An updated review on the safety of N, N-diethyl-meta-toluamide insect repellent use in children and the efficacy of natural alternatives.

N, N-diethyl-meta-toluamide (DEET) has been considered the 'gold standard' for insect repellent use since the 1950s and constitutes most insect repellents on the market. However, conflicting data in the scientific literature and confusing information in the media are at the core of debates about the safety of DEET insect repellents for the protection of children against arthropod bites. The few fatal occurrences involving DEET insect repellents and complications of their use in the pediatric population are typically the result of accidental overdoses or misuse of insect repellents that disregard warnings on product labels. With appropriate application, the safety record of DEET insect repellents continues to be excellent with few side effects. The purpose of this review is to provide a summary of the literature on safety outcomes of DEET insect repellent use in children; outline the pediatric recommendations relating to DEET insect repellents; and provide an overview of EPA-approved and naturally derived alternatives to DEET that possess low toxicity while providing a similar level of protection to synthetic insect repellents.

Encapsulation of carvacrol and thymol with yeast cell wall and its repellent activity against Amblyomma sculptum and Rhipicephalus sanguineus (Sensu Lato).

The main way to avoid contact with ticks and consequently tick-borne disease is the use of synthetic repellents. The search of new repellent compounds to increase the possibilities of use in strategies controls are necessary. The present study evaluated the repellent activity of two natural terpenes carvacrol and thymol in each one two different formulation (encapsulated and nonencapsulated with yeast cell wall) against the ticks Amblyomma sculptum and Rhipicephalus sanguineus sensu lato nymphs. Nymphs of A. sculptum and R. sanguineus s.l. of a single generation were used. The vertical filter paper repellency assay were performed with different concentration of both terpenes encapsulated and nonencapsulated in yeast cell wall. The repellent concentration 50% (RC50) were calculated to each compound formulation. Both carvacrol and thymol (encapsulated and nonencapsulated), had a repellent activity against A. sculptum and R. sanguineus s.l nymphs. Amblyomma sculptum was more sensitive to nonencapsulated carvacrol (RC50 values: 0.0032 to 0.0082 mg/cm2 after 1 and 15 min) (P < 0.05), while R. sanguineus s.l. was more sensitive to encapsulated carvacrol (RC50 values: 0.00008 to 0.0035 mg/cm2 after 1 and 15 min) (P < 0.05). Among tick species, R. sanguineus s.l. was more sensitive for most compounds than A. sculptum (P < 0.05). Although with distinct repellent activities, carvacrol and thymol encapsulated can be a promising alternative to synthetic repellents against A. sculptum and R. sanguineus s.l.

Recent advances in degradation of N,N-diethyl-3-toluamide (DEET)-an emerging environmental contaminant: a review.

N,N-Diethyl-3-toluamide (DEET) is a commonly used insect repellent, which acts as an organic chemical contaminant in water and considered as an emerging contaminant which has been observed worldwide. It gets discharged into the environment through sewage waste. The various methods have been used to degrade DEET, such as UV based, ozonation, photocatalytic degradation, and biodegradation (based on the metabolic activity of fungi and bacteria). However, less research has been done on the degradation of DEET by deploying nanoparticles. Therefore, biodegradation and nanotechnology-based methods can be the potential solution to remediate DEET from the environment. This review is an attempt to analyze the routes of entry of DEET into the atmosphere and its environmental health consequences and to explore physical, chemical, and biological methods of degradation. Furthermore, it focuses on the various methods used for the biodegradation of the DEET, including their environmental consequences. Future research is needed with the application of biological methods for the degradation of DEET. Metabolic pathway for biodegradation was explored for the new potent microbial strains by the application of physical, chemical, and microbial genomics; molecular biology; genetic engineering; and genome sequencing methods.

Insect repellents: An updated review for the clinician.

Malaria, Zika virus, West Nile virus, Dengue fever, and Lyme disease are common causes of morbidity and mortality around the world. While arthropod bites may cause local inflammation and discomfort, a greater concern is the potential to develop deadly systemic infection. The use of insect repellents (IRs) to prevent systemic infections constitutes a fundamental public health effort. Cost effectiveness, availability, and high efficacy against arthropod vectors are key characteristics of an ideal IR. Currently, numerous IRs are available on the market, with N,N-diethyl-3-methylbenzamide (DEET) being the most widely used. DEET has an excellent safety profile and remarkable protection against mosquitoes and various other arthropods. Other Environmental Protection Agency-registered IR ingredients (eg, permethrin, picaridin, IR3535, oil of lemon eucalyptus, oil of citronella, catnip oil, and 2-undecanone) are alternative IRs of great interest because some of these ingredients have efficacies comparable to that of DEET. These alternative IRs possess low toxicity and favorable customer experiences in use (eg, cosmetically pleasant, naturally occurring). This review summarizes the currently available Environmental Protection Agency-registered IRs, including their origins, mechanisms of action, side effect profiles, and available formulations. This review will enable the clinician to select the best IR option to meet a patient's needs and provide the greatest protection from arthropod bites and their sequelae.

Biomonitoring Equivalents for N,N-Diethyl-meta-toluamide (DEET).

N,N-Diethyl-meta-toluamide (DEET) is widely used as an effective mosquito and tick repellent. DEET is absorbed systemically after applications to skin. Once absorbed, DEET is rapidly metabolized with the predominant metabolite being m-dimethylaminocarbonyl benzoic acid (DBA). DEET and metabolites are predominantly excreted in urine after being absorbed systemically. Exposures to DEET are typically biomonitored via measures of DEET and DBA in urine. In this evaluation, we review available health-based risk assessments and toxicological reference values (TRVs) for DEET and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. BEs were derived based on existing TRVs derived by Health Canada, yielding 38 and 23 mg/L DBA in urine for adults and 57 and 34 mg/L DBA in urine in children for the acute oral and intermediate dermal TRVs, respectively. The BEs for unchanged DEET in urine are 21 and 12 mg/L in adults and 4.5 and 2.7 mg/L in children for the acute oral and intermediate dermal TRVs. The BE values derived in this manuscript can serve as a guide to help public health officials and regulators interpret population based DEET biomonitoring data in a public health risk context.

A rapid method for screening mosquito repellents on Anopheles pseudopunctipennis and Aedes aegypti.

One of the main vectors for malaria in Latin America is Anopheles pseudopunctipennis (Theobald), whereas Aedes aegypti (L.) is the primary vector of dengue, yellow fever, Zika and chikungunya viruses. The use of repellents is recommended as a personal protection method against these mosquitoes. However there are very few studies evaluating the effect of repellents on An. pseudopunctipennis. The use of a Petri dish to study repellence has been applied by several authors on flies, cockroaches, kissing bugs and mosquitoes, being a valuable technique for species that are difficult to breed under laboratory conditions, such as An. pseudopunctipennis. In the present study, we evaluated the repellence of the essential oil of the Eucalyptus nitens (Shining gum), its main component (1,8-cineole) and the commercial repellent DEET on An. pseudopunctipennis and Ae. aegypti adult females using the plaque repellency method coupled to EthoVision XT10.1 video-tracking software. Repellent effect and locomotor activity were studied through a repellence index (RI) together with an axis constructed from the behavioural variables obtained using the tracking software. DEET repellent effect was observed at 0.01 mg/mL for Ae. aegypti and 0.01 and 0.1 mg/mL for An. pseudopunctipennis. In addition, the essential oil showed significant repellence at 1 and 10 mg/mL for Ae. aegypti, and 1, 5, 10 and 25 mg/mL for An. pseudopunctipennis. Neither of these species were repelled at any concentration of 1,8-cineole. This is the first study that evaluates these compounds on An. pseudopunctipennis females and quantifies their effects on the activity of both species.

In Search of Synergistic Insect Repellents: Modeling of Muscarinic GPCR Interactions with Classical and Bitopic Photoactive Ligands.

Insect vector-borne diseases pose serious health problems, so there is a high demand for efficient molecules that could reduce transmission. Using molecular docking and molecular dynamics (MD) simulation, we studied a series of compounds acting on human and insect muscarinic acetylcholine receptors (mAChRs), a novel target of synergistic agents in pest control. We characterized early conformational changes of human M1 and fruit fly type-A mAChR G protein-coupled receptors (GPCRs) in response to DEET, IR3535, and muscarine binding based on the MD analysis of the activation microswitches known to form the signal transduction pathway in class A GPCRs. We indicated groups of microswitches that are the most affected by the presence of a ligand. Moreover, to increase selectivity towards insects, we proposed a new, bitopic, photoswitchable mAChR ligand-BQCA-azo-IR353 and studied its interactions with both receptors. Modeling data showed that using a bitopic ligand may be a promising strategy in the search for better insect control.

Single blinded semi-field evaluation of MAÏA® topical repellent ointment compared to unformulated 20% DEET against Anopheles gambiae, Anopheles arabiensis and Aedes aegypti in Tanzania.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (DEET) topical mosquito repellents are effective personal protection tools. However, DEET-based repellents tend to have low consumer acceptability because they are cosmetically unappealing. More attractive formulations are needed to encourage regular user compliance. This study evaluated the protective efficacy and protection duration of a new topical repellent ointment containing 15% DEET, MAÏA® compared to 20% DEET in ethanol using malaria and dengue mosquito vectors in Bagamoyo Tanzania. METHODS: Fully balanced 3 × 3 Latin square design studies were conducted in large semi-field chambers using laboratory strains of Anopheles gambiae sensu stricto, Anopheles arabiensis and Aedes aegypti. Human volunteers applied either MAÏA® ointment, 20% DEET or ethanol to their lower limbs 6 h before the start of tests. Approximately 100 mosquitoes per strain per replicate were released inside each chamber, with 25 mosquitoes released at regular intervals during the collection period to maintain adequate biting pressure throughout the test. Volunteers recaptured mosquitoes landing on their lower limbs for 6 h over a period of 6 to 12-h post-application of repellents. Data analysis was conducted using mixed-effects logistic regression. RESULTS: The protective efficacy of MAÏA® and 20% DEET was not statistically different for each of the mosquito strains: 95.9% vs. 97.4% against An. gambiae (OR = 1.53 [95% CI 0.93-2.51] p = 0.091); 96.8% vs 97.2% against An. arabiensis (OR = 1.08 [95% CI 0.66-1.77] p = 0.757); 93.1% vs 94.6% against Ae. aegypti (OR = 0.76 [95% CI 0.20-2.80] p = 0.675). Average complete protection time (CPT) in minutes of MAÏA® and that of DEET was similar for each of the mosquito strains: 571.6 min (95% CI 558.3-584.8) vs 575.0 min (95% CI 562.1-587.9) against An. gambiae; 585.6 min (95% CI 571.4-599.8) vs 580.9 min (95% CI 571.1-590.7) against An. arabiensis; 444.1 min (95% CI 401.8-486.5) vs 436.9 min (95% CI 405.2-468.5) against Ae. aegypti. CONCLUSIONS: MAÏA® repellent ointment provides complete protection for 9 h against both An. gambiae and An. arabiensis, and 7 h against Ae. aegypti similar to 20% DEET (in ethanol). MAÏA® repellent ointment can be recommended as a tool for prevention against outdoor biting mosquitoes in tropical locations where the majority of the people spend an ample time outdoor before going to bed.

Avoidance of the Plant Hormone Cis-Jasmone by Aedes aegypti Depends On Mosquito Age in Both Plant and Human Odor Backgrounds.

Adults of many mosquito species feed on plants to obtain metabolic energy and to enhance reproduction. Mosquitoes primarily rely on olfaction to locate plants and are known to respond to a range of plant volatiles. We studied the olfactory response of the yellow fever mosquito Aedes aegypti to methyl jasmonate (MeJA) and cis-jasmone (CiJA), volatile compounds originating from the octadecanoid signaling pathway that plays a key role in plant defense against herbivores. Specifically, we investigated how Ae. aegypti of different ages responded to elevated levels of CiJA in two attractive odor contexts, either derived from Lima bean plants or human skin. Aedes aegypti females landed significantly less often on a surface with CiJA and MeJA compared to the solvent control, CiJA exerting a stronger reduction in landing than MeJA. Odor context (plant or human) had no significant main effect on the olfactory responses of Ae. aegypti females to CiJA. Mosquito age significantly affected the olfactory response, older females (7-9 d) responding more strongly to elevated levels of CiJA than young females (1-3 d) in either odor context. Our results show that avoidance of CiJA by Ae. aegypti is independent of odor background, suggesting that jasmonates are inherently aversive cues to these mosquitoes. We propose that avoidance of plants with elevated levels of jasmonates is adaptive to mosquitoes to reduce the risk of encountering predators that is higher on these plants, i.e. by avoiding 'enemy-dense-space'.

Wilderness Medical Society Clinical Practice Guidelines for the Prevention and Management of Tick-Borne Illness in the United States.

The Wilderness Medical Society convened an expert panel to develop evidence-based guidelines for the prevention and management of tick-borne illness (TBI). Recommendations are graded based on quality of supporting evidence according to criteria put forth by the American College of Chest Physicians. The guidelines include a brief review of the clinical presentation, epidemiology, prevention, and management of TBI in the United States, with a primary focus on interventions that are appropriate for resource-limited settings. Strong recommendations are provided for the use of DEET, picaridin, and permethrin; tick checks; washing and drying clothing at high temperatures; mechanical tick removal within 36 h of attachment; single-dose doxycycline for high-risk Lyme disease exposures versus "watchful waiting;" evacuation from backcountry settings for symptomatic tick exposures; and TBI education programs. Weak recommendations are provided for the use of light-colored clothing; insect repellents other than DEET, picaridin, and permethrin; and showering after exposure to tick habitat. Weak recommendations are also provided against passive methods of tick removal, including the use of systemic and local treatments. There was insufficient evidence to support the use of long-sleeved clothing and the avoidance of tick habitat such as long grasses and leaf litter. Although there was sound evidence supporting Lyme disease vaccination, a grade was not offered as the vaccine is not currently available for use in the United States.

Carboxylic acid responses by a conserved odorant receptor in culicine vector mosquitoes.

Many mosquito behaviours that are critical for survival and reproduction depend upon timely responses to chemical cues. Of interest are the effects of volatile organic compounds like carboxylic acids (CAs) that are released by potential blood meal hosts. Short chain CAs are among the primary attractants for host-seeking females and influence host selection in vector species. Although the behavioural relevance of CA's has been established, less is known about the molecular receptive events that evoke responses to specific compounds, with the Ir family of chemoreceptors being broadly implicated in their detection. In this study, we demonstrate that Or orthologs from two vector species, Aedes aegypti (L.) and Aedes albopictus (Skuse), are selectively activated by straight chain carboxylic acids and that these responses are attenuated by the commercial insect repellant N,N-Diethyl-meta-toluamide. Our results suggest that multiple chemoreceptors, representing diverse families, are able to mediate molecular responses to CAs and may therefore underlie important behaviours that directly impact disease-transmission cycles.

Who's afraid of DEET? Fearmongering in papers on botanical repellents.

DEET (N,N-Diethyl-meta-toluamide) is considered the gold standard in mosquito repellents, not only for its effectiveness, but also for its safety. DEET has been more extensively studied for safety than any other repellent, and is accepted as completely safe when used correctly (i.e. not consumed or bathed in). Researchers studying botanical repellents, however, often paint DEET as far more toxic than it really is, falsely claiming it is a menace to the public health or even the environment. These claims are unfounded, and often the only evidence given by such publications are references to other publications also studying botanical repellents. Such publications are biased, and may be attacking DEET's excellent safety record to justify their existence and the need for their research. The inconvenient yet undisputable fact is that no botanical repellent has been proven to be as safe as DEET, and the majority never had any safety testing whatsoever. The automatic assumption that botanical repellents are safer than DEET is the 'appeal to nature fallacy,' which also drives most of the market for "natural" repellents, yet natural repellents have side effects and even a body count. Finding a botanical repellent that works as well as DEET and is equally safe is a legitimate research goal on its own, and need not be justified by fear-mongering and irrational chemophobia. Researchers studying these alternatives should strive for integrity, raising the real issue of the lack of safety testing for botanical repellents rather than denying the proven safety of DEET.

Activation of the ambusher tick Rhipicephalus microplus (Acari: Ixodidae) exposed to different stimuli.

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO2 alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO2 combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO2 was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO2 activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO2 , larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO2 . The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.

Role of long non-coding RNA in DEET- and fipronil-mediated alteration of transcripts associated with Phase I and Phase II xenobiotic metabolism in human primary hepatocytes.

Human exposure to environmental chemicals both individually and in combination occurs frequently world-wide most often with unknown consequences. Use of molecular approaches to aide in the assessment of risk involved in chemical exposure is a growing field in toxicology. In this study, we examined the impact of two environmental chemicals used in and around homes, the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels of enzymes potentially involved in xenobiotic metabolism and on long non-coding RNAs (lncRNAs). Primary human hepatocytes were treated with these two chemicals both individually and in combination. Using RNA-Seq, we found that 10 major enzyme categories involved in phase 1 and phase 2 xenobiotic metabolism were significantly (α = 0.05) up- and down-regulated (i.e., 100 µM DEET-19 transcripts, 89% up and 11% down; 10 µM fipronil-52 transcripts, 53% up and 47% down; and 100 µM DEET +10 µM fipronil-69 transcripts, 43% up and 57% down). The altered genes were then mapped to the human genome and their proximity (within 1,000,000 bp) to lncRNAs examined. Unique proximities were discovered between altered lncRNA and altered P450s (CYP) and other enzymes (DEET, 2 CYP; Fipronil, 6 CYP and 15 other; and DEET + fipronil, 7 CYP and 21 other). Many of the altered P450 transcripts were in multiple clusters in the genome with proximal altered lncRNAs, suggesting a regulator function for the lncRNA. At the gene level there was high percent identity for lncRNAs near P450 clusters, but this relationship was not found at the transcript level. The role of these altered lncRNAs associated with xenobiotic induction, human diseases and chemical mixtures is discussed.

In vitro skin irritation assessment using EpiDerm™: applicability for updating toxicity information of oxybenzone and N,N-diethyl-m-toluamide.

A skin irritation test using in vitro reconstructed human epidermis (RhE) models was established for hazard identification of irritant chemicals in accordance with UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) category. In this study, EpiDerm™ was used to assess skin irritation by oxybenzone and N,N-diethyl-m-toluamide (DEET), which are widely used sunscreen and insect repellent components, respectively. EpiDerm™ was applied with oxybenzone and DEET, combined and sequentially with each single dose. Epidermal morphology and differentiation/proliferation were examined microscopically. Oxybenzone and sequential administration groups were determined as nonirritant with cell viability >50% and the morphology was comparable to the human epidermis. Contrastingly, the DEET and coadministration groups exhibited cell viability <50% and poor epidermal morphology. Interleukin (IL)-1α release from substance-treated EpiDerm™ increased inversely to cell viability, suggesting the pro-inflammatory reaction was initiated by DEET. CK-10, E-cadherin, Ki-67, laminin, and ceramide were identified as relevant markers to assess oxybenzone- or DEET-induced epidermal injury. In conclusion, these results may indicate to be aware of the possible skin irritation by indiscriminate use of oxybenzone and DEET without animal testing.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET).

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5 mM (0.02%-0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

Quantification of DEET and neonicotinoid pesticide biomarkers in human urine by online solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry.

Neonicotinoid insecticides are widely used replacements for organophosphate and carbamate insecticides, but the extent of human exposure is largely unknown. On the other hand, based on urinary concentrations of DEET metabolites, human exposure to N,N-diethyl-m-toluamide (DEET) appears to be widespread. We developed a fast online solid-phase extraction high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-MS/MS) method to measure in 200 µL of human urine the concentrations of six neonicotinoid biomarkers (acetamiprid, N-desmethyl-acetamiprid, clothianidin, imidacloprid, 5-hydroxy-imidacloprid, thiacloprid), and two DEET biomarkers (3-diethyl-carbamoyl benzoic acid, 3-ethyl-carbamoyl benzoic acid). Limits of detection ranged from 0.01 to 0.1 µg/L, depending on the biomarker. Accuracy ranged from 91 to 116% and precision ranged from 3.7 to 10 %RSD. The presented method can be used to increase our understanding of exposure to neonicotinoid insecticides and DEET, and to evaluate the potential health effects from such exposures.

High-throughput screening method for evaluating spatial repellency and vapour toxicity to mosquitoes.

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high-throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (µg). In a horizontal tube orientation, DEET (N,N-diethyl-meta-toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC50 ) values of 32, 32 and 298 µg/cm2 , respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC50 values by approximately two-fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration-dependent spatial repellency (1 h EC50 = 0.5 µg/cm2 ) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 µg/cm2 and 50% mortality at 0.15 µg/cm2 in horizontal tubes. In conclusion, this high-throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi-field and field studies.

Bug off! Severe toxicity following inhalational exposure to N, N-diethyl-meta-toluamide (DEET).

N, N-diethyl-meta-toluamide (DEET) is an insect repellent currently used by millions of people since 1956. DEET has an excellent safety profile and has remarkable protection against mosquitoes, ticks and various other arthropods. Toxicity is unusual, and is generally associated with incorrect, or overuse of the product. We report a patient with severe toxicity following inhalational exposure to a "bug bomb". containing 98% DEET.

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition.

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1•Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin's equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D 1H-15N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.