kg bw/day of 3-PBA were obtained. None of the workers presented values above these biological reference values.

Simultaneous determination of cis-permethrin and trans-permethrin in rat plasma and brain tissue using gas chromatography-negative chemical ionization mass spectrometry.

A sensitive method for the simultaneous determination of cis-permethrin (cis-PERM) and trans-permethrin (trans-PERM) in small volumes (100µL) of rat plasma and brain homogenate was developed, using a liquid-liquid extraction for sample preparation and gas chromatography-negative chemical ionization mass spectrometry (GCNCI-MS) for detection. Quantitation of trace levels of the insecticide in small volumes of biological samples is essential to support toxicokinetic studies in small animals. There are currently no validated methods in the literature for determining cis-PERM and trans- PERM in volumes as low as 100µL of rat plasma or brain homogenate. The method provided a linear range of 0.2-150.0ng/mL for analytes in both matrices. The intra- and inter-batch precision (as% relative standard deviation, RSD) and accuracy (as relative error, RE) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range. The validated method was applied in a toxicokinetic study in adult rats with oral dosing of 10mg/kg (cis-PERM) and 100mg/kg (trans-PERM) in corn oil. cis-PERM and trans- PERM were monitored in rat plasma and brain tissue samples for 6h following dosing, and both analytes were detected in all plasma and brain samples.

Influence of Maturation on In Vivo Tissue to Plasma Partition Coefficients for Cis- and Trans-Permethrin.

Permethrin, the most widely used household insecticide in the United States, is marketed as a mixture of its cis (CIS) and trans (TRANS) isomers. The major objective of this investigation is to develop and utilize a reliable approach to determine in vivo partition coefficients (PCs) for CIS and TRANS in immature and adult Sprague-Dawley rats. Adult, postnatal day (PND) 21, and PND 15 rats were infused with environmentally relevant concentrations of CIS or TRANS via a subcutaneous osmotic pump for 48 or 72 h. The adult and PND 21 rats also received an oral loading dose. Systemic steady-state or equilibrium was attained in each age group within 72 h of the protocol. CIS and TRANS were both distributed to tissues according to their neutral lipid content, with adipose tissue exhibiting much higher tissue:plasma PCs than skeletal muscle, liver, or brain. Liver:plasma and brain:plasma PCs were consistently at or lower than unity. Tissue:plasma PCs were generally higher for CIS than for TRANS, although the isomers are of comparable lipophilicity. Significantly higher blood levels of CIS apparently saturate plasma binding, resulting in greater tissue deposition of the isomer. CIS and TRANS tissue:plasma PCs were found to be inversely related to the rats' age, although TRANS brain:plasma PCs were comparable in immature and mature animals. These data support the conclusion that age-dependent partitioning is an important determinant of the pharmacokinetics of permethrin. Such partitioning could influence the risk assessment of these insecticides in infants and children when incorporated into physiologically based pharmacokinetic models.

Biomonitoring and evaluation of permethrin uptake in forestry workers using permethrin-treated tick-proof pants.

We conducted a randomized case-control trial to analyze uptake of the insecticide/arcaricide permethrin in wearers of permethrin-impregnated and non-impregnated pants in German forestry. Eighty-two male workers were each equipped for a 16-week period with permethrin-treated (test group) or with non-treated work pants (control group). Pants with or without lining to protect against cuts, obtained from two different distributors, were worn in each group. Urinary permethrin metabolite levels were measured by GC-MS/MS before, during and after wearing of the pants. Permethrin uptake was calculated using additional questionnaire data. In the control group, metabolite levels in the range of environmental background exposure (median: ~0.5 µg/l) were measured. Subjects wearing impregnated pants showed consistently significantly higher exposure levels even before the first use of the pants with a maximum after 1 week of wearing the pants (median: ~12.5 µg/l). Significant differences in internal exposure were found depending on which of the distributors the pants came from. Metabolite levels decreased probably due to permethrin losses associated with laundering the pants. Calculated permethrin uptake is below the value corresponding to the WHO-proposed acceptable daily intake. Based on our data, a marginally increased cancer risk compared with the general population cannot be excluded when wearing impregnated pants over a working-lifetime period.

Single-dose toxicokinetics of permethrin in broiler chickens.

Single-dose toxicokinetics of permethrin was investigated in broiler chickens. A total of 20 male broiler chickens were assigned at random to two groups of 10 at 30 days of age. A single dose of 10 mg/kg body weight of permethrin was administered intravenously to the first group; in the second group, the same dose was administered into the crop. Serum permethrin was measured using an electron capture detector and gas chromatography equipment. The derived serum permethrin concentration/time curve demonstrated that the distribution kinetics of permethrin was well described by a two-compartment open model. For intravenous permethrin administration, the half-life at λ phase (t1/2λ), mean residence time (MRT) and area under the concentration-time curve in 0→∞ (AUC0→∞) values respectively were 4.73 ± 1.00 h, 5.06 ± 1.05 h and 16.45 ± 3.28 mg/h/l. In contrast, the Cmax, tmax, t1/2λ, MRT and AUC0→∞ values respectively of the group given intra-crop permethrin were 0.60 ± 0.42 µg/ml, 0.55 ± 0.19 h, 5.54 ± 0.78 h, 7.06 ± 0.63 h and 1.95 ± 0.97 mg/h/l. The bioavailability of permethrin was 0.11. For both administration routes, the residence time of permethrin in the body was short and the bioavailability of permethrin was low. These results are relevant for assessing the use and safety of permethrin.

Time profiles and toxicokinetic parameters of key biomarkers of exposure to cypermethrin in orally exposed volunteers compared with previously available kinetic data following permethrin exposure.

Biomonitoring of pyrethroid exposure is largely conducted but human toxicokinetics has not been fully documented. This is essential for a proper interpretation of biomonitoring data. Time profiles and toxicokinetic parameters of key biomarkers of exposure to cypermethrin in orally exposed volunteers have been documented and compared with previously available kinetic data following permethrin dosing. Six volunteers ingested 0.1 mg kg(-1) bodyweight of cypermethrin acutely. The same volunteers were exposed to permethrin earlier. Blood samples were taken over 72 h after treatment and complete timed urine voids were collected over 84 h postdosing. Cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids (trans- and cis-DCCA) and 3-phenoxybenzoic acid (3-PBA) metabolites, common to both cypermethrin and permethrin, were quantified. Blood and urinary time courses of all three metabolites were similar following cypermethrin and permethrin exposure. Plasma levels of metabolites reached peak values on average ≈ 5-7 h post-dosing; the elimination phase showed mean apparent half-lives (t½ ) for trans-DCCA, cis-DCCA and 3-PBA of 5.1, 6.9 and 9.2 h, respectively, following cypermethrin treatment as compared to 7.1, 6.2 and 6.5 h after permethrin dosing. Corresponding mean values obtained from urinary rate time courses were peak values at ≈ 9 h post-dosing and apparent elimination t½ of 6.3, 6.4 and 6.4 h for trans-DCCA, cis-DCCA and 3-PBA, respectively, following cypermethrin treatment as compared to 5.4, 4.5 and 5.7 h after permethrin dosing. These data confirm that the kinetics of cypermethrin is similar to that of permethrin in humans and that their common biomarkers of exposure may be used for an overall assessment of exposure.

Measurement of plasma protein and lipoprotein binding of pyrethroids.

INTRODUCTION: A simple, reliable procedure was developed to measure binding of pyrethroid insecticides to total proteins and lipoproteins of rat and human plasma. METHODS: The extent of binding of (14)C-labeled deltamethrin (DLM), cis-permethrin (CIS) and trans-permethrin (TRANS) was quantified by a 3-step organic solvent extraction technique. Rat and human plasma samples, containing NaF to inhibit esterases, were spiked with a range of concentrations of each radiolabeled pyrethroid. Protein binding reached equilibrium within ~1h of incubation at 37°C. The samples were extracted in turn with: isooctane to collect the unbound fraction; 2-octanol to extract the lipoprotein-bound fraction; and acetonitrile to obtain the protein-bound fraction. RESULTS: Absolute recoveries of DLM, CIS and TRANS ranged from 86 to 95%. Adherence of these very lipophilic chemicals to glass and plastic was minimized by using silanized glass vials and LoBind® plastic pipettes. The method's ability to distinguish lipoprotein from protein binding was confirmed by experiments with diazepam and cyclosporine, drugs that bind selectively to albumin and lipoproteins, respectively. DISCUSSION: This procedure was effectively utilized for studies of the species-dependence of plasma protein and lipoprotein binding of three pyrethroids for inclusion in physiologically-based pharmacokinetic models of pyrethroids for use in health risk assessments of the insecticides in children and adults.

Determination of cis-permethrin, trans-permethrin and associated metabolites in rat blood and organs by gas chromatography-ion trap mass spectrometry.

An analytical method was developed to measure cis-permethrin and trans-permethrin in different biological rat matrices and fluids (whole blood, red blood cells, plasma, brain, liver, muscle, testes, kidneys, fat and faeces). The method was also suitable for the simultaneous quantification of their associated metabolites [cis-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis-DCCA), trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (trans-DCCA) and 3-phenoxybenzoic acid (3-PBA)] in blood (whole blood, red blood cells, plasma) and liver. The target analytes were derivatised in samples using a methanolic/hydrochloric acid solution and then extracted with toluene. The analysis was performed by gas chromatography, and detection using ion trap tandem mass spectrometry. The selectivity obtained for complex matrices such as rat organs allowed the use of a purification step to be avoided for most of the matrices investigated. In the case of fat, where permethrin is suspected to accumulate, a dedicated purification step was developed. In fluids, the limits of quantification were at the 50 ng/mL level for the parent compounds and 3-PBA and at 25 ng/mL for cis-DCCA and trans-DCCA. For solid matrices excluding fat, the limits of quantification ranged from 50 ng/g for muscle to 100 ng/g for brain and testes for both cis-permethrin and trans-permethrin. The extraction recoveries ranged primarily between 80 and 120% for the matrix tested. The stability of blood samples was tested through the addition of 1% v/v formic acid. The methods developed were applied in a toxicokinetic study in adult rats. cis-Permethrin and the metabolites were detected in all corresponding matrices, whereas trans-permethrin was detected only in blood, plasma and faeces.

Blood plasma metabolites and the risk of developing lung cancer in Russia.

Lung cancer is one of the most common types of cancer in men, and is a leading cause of cancer-related deaths. Therefore, the identification of specific markers associated with a risk of lung cancer development, particularly metabolites that are more easily assayed, would be very valuable. To this end, а comparative metabolomics study of blood plasma samples collected from patients with lung cancer (n=100) and controls (n=100) recruited in Moscow was carried out. After the extraction of blood plasma proteins with methanol, the remaining plasma metabolite fractions were analyzed directly using mass spectrometry. Hundreds of cancer-associated metabolites were detected, and at least 70 metabolite ions with odds ratio values of 10-288 were found to be associated with the presence of cancer. Although these metabolites were present at higher levels in cancer patients, particularly in the early stages of disease, they did not correlate positively with cancer progression. On the basis of these findings, this metabolomics study of blood plasma samples from cancer patients shows that numerous cancer-associated metabolites were present in the studied population, and these could be used as factors for calculating the risk of lung cancer development in addition to currently used risk factors.

Applying biofluid metabonomic techniques to analyze the combined subchronic toxicity of propoxur and permethrin in rats.

BACKGROUND: NMR combined with pattern recognition was recently introduced as a new technique for rapid xenobiotic toxicity evaluation. In this article, metabolic changes in the biofluid of rats after 90-day oral treatment with propoxur, permethrin and a combination of these two pesticides were investigated. RESULTS: Propoxur dosing induced increased urinary taurine, creatinine and glucose, whereas urinary lactate and acetate were increased in the highest permethrin dose group. Urinary acetate, alanine, lactate and trimethylamine levels were increased in the mixture group, accompanied by decreased urinary tricarboxylic acid cycle intermediates. In addition, the highest dose of the mixture displayed raised 3-D-hydroxybutyrate, acetate and lactate levels in the serum sample. CONCLUSION: Chronic exposure to a combination of propoxur and permethrin may induce hepatotoxicity and nephrotoxicity. An increase in acetate, alanine and formate in the urine could be a potentially sensitive biomarker of the chronic, combined effects of permethrin and propoxur.

Regional variation in pesticide concentrations in plasma of delivering women residing in rural Indian Ocean coastal regions of South Africa.

Exposure to pesticides places pregnant women and the developing foetus at the highest risk. The objective of this study is to obtain an exposure assessment by investigating levels of pesticides in blood plasma of delivering women. We report on the concentrations of α, ß, γ HCH, endosulfan, HCB and the pyrethroids: cis-permethrin, cyfluthrin, cypermethrin and deltamethrin found in the maternal blood plasma of delivering women (n = 241) in three coastal sites of KwaZulu Natal. γ-HCH and endosulfan 1 and 2 were the most dominant pesticides in all three sites. Significantly, higher levels of γ-HCH and endosulfan were found in site 3 (vicinity of Empangeni) compared to the other two sites (p < 0.05). The GM levels for γ-HCH, endosulfan 1 and 2 were 956, 141 and 21 ng g(-1) lipids in site 3, respectively. The pyrethroid pesticides, HCB, α-HCH and ß-HCH were detected in less than 31% of the samples in all sites. γ-HCH correlated positively and strongly to both endosulfan 1 and 2 (r > 0.47), indicating a common source of exposure. The high levels of γ-HCH and endosulfan in maternal plasma samples in site 3 indicate the current and on-going exposure, which is of great concern for reproductive health and prenatal exposure.

Fetal exposure to chlordane and permethrin mixtures in relation to inflammatory cytokines and birth outcomes.

We sought to characterize the relationships between cord serum concentrations of chlordane and permethrin pesticides, inflammatory cytokines, gestational age, and size at birth. Umbilical cord serum levels of trans-nonachlor, oxychlordane, cis- and trans-permethrin, piperonyl butoxide, and cytokines (TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, GMCSF) were quantified in 300 newborns at the Johns Hopkins Hospital in Baltimore, MD (2004-2005). Principal component analyses were used to quantitate chlordane and permethrin mixtures and to identify independent cytokine components. Five cytokine components described 87% of the variance in cord serum cytokine levels; these (and predominant loadings) were as follows: (1) all 9 cytokines; (2) acute phase (IL-1ß, IL-6); (3) anti-inflammatory (IL-10); (4) TNF-α; and (5) IL-1ß. Of these, the TNF-α component was significantly associated with a 2-day decrease in gestational age. Chlordane was associated with lower levels of the pro-inflammatory IL-1ß [ß: -0.11 (-0.20, -0.02)]. Permethrin was negatively associated with the anti-inflammatory cytokine IL-10 [ß: -0.14 (-0.22, -0.05)]. Neither pesticides nor cytokines were significantly associated with birthweight, length, or head circumference, and pesticides were not associated with gestational age. Our findings suggest that chlordane and permethrin concentrations in cord blood may be associated with levels of inflammatory cytokines in the fetus.

In vitro metabolism and interactions of pyridostigmine bromide, N,N-diethyl-m-toluamide, and permethrin in human plasma and liver microsomal enzymes.

1. The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide (PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide (DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination were investigated. 2. The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET. The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB. Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma. Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems. 3. The present study evidences that PB and permethrin are metabolized by both human plasma and liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined exposure to test chemicals increases their neurotoxicity by impeding the body's ability to eliminate them because of the competition for detoxifying enzymes.

Pyridostigmine, diethyltoluamide, permethrin, and stress: a double-blind, randomized, placebo-controlled trial to assess safety.

OBJECTIVE: To determine whether short-term human exposure to pyridostigmine bromide, diethyltoluamide, and permethrin, at rest or under stress, adversely affects short-term physical or neurocognitive performance. PARTICIPANTS AND METHODS: A multicenter, prospective, double-blind, placebo-controlled crossover trial exposing 64 volunteers to permethrin-impregnated uniforms, diethyltoluamide-containing skin cream, oral pyridostigmine, and corresponding placebos was performed. Each participant had 4 separate sessions, ensuring exposure to all treatments and placebos under both stress and rest conditions in random order. Outcomes Included physical performance (handgrip strength and duration, stair climbing, and pull-ups [males] or push-ups [females]), neurocognitive performance (computerized tests), and self-reported adverse effects. RESULTS: Permethrin was undetectable in the serum of all participants; pyridostigmine levels were higher Immediately after stress (41.6 ng/mL; 95% confidence Interval, 35.1-48.1 ng/mL) than rest (23.0 ng/mL; 95% confidence Interval, 19.2-26.9 ng/mL), whereas diethyltoluamide levels did not significantly differ by stress condition. Heart rate and systolic blood pressure increased significantly with stress compared with rest but did not vary with treatment vs placebo. Physical and neurocognitive outcome measures and self-reported adverse effects did not significantly differ by exposure group. CONCLUSION: Combined, correct use of pyridostigmine, diethyltoluamide, and permethrin is well tolerated and without evidence of short-term physical or neurocognitive impairment.

Rapid determination of N,N-diethyl-m-toluamide and permethrin in human plasma by gas chromatography-mass spectrometry and pyridostigmine bromide by high-performance liquid chromatography.

A rapid and highly sensitive gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of N,N-diethyl-m-toluamide (DEET) and permethrin with (2)H(10)-phenanthrene (98 atom %) as an internal standard and a separate external standard high-performance liquid chromatography (HPLC) method for pyridostigmine bromide (PB) determination in human plasma were developed and validated. The GC-MS method for DEET and permethrin quantification utilizes a one-step extraction with tert-butylmethylether. The HPLC method for PB quantification involves a solid-phase extraction and UV detection. The range of the analytical method for DEET and permethrin was 1 ng/mL to 100 ng/mL and for PB was 5 ng/mL to 100 ng/mL. Recovery from plasma proved to be more than 80%. The intraday precision ranged from 1.3% to 8% for DEET, from 2.1% to 11.4% for permethrin, and from 3.0% to 4.8% for PB. The interday precision was 3% for DEET, ranged from 5% to 9% for permethrin, and from 5% to 9% for PB. The accuracy for the limit of quantification was 92% +/- 8% relative standard deviation (RSD) for DEET, 112% +/- 11% RSD for permethrin, and 109% +/- 5% RSD for PB. All 3 compounds were stable in human plasma at -80 degrees C for at least 12 months and after 2 freeze-thaw cycles with RSD values ranging from 7.1% (DEET, 80 ng/mL) to 8.1% (DEET, 8 ng/mL), from 2.3% (permethrin, 80 ng/mL) to 11.6 % (permethrin, 8 ng/mL), and from 0.2% (PB, 80 ng/mL) to 3.6% (PB, 8 ng/mL). Both methods were successfully applied to pharmacokinetic/ pharmacodynamic studies of combined exposure of DEET (skin application), permethrin (treated uniforms), and PB (30 mg orally three times/day for four doses) in healthy volunteers (n = 81).