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Deet Safety Research Paper

Abstract

Reducing the number of host-vector interactions is an effective way to reduce the spread of vector-borne diseases. Repellents are widely used to protect humans from a variety of protozoans, viruses, and nematodes. DEET (N,N-Diethyl-meta-toluamide), a safe and effective repellent, was developed during World War II. Fear of possible side effects of DEET has created a large market for “natural” DEET-free repellents with a variety of active ingredients. We present a comparative study on the efficacy of eight commercially available products, two fragrances, and a vitamin B patch. The products were tested using a human hand as attractant in a Y-tube olfactometer setup with Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), both major human disease vectors. We found that Ae. albopictus were generally less attracted to the test subject’s hand compared with Ae, aegypti . Repellents with DEET as active ingredient had a prominent repellency effect over longer times and on both species. Repellents containing p-menthane-3,8-diol produced comparable results but for shorter time periods. Some of the DEET-free products containing citronella or geraniol did not have any significant repellency effect. Interestingly, the perfume we tested had a modest repellency effect early after application, and the vitamin B patch had no effect on either species. This study shows that the different active ingredients in commercially available mosquito repellent products are not equivalent in terms of duration and strength of repellency. Our results suggest that products containing DEET or p-menthane-3,8-diol have long-lasting repellent effects and therefore provide good protection from mosquito-borne diseases.

Mosquitoes are hosts for an array of different protozoan parasites, nematodes, and viruses ( Marquardt 2004 , Enserink 2007 , Murray et al. 2010 ). Controlling mosquito populations is an effective tool for the fight against such pathogens. Several different methods for mosquito control have been developed, e.g., source reduction, physical exclusion (nets, screens, etc.), pesticide application, biological control methods, sterile insect technique, and release of genetically modified mosquitoes ( Rose 2001 , Phuc et al. 2007 , Alphey et al. 2010 ). Unfortunately and for a variety of reasons, these approaches can be difficult to implement in many locations ( Peter et al. 2005 ). Widespread insecticide resistance in disease-carrying mosquito populations also poses a significant problem.

On an individual level, mosquito repellents are widely used to avoid disease exposure ( Barnard 2000 , Barnard and Xue 2004 ). Repellents, even though they can never guarantee complete protection, can significantly lessen the chance of contracting vector-borne diseases ( Kahn et al. 1975 , Barnard et al. 1998 , Barnard and Xue 2004 , Rowland et al. 2004 , Hill et al. 2007 ). They are especially useful when used where human activity coincides with the diurnal activity patterns of mosquitoes, e.g., outdoor activities that take place at dusk and dawn, e.g., hunting and fishing.

The sense of smell is one of the most important senses that mosquitoes use for long range host seeking ( Potter 2014 ). Insect olfaction has been extensively studied leading to the identification of the key proteins involved: odorant receptors, odorant receptor co-receptors, gustatory receptors, and odorant binding proteins ( Kaupp 2010 , Suh et al. 2014 ). The processing of olfactory information in different regions of the insect brain has also attracted a lot of research interest ( Galizia 2014 ). Various mosquito attractants and repellents have been identified, many of which are produced by human metabolism or the bacterial degradation of sweat components. Lactic acid and 1-octen-3-ol are two components that act as strong mosquito attractants ( Zwiebel and Takken 2004 ). Carbon dioxide from breath is another strong attractant that sensitizes mosquitoes to other odorants ( Dekker et al. 2005 ). Studies have shown that different insect repellents use a similar mode-of-action. Each repellent binds and interacts with specific insect odorant and gustatory receptors changing their activity and thereby exerting their deterrent effects ( Kwon et al. 2010 , Dickens and Bohbot 2013 , Xu et al. 2014 ). The most widely used insect repellent, DEET (N,N-diethyl-m-toluamide), has been in use for about 70 yr. DEET is considered a very safe repellent ( Osimitz and Grothaus 1995 , Koren et al. 2003 , Sudakin and Trevathan 2003 ). Nevertheless, fear of possible side effects of DEET and general chemophobia has resulted in the development of a multitude of “DEET-free” mosquito repellents with a variety of active ingredients. Plant-based repellents usually contain essential plant oils as active ingredients.

There are several approaches for evaluating the efficacy of insect repellents. Some of the bioassays that have been used include spatial repellency assay, host attraction-inhibition assay, landing inhibition assay, effective dose and duration assays, taxis cage assays, etc. ( Lorenz et al. 2013 , Afify et al. 2014 , Menger et al. 2014 ). Olfactometers are useful tools used in attraction-inhibition assays to test repellent efficacy. They allow the experimenter to perform the tests under very controlled conditions, thereby eliminating many variables that may alter experimental results in more open systems ( McIndoo 1926 ).

Here, we report experiments performed with a Y-tube olfactometer that was constructed according to a blueprint published by the World Health Organization in its publication “Guidelines for efficacy testing of spatial repellents” ( World Health Organization [WHO] 2013 ). We performed host attraction-inhibition assays and tested the efficacy of eight commercially available mosquito repellent products, a perfume, a bath oil, and a vitamin B patch.

Materials and Methods

Mosquito Culture

Ae . aegypti UGAL strain and Ae. albopictus F10 strain were acquired from the Malaria Research and Reference Reagent Resource Center (ATCC 2015). Mosquito culture was executed as described in Marquardt(2004), using chicken for a blood meal source. Larvae were reared in 13” by 20” pans filled with 2 liters of deionized water held at 27°C. Larvae were given five dry cat food pellets (Special Kitty-Wal-Mart Stores, Inc. Bentonville, AR) every 3 d; water was changed after 5 d. The mosquitoes were allowed to mature for 5 d in a BugDorm-1 Insect Rearing Cage (30 by 30 by 30 cm, BugDorm Store, Taichung, Taiwan) before the experiment commenced. The adult mosquitoes were maintained on 20% sucrose solution, ad libitum, up to 24 h before the experiment began. The cages were placed in an incubation room that was maintained at 80% humidity and 27°C with a photoperiod of 14:10 (L:D) h. Mosquitoes were starved 24 h before each Y-tube assay.

Institutional Review Board Approval

This study has been approved by the New Mexico State University Institutional Review Board. Title: “Efficacy of different insect repellents,” study no. 11505A.

Selection of Commercially Available Repellents

Repellents were purchased locally in Las Cruces, NM, or ordered online via Amazon. Table 1 lists the products tested, active ingredients, manufacturer and location, and the manufacturer’s estimated protection times. The Mosquito Skin Patch was applied 2 h before the start of the experiment.

Table 1.

Active ingredients, manufacturers, and estimated protection time of the repellents, fragrances, and patch

Product name Product type Active ingredient(s) Manufacturer/distributor  Estimated protection time a
Repel 100 insect repellentRepellent spray DEET (98.11%) WPC Brands, Inc. 10 h 
OFF deep woods insect repellent VIIIRepellent spray DEET (25%) S.C. Johnson & Son, Inc. Not provided 
Cutter skinsations insect repellentRepellent spray DEET (7.0%) Spectrum Division of United Industries Corporation Not provided 
Cutter natural insect repellentRepellent spray Geraniol (5%) Spectrum Division of United Industries Corporation 2 h 
Soybean oil (2%) 
EcoSmart organic insect repellentRepellent spray Geraniol (1.0%) EcoSMART Technologies Inc. 2 h 
Rosemary oil (0.5%) 
Cinnamon oil (0.5%) 
Lemongrass oil (0.5%) 
Cutter lemon eucalyptus insect repellentRepellent spray Oil of lemon eucalyptus (30%) Spectrum Division of United Industries Corporation 6 h 
This oil contains 65% p-menthane-3-8-diol 
Avon Skin So Soft Bug GuardRepellent spray Oil of citronella (10%) Avon Products, Inc. 2 h 
Avon Skin So Soft Bath OilFragrance Unknown Avon Products, Inc. Not Recommended 
Victoria Secret BombshellFragrance Unknown Victoria Secret Not Recommended 
Mosquito skin patchPatch Thiamin B1 (300 mg) AgraCo Technologies International, LLC 36 h 
Product name Product type Active ingredient(s) Manufacturer/distributor  Estimated protection time a
Repel 100 insect repellentRepellent spray DEET (98.11%) WPC Brands, Inc. 10 h 
OFF deep woods insect repellent VIIIRepellent spray DEET (25%) S.C. Johnson & Son, Inc. Not provided 
Cutter skinsations insect repellentRepellent spray DEET (7.0%) Spectrum Division of United Industries Corporation Not provided 
Cutter natural insect repellentRepellent spray Geraniol (5%) Spectrum Division of United Industries Corporation 2 h 
Soybean oil (2%) 
EcoSmart organic insect repellentRepellent spray Geraniol (1.0%) EcoSMART Technologies Inc. 2 h 
Rosemary oil (0.5%) 
Cinnamon oil (0.5%) 
Lemongrass oil (0.5%) 
Cutter lemon eucalyptus insect repellentRepellent spray Oil of lemon eucalyptus (30%) Spectrum Division of United Industries Corporation 6 h 
This oil contains 65% p-menthane-3-8-diol 
Avon Skin So Soft Bug GuardRepellent spray Oil of citronella (10%) Avon Products, Inc. 2 h 
Avon Skin So Soft Bath OilFragrance Unknown Avon Products, Inc. Not Recommended 
Victoria Secret BombshellFragrance Unknown Victoria Secret Not Recommended 
Mosquito skin patchPatch Thiamin B1 (300 mg) AgraCo Technologies International, LLC 36 h 

View Large

Attraction-Inhibition Assay

The Y-tube (see Fig. 1 and Supp Material 1 [online only]) was constructed according to the WHO schematic with modifications ( WHO 2013 ). A constant air current was produced by a computer fan that was placed at the bottom of the Y-tube. The air flow was adjusted to 0.4 m/s in the base leg (0.2 m/s in each port) by placing the probe of an anemometer within the different shafts of the Y-tube and moving the fan in relation to the tube opening until the correct air flow speed was achieved. Experiments were all performed between 0800 and 1200 to avoid diurnal changes in mosquito activity. One of the co-authors was selected as the attractant for Y-tube assays based on preliminary attraction studies that found her to be a superior attractant. The volunteer was not allowed to wash her hands, wear perfume, or take a shower in the morning prior to the experiments. One of the attractant’s hands was sprayed with approximately 0.5 ml of liquid repellent; the other hand was covered with a nitrile glove. The hand was sprayed on both sides and allowed to air dry. Trap doors 1 and 2 were opened and the mosquitoes were placed in the holding chamber of the Y-tube. The treated hand was then placed in one of the decision ports (from here on referred to as the “hand port”); the other, untreated gloved hand was inserted into the other port (control port). Alternating decision ports were used for the biological replicates to ensure there was no bias. The mosquitoes were given 30 s to acclimate to their environment while exposed to the odor on the hand before they were released from the holding chamber by opening trap door 3. The mosquitoes were given 2 min to relocate within the tube. After a 2-min period, all trap doors were closed. Three groups of mosquitoes were counted and recorded: the ones that stayed in the holding port, the ones that arrived in either decision port, and the ones that stayed in the shaft of the Y-tube. The mosquitoes that were not captured at either decision port or in the holding port were considered wandering. For each replicate, there were a total of 20 mosquitoes placed in the holding chamber. We evaluated efficacy of the repellent over a 4-h time period with evaluation points at: 0 min, 30 min, 120 min, and 240 min post application. The experiments commenced in the early morning and ran for 4 h, the time of commencement represented the laboratory mosquitoes’ dawn. Five replicates were performed for each time point, and the experiments were performed over a 3-mo period. Attraction rate (%) was calculated as the number of mosquitoes in the treated hand port divided by the total number of mosquitoes in the replicate.

Statistical Methods

To evaluate the efficacy of each repellent, a one-way repeated measures analysis of variance was used. The dependent variable was the rank-transformed ratio of the number of mosquitoes that ended up in the port containing the hand or the holding port vs. the total number of mosquitoes in the test. One test was performed for the mosquitoes in the hand port and a second for those in the holding chamber. Dunnett’s multiple comparison procedure was used, with the untreated time zero control group as the control.

Results

Ae. aegypti Attraction-Inhibition Assays

Table 2 lists the overall attraction rates of Ae. aegypti females to an untreated control hand and hands treated with various repellents. The overall attraction rate was determined as an average of five replicates, calculating the number of mosquitoes in the treated hand port out of the total mosquitoes in the replicate.

Table 2.

Average percentage of Ae. aegypti mosquitoes trapped in the port with the hand

Treatments ( N = 5)  Initial (±SE) 30 min (±SE) 120 min (±SE) 240 min (±SE) 
Control61 (±4.30) 61 (±4.00) 58 (±2.00) 68 (±3.39) 
Repel 100 insect repellent 10 ** (±1.58)   18 ** (±3.39)   15 ** (±5.24)   14 ** (±4.85)  
OFF deep woods insect repellent VIII 6 ** (±1.87)   17 ** (±1.22)   14 ** (±1.00)   29 ** (±3.32)  
Cutter skinsations insect repellent 11 ** (±3.67)   22 ** (±5.15)   17 ** (±2.55)   30 ** (±5.24)  
Cutter natural insect repellent57 (±3.39) 47 (±4.06) 64 (±1.87) 65 (±6.12) 
EcoSmart organic insect repellent 9 ** (±1.87)  55 (±3.16) 68 (±2.00) 67 (±3.39) 
Cutter lemon eucalyptus insect repellent 9 ** (±2.45)   8 ** (±3.00   13 ** (±4.64)   18 ** (±3.74)  
Avon Skin So Soft Bug Guard48 (±4.06)  42 * (±2.55)  52 (±2.55) 67 (±5.15) 
Avon Skin So Soft Bath Oil 31 ** (±1.00)   35 ** (±5.70)   43 * (±6.04)  53 (±8.00) 
Victoria Secret Bombshell 17 ** (±5.39)   15 ** (±5.24)   18 ** (±3.00)  47 (±5.39) 
Mosquito skin patch68 (±5.10) 67 (±5.61) 48 (±4.90) 68 (±5.15) 
Treatments ( N = 5)  Initial (±SE) 30 min (±SE) 120 min (±SE) 240 min (±SE) 
Control61 (±4.30) 61 (±4.00) 58 (±2.00) 68 (±3.39) 
Repel 100 insect repellent 10 ** (±1.58)   18 ** (±3.39)   15 ** (±5.24)   14 ** (±4.85)  
OFF deep woods insect repellent VIII 6 ** (±1.87)   17 ** (±1.22)   14 ** (±1.00)   29 ** (±3.32)  
Cutter skinsations insect repellent 11 ** (±3.67)   22 ** (±5.15)  

DEET is a popular insect repellent—an estimated 30 percent of Americans use it every year—yet it sounds like a good number of people are wary of it, too. A Google search of "DEET dangers" found a Mercola article saying DEET kills mosquitoes, although it doesn’t (Perhaps they’ve confused DEET with DDT?). A site called FitSugar says “DEET pumps through your nervous system and has been proven to kill brain cells,” neither of which is true. Weird! Guess DEET does really suffer from a "perception problem," as the Los Angeles Times reported in 2011.

Now, recent review of DEET’s effects on human health has found the insect repellent is safe to use on the skin. The review is one of several that researchers have done over the past 20 years, all finding that DEET is generally safe. In fact, there’s no direct evidence using DEET normally harms the nervous system. Bad reactions to DEET, when applied to the skin in bug sprays, are extremely rare, but not non-existent.

Why use DEET if there’s any risk of health problems at all? The chemical is the best insect repellent people have ever invented. Nothing else has the same history of efficacy, although afewothers have strong evidence for them, too. Still, in regions where people are at risk for serious insect-borne diseases such as malaria or West Nile virus, DEET is what doctors recommend. In addition, doctors recommend using DEET in concentrations between 20 percent and 50 percent. The idea is that lower concentrations require people to reapply more frequently than they're apt to (while concentrations higher than 50 percent don't necessarily work better or longer). This means if you want DEET protection, you’ll have to resign yourself to using pretty high concentrations of the stuff.

Let’s start with the evidence for neurological problems from DEET, which FitSugar was so afraid of. In the scientific literature—in all of scientific literature since 1957, when DEET first appeared on store shelves—researchers have reported 14 cases of kids who suffered encephalopathy, including seizures, after using DEET on their skin. All but one were kids under age 8. Three children died. The others recovered fully. In each of the cases, it was difficult to determine whether DEET caused the brain symptoms. Such data are just hard to come by; the kids could have been exposed to other things, but it could have been the DEET, too.

DEET is the best insect repellent humans have ever invented.

More straightforward is one study researchers conducted, following women in Thailand who used DEET from their second trimester of pregnancy onward. DEET reduced the incidences of malaria the women suffered, the study found. In addition, babies born to moms who used DEET didn’t differ from babies born to moms who didn’t use DEET. Babies in the two groups had the same weights and lengths and had the same head circumferences. All the babies also performed the same in neurological tests.

The U.S. Agency for Toxic Substances and Disease Registry has a page of reports in the U.S. of health problems in people after using DEET. Among the cases are two deaths in adults and three cases in which pregnant women who used large amounts of DEET gave birth to babies with problems. One baby died. As with the kids with encephalopathy, in the nearly all of the ATSDR-reported cases, it’s difficult to know if DEET was the culprit. Compared to how often people use DEET around the world, those cases are very rare. Researchers estimate people around the world put on DEET 200 million times a year.

These figures are all for people who use DEET on their skin. Doctors definitely don’t recommend ingesting a lot of DEET or getting it in your eyes. Don’t leave bug sprays in a place where a kid could do either by accident. MedlinePlus lists symptoms of DEET ingestion, plus recommendations for what to do if someone does consume a lot of DEET or get it in his eyes.

We’ll leave you with what’s perhaps science’s strangest report of DEET-related problems. From the Agency for Toxic Substances and Disease Registry:

Happy camping, dear readers.

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