Article in Press. History of Insect Repellents. Historical Review. Sarah J. Moore and Mustapha Debboun

Transcription

1 History of Insect Repellents Sarah J. Moore and Mustapha Debboun CONTENTS Historical Review...3 Traditional Repellent Use Today...5 Pyrethrum, Mosquito Coils and Area Repellents...5 The Development of Modern Synthetic Repellents...6 Deet A Breakthrough in Repellents...8 Recent Repellent Discoveries...8 DEPA...8 IR Piperidine Compounds...9 KBR AI and AI SS Repellent Delivery Methods...11 Area Repellents...13 The Evolution of Repellent Testing...14 Kairomones...14 Choice...14 In Vitro and Animal Tests...15 Test Standardization...15 References...17 Historical Review It is likely that the use of repellents against biting arthropods developed thousands possibly even millions of years ago. Several species of primate have been observed anointing their pelage by rubbing it with millipedes and plants including Citrus spp., Piper marginatum, and Clematis dioica. 1 4 Wedgecapped capuchins (Cebus olivaceus) were observed rubbing the millipede Orthoporus dorsovittatus onto their coat during the period of maximum mosquito activity. 5 The O. dorsovittatus species contains insect-repellent chemicals called benzoquinones, and it was hypothesized that the anointing behavior was designed to deter biting insects. Laboratory studies went on to show a significant repellent effect of 3

2 4 Insect Repellents: Principles, Methods, and Uses benzoquinones against Aedes (Stegomyia) aegypti (the yellow fever mosquito) 6 and Amblyomma americanum (the lone star tick). 7 Anointing behavior to deter blood-feeding arthropods is also common among birds, 8 and it may be genetically expressed as an extended phenotype because it has obvious adaptive benefit. 9 Evidence for this lies in the fact that benzoquinones applied to filter paper elicited anointing activity among captive-born capuchins. 6 The first recorded use of repellents may be found among the writings of Herodotus (484 BCE ca. 425 BCE), who observed Egyptian fishermen. 10 Herodotus stated that, The Egyptians who live in the marsh-country use oil extracted from the castor-oil plant. This plant, which grows wild in Greece, they call Kiki, and the Egyptian variety is very prolific and has a disagreeable smell. Their practice is to sow it along the banks of rivers and lakes, and when the fruit is gathered it is either bruised and pressed, or else boiled down, and the liquid thus obtained is of an oily nature and quite as good as olive oil for burning in lamps, although the smell is unpleasant. It was argued that the oils acted as an area repellent because high densities of nuisance mosquitoes are active in the evenings in this region. This would have driven the Egyptians to their beds (where Herodotus also observed that they slept under rudimentary bed nets) had the lamp not provided protection from biting insects. 11 The Romans also recorded methods of repelling flying insects (gnats) that would have included mosquitoes, as much of Italy was once swampland where the malaria vectors Anopheles labranchiae, Anopheles sacharovi, and Anopheles superpictus were abundant prior to the malaria eradication program of The Geoponika is a collection of Roman agricultural lore, compiled during the tenth century for the Byzantine emperor Constantine VII Porphyrogenitus, that was heavily based upon the writings of Vindonius Anatolius (fourth century), as well as earlier writers, including Pliny. 13 The text suggests rubbing a concoction of vinegar, manna, and oil on the body, especially the head and feet, to repel gnats. 13 This may have had an effect on nuisance insects, especially mosquitoes, as natural vinegars contain acetic acid and smaller amounts of tartaric and citric acids. These acids may have had a mild antibacterial effect on the skin and therefore reduced the production of bacterial metabolites that mosquitoes use to locate human hosts, 14 particularly those produced by the feet. 15 In addition, some oils have a mild repellent action, 16 perhaps by reducing the emanation of host odor. In addition, Geoponika describes burning herbs such as black cumin (Nigella sativa), bay (Laurus nobilis), galbanum (Ferula gummosa), and oregano (Origanum vulgare) to drive away nuisance insects. 13 Writings (ca. seventeenth century) derived from the ancient Sanskrit Yoga Ratnakara also contain references to the burning of plants to repel biting insects, including Vaca (Acorus calamus), Marica (Piper nigrum), asafoetidia (Ferula asafoetida), and Nimba or Neem (Azadirachta indica). 17 Other remedies suggested in Geoponika and Yoga Ratnakara included burning fish, shells, various bones, dung, snakeskin, and peacock feather. This would have created a thick noxious smoke, as would have burning asafoetidia that has the colloquial name of Devil s Dung in old French. This may have been perceived to work, as the smoke generated was thick and noxious to humans, although smoke does have some repellent effects on mosquitoes. 18 The smoke may mask human kairomones, particularly carbon dioxide, and the convection currents that mosquitoes need for short-range host location. Smoke production also lowers humidity by reducing the moisture-carrying capacity of the air. This makes mosquitoes susceptible to desiccation and reduces sensory input because mosquito chemoreceptors are more responsive in the presence of moisture. 19 In North America, native cultures relied heavily on plants, and many used plants to repel biting insects. 20 The Southern Carrier Tribe, or Dakelh, meaning people who go around in boats, live near rivers in British Columbia where mosquito densities are extremely high. This group used an infusion of common cow parsnip blossoms (Heracleum maximum) rubbed on the body to repel flies and mosquitoes; however, the more common mode of use was burning. For instance, the Colville Indians based around the Columbia River used leaves and stems of Common Yarrow (Achillea millefolium) as a smudge to keep

3 History of Insect Repellents away mosquitoes. The Blackfoot tribe, whose territory stretched along the Saskatchewan River, put the fringed sagewort (Artemesia frigida) plant on campfire coals to drive away mosquitoes. Apparently, it was so effective that wild horses sheltered from insect pests in the smoke; consequently, the Indians used it to attract horses. The use of smoke against biting insects was carried on by European settlers as recorded by settlers to The Black Swamp in Ohio 21 : They [first settlers in Wood County] were subject to all kinds of deprivations. The most distressing of all the rest was their being subject to epidemics that swept through the country every summer and fall in the shape of malarial fevers. The warm months gave way to unrelenting swarms of gnats and mosquitoes. The most effective tool available [to fight the mosquito] was the smudge pot. These pots and their accompanying clouds of dark smoke discouraged the insects and were useful throughout most of the day; they were next to the cow while milking, under the table while eating, and even beside the bed while sleeping. Traditional Repellent Use Today Smoke is still the most widely used means of repelling mosquitoes utilized throughout the rural tropics. Waste plant materials are frequently burned in Sri Lanka as a mosquito repellent, even though indoor residual spraying has been carried out by the government for many years. 22 In rural Guinea-Bissau, 86% of residents used an unimpregnated bednet in conjunction with mosquito coils or plant-based smoke. 23 In the Solomon Islands, a recent survey revealed that fire with coconut husks and papaya leaves was the most prevalent form of personal protection from mosquitoes, being used by 52% of residents. 24 Surveys from South America found that 69 and 90% of respondents from Mexico 25 and Guatemala, 26 respectively, burned waste materials to drive away mosquitoes. Smoke is also used to drive away biting insects in Southeast Asia: wood-fires and smudge pots are used in Myanmar, 27 whereas herbs are thrown on the fire in Yunnan, China. 28 Although these methods are crude, many traditional repellents do have a repellent effect. A recent controlled field trial showed a comparable repellent effect produced by a 0.2% pyrethrin mosquito coil and lemon gum (Corymbia citriodora) volatiles expelled by heating on metal plates. 29 Several field evaluations, where plants were burned to repel mosquitoes, have shown good reduction in mosquito landings. 23,30,31 One well-designed study in Papua New Guinea showed that burning local wood and leaves (mango wood, coconut husks, wild ginger leaves, and betelnut leaves) repelled between 57 and 75% of mosquitoes. 31 Smoke also reduced indoor sand fly density by 1.7 times in East Africa. 32 The use of smoke, although effective, requires continuous production in order to repel biting insects when used as an area repellent outdoors. 33 Although smoke does have a residual repellent effect when used within houses, 29 the indoor combustion of biomass has severe health consequences. 34 Therefore, safer and more modern methods of repelling mosquitoes are desirable. Pyrethrum, Mosquito Coils and Area Repellents Pyrethrum is natural plant oil that occurs in the two species of pyrethrum daisy: Chrysanthemum cinerariifolium from the Dalmatian region and Chrysanthemum coccineum of Persian origin. The insecticidal component, comprising six esters (pyrethrins), is found in tiny oil-containing glands on

4 6 Insect Repellents: Principles, Methods, and Uses the surface of the seed case in the flower head. It is a highly effective insecticide that although it has been used for centuries against all manner of insect pests, is relatively harmless to mammals. 35 Pyrethrum is thought to have originally been used in China and was introduced to the Middle East along the trade routes through Central Asia, 36 from where it was introduced into Europe during the nineteenth century. 37 It is currently incorporated into mosquito coils to repel insects, and this practice probably derived from the incense used in religious ceremonies by Hindus, Buddhists, and the followers of Confucius. In Java today, the same incense used in ceremonies to honor ancestors is also used on a daily basis to repel mosquitoes. 38 Pyrethrum powders were used by armies from the time of Napoleon to the World War II to combat head and body lice. Before World War II, Japan was the major growing area, 37 and exported pyrethrum powder that was mainly used directly in its unrefined form as a powder for killing fleas. At that time in Japan, people usually mixed pyrethrum powder with sawdust and burned it in a brazier or incense burner to repel mosquitoes. Around 1890, the businessman Eiichiro Ueyama improved the pyrethrum powder and successfully developed a spiral-shaped mosquito repellent. 39 He formulated that idea when he met the son of an incense dealer at an inn in Tokyo. While talking with him, he came up with the idea of mixing starch powder with pyrethrum powder, then kneading it into the shape of stick incense. After several failures, Mr. Ueyama employed the workers of incense makers in Sakai, and thereby succeeded in creating a viable commercial product: a mixture of starch powder, dried mandarin orange skin powder, and pyrethrum powder. It was thoroughly mixed and kneaded, and placed into a wooden mortar, extruded, and cut into the form of stick incense. Ueyama then replaced the wooden mortar with a compressing machine and was able to realize mass production. However, the bar-shaped mosquito stick burned rapidly, and several sticks had to be burned at once to obtain sufficient smoke to repel insects. In 1895, Yuki, the wife of Eiichiro, proposed making the stick thicker and longer, and curling it into a spiral shape. Eiichiro acted immediately on her proposal, but it was not until 1902, after years of experimentation, that he was finally able to complete a mosquito repellent with a spiral shape that was worthy of marketing. The final method involved cutting a thick bar of incense to a certain length and manually winding it. This same method continued to be used until 1957, when it was improved through machine punching, making mass production possible on a far larger scale. 39 Mosquito coils are widely used today: 29 billion mosquito coils are sold each year, 95% of them in Asia, 40 and household expenditure on these methods in the developing countries is substantial. 41,42 There is ample evidence that mosquito coils effectively repel mosquitoes. 43 Pyrethrum affects the central nervous systems of all types of flying and crawling insects, blocking sodium-gated nerve junctions so that nervous impulses fail, 44 and the insect is knocked down and may die. In the lowest concentrations, pyrethrum affects insect behavior, producing a so-called avoidance reaction or excito-repellency that results in the insect fleeing the source of the chemicals. 45 Synthetic analogues of pyrethrum were developed from the 1940s onwards. They exhibit a similar mode of action to pyrethrum, but are more potent and photostable. 46,47 The insecticides broadly act in two ways: (1) the choreoathetosis/salivation (CS) pathway, and (2) the tremor (T) pathway. 48 Importantly, these effects result in deterrency from entering a room where coils are burning, expellency of mosquitoes from within, interference with host-finding, bite inhibition, knockdown, and kill. 49 These repellence and bite-inhibition effects have been exploited to produce highly-efficacious repellents that combine permethrin (a synthetic pyrethroid) and deet, a synthetic repellent discussed extensively in this volume. 50,51 The Development of Modern Synthetic Repellents The military has conducted significant research into modern repellents to protect their troops from insect-borne disease. The first military repellents contained essential oils derived from plants.

5 History of Insect Repellents Q1 FIGURE 1.1 Repellents distributed to U.S. troops, Bombay 1945 ( q Office of the Army Surgeon General, Public Affairs, and the Directorate of Information Management, Fort Detrick, Maryland, U.S.A.). For instance, the Indian Army was issued a repellent comprised of citronella, camphor, and paraffin. 52 However, these repellents had limited duration, and intensive research began during World War II to find long-lasting repellents. The enormous burden of disease suffered by troops fighting in endemic areas motivated this research. For instance, 821,184 cases of malaria were recorded among U.S. troops involved in overseas campaigns, resulting in 302 deaths, 53 and over 12 million lost duty days. 54 With the advent of large-scale jungle warfare, chigger-borne scrub typhus became an important medical problem for troops in the Far East. Indeed, approximately 6,000 cases were to appear in U.S. forces alone during the campaigns that followed the outbreak of war with Japan. 55 Chiggers were also the cause of considerable discomfort for soldiers training in the U.S.; this resulted in the Surgeon General requesting the Orlando laboratory of the United States Department of Agriculture (USDA) to study means and methods for controlling chiggers by repellents or insecticides in the summer of Between 1942 and 1945, over 7,000 potentially repellent compounds were tested by the USDA. 57 One of the first chemical repellents to be developed was dimethyl phthalate (DMP; it was patented in 1929 as a fly repellent), followed by Indalone w (butyl-3,3-dihydro-2,2-dimethyl-4-oxo-2h-pyran-6-carboxylate; patented in 1937), and ethyl hexanediol (2-ethyl-1,3-hexanediol), also called Rutgers 612, that became available in In 1942, DMP and Indalone demonstrated significant protection against chiggers when tested by Madden, Lindquist, and Knipling of the Orlando laboratory for troops in Louisiana. 59 The result was corroborated by field trials in New Guinea against scrub chiggers (Leptotrombidium) 60 and a range of other species. 61 The introduction of chemical repellents dramatically lowered incidence of scrub typhus, 55,60 and allowed less-restrictive battle uniforms. Prior to the introduction of DMP and Indalone for impregnation of uniforms and application to exposed skin, prevention of insect bites had relied on long clothing plus head nets and mosquito gloves. 56 The head nets were uncomfortably hot and restricted vision, making them unpopular with troops and therefore rarely used. The introduction of repellents for exposed parts of the body proved more popular (Figure 1.1). 56 After the war, a repellent known as or M-250, containing 6 parts DMP, 2 parts Indalone, and 2 parts ethyl hexanediol, became popular in the U.S.A. However, products containing ethyl hexanediol were voluntarily removed from the U.S. and Canadian markets in 1991 in response to an unpublished study by a manufacturer showing poor lung expansion in the offspring of exposed animals. 62 Additional studies showed mild developmental toxicity after cutaneous administration to pregnant rats. 63

6 8 Insect Repellents: Principles, Methods, and Uses Deet A Breakthrough in Repellents Prior to the removal of from the marketplace, its use was eclipsed by the discovery of deet (N,N-diethyl-3-methlybenzamide or N,N-diethyl-m-toluamide) in This was perhaps the single most important event in the evolution of repellents, and deet remains the principal, and the most effective repellent in use today 65 more than 50 years after its discovery. Deet is a broad-spectrum repellent that is highly effective against all mosquitoes: Aedes spp., including the dengue vectors Aedes aegypti 70,71 and Aedes albopictus 72,73 ; Culex spp. 71,73 76 ; Mansonia spp. 71,74,77 ; and Anopheles malaria vectors, including the Afrotropical Anopheles gambiae, 74,78,79 and Anopheles arabiensis, Southeast Asian Anopheles dirus, 71,83,84 and Anopheles minimus 30 ; South American Anopheles darlingi, 85 and Western Pacific Anopheles farauti. 86,87 Other insects of medical importance repelled by deet include sand flies (Psychodidae, both Old World and New World) ; black flies (Simulidae) 67,91 ; chiggers (Trombiculidae) ; hard and soft ticks (Ixodidae) ; bedbugs (Cimex hemipterus) 99 ; and fleas (Siphonaptera). 100 It is, therefore, now used as the gold-standard repellent against which other substances are compared in laboratory and field trials. An estimated 15 million people in the U.K., 78 million people in the U.S.A., 101 and 200 million people globally use deet each year. 102 There has been much speculation on the safety of deet following reports linking it to seizures and encephalopathy, particularly in children, as well as neurotoxicity, 107 especially in combination with other pesticides. 108 However, deet has been used for 50 years with a tiny number of reported adverse effects, many of which had a history of excessive or inappropriate use of repellent. 109,110 Nonetheless, its toxicology has been more closely scrutinized than any other repellent, but it has been deemed safe for human use, 101,111 including use on children 106 and pregnant women. 112 The use of a deet/permethrin repellent has recently been proven to reduce malaria incidence amongst users. 113 Recent Repellent Discoveries DEPA Recently, DEPA (N,N-diethyl phenyl acetamide), a compound developed around the same time as deet, 64 has received renewed attention. It has similar cosmetic properties to deet, similar dermal absorption and excretion, plus the symptoms of acute poisoning with DEPA are similar to deet. 114 However, its dermal toxicity to rats has been reported as LD g/kg, 114 and 3 4 g/kg, 115 which may require further clarification. In a field study, 0.3 mg/cm 2 DEPA in alcohol provided complete protection against Culex quinquefasciatus mosquitoes at a mean landing rate of 9.22 mosquitoes/person/h. 116 Another field test of DEPA with Culex quinquefasciatus, Simulium himalayense, and the leech Haemadipsa zeylanica showed 1.5, 2, and 1.5 h of complete protection, respectively. 117 However, control numbers were not given in this publication. Laboratory tests using rabbits showed that there was no significant difference in the response of the sand fly Phlebotomus papatasi to DEPA or deet. 90 Furthermore, in vitro application of repellents to a membrane blood feeding system, for Aedes aegypti, has shown that two analogues of DEPA, DM156 and DM34, show promising repellency and low toxicity, warranting further evaluation. 118 DEPA is an extremely cheap repellent, costing Rs (U.S. $25.40) per kg compared to Rs (U.S. $48.40) for deet. 116 This is because one of the precursors of DEET, (3-methylbenzoic acid) is not readily available in India. 119 DEPA has now been formulated in a commercial preparation by the Defence Research and Development Establishment (DRDE) and has been granted approval by the

7 History of Insect Repellents Drug Controller of India. 120 This repellent may prove useful, particularly among residents of the developing world, for whom cost is the main motivator in personal repellent choice. 121 IR 3535 Insect repellent 3535 (IR 3535), [3-(N-acetyl-N-butyl) aminopropionic acid ethyl ester], also known as MERCK 3535, was developed in 1975 by Merck, 122 and has been on the market in Europe for the past twenty years. It has low toxicity, although it is irritating to the eyes and sometimes the skin. 123 It became available in the U.S. in 1999 after being passed by the EPA, classified as a biopesticide, as it is a substituted B-amino acid, structurally similar to naturally occurring B-alanine. 124 Efficacy data for IR 3535 is variable, but it is generally comparable with DEET. Data from the laboratory showed IR 3535 to be equal to deet against Aedes aegypti, Culex quinquefasciatus, 69,125 and Culex taeniorhynchus, but not Anopheles dirus. 125 However, another laboratory study with Aedes aegypti and Anopheles maculatus showed IR 3535 to be significantly inferior to deet. 126 Field trials in Southeast Asia against Aedes albopictus, and Culex gelidus 125 ; and Aedes albopictus, Culex quinquefasciatus, and Culex bitaeniorhynchus 127 found that IR 3535 and deet offered similar protection. However, a test against Aedes cantans and Aedes annulipes under initial biting pressures of 714 landings/person/h produced data that indicated that deet had a duration twice that of IR 3535 (4.8 vs 9.7 h). 128 A further test against Aedes (Ochlerotatus) taeniorhynchus in the Everglades, also under high biting pressure, measured no significant difference between the protection offered by DEET and IR A comprehensive field test against Anopheles gambiae showed that IR 3535 decayed at a similar rate to deet, 78 and the World Health Organization (WHO) has declared it a safe and effective repellent for human use. 123 In fact, there is not a single recorded incidence of an adverse reaction to this compound. Piperidine Compounds There has been a flurry of renewed interest in the piperidine-based compounds, leading to the discovery of several new and highly effective repellents. Piperidines, as a chemical class, are cyclic amines. The piperidine skeleton is present in piperine, the main active chemical agent in pepper (Piper sp.). During the 1970s, approximately 600 synthetic compounds related to piperidines were developed by scientists at the Gainesville and Beltsville research centers of the USDA. The data from these experiments is now being re-examined using new, high-tech methodologies coupled with rapid-screening bioassays. This interest in finding deet alternatives has been motivated by the controversy around the safety of deet, its low user acceptability, and its plasticizing effect. KBR 3023 The repellent 1-piperidine carboxylic acid-2(2-hydroxyethyl)-1-methylpropylester was developed by Bayer in the 1980s using molecular modelling. 129 It has several synonyms: Picaridin is its common name, Bayrepel w is its Bayer trademark name, Icaridin was used by WHO, and KBR 3023 is another trade name. This compound, the most recent piperidine derivative, is registered in many European, South American, Asian and African countries as well as Japan, Canada, and the U.S. Its most important new feature is its very low toxicity (EPA Grade III). Most importantly, it elicits practically no dermal or eye irritation (EPA Grade IV) nor skin sensitization. 130 Furthermore, it does not have a significant plasticizing effect, which is a major drawback of deet. Cosmetically, it is superior to deet as it is colorless, odorless and has a pleasant feel on the skin. 131 A user acceptability study showed a distinct preference for KBR 3023 among Australian troops when compared to deet, which was uncomfortably oily or caused irritation to half of respondents. 132 The efficacy of Picaridin is excellent, and it is generally superior to deet in terms of longevity. In a carefully designed field evaluation against Anopheles gambiae and Anopheles funestus, KBR 3023 in

8 10 Insect Repellents: Principles, Methods, and Uses ethanol outperformed deet after a 10-h exposure, and the half-life of the repellent was one hour longer than that calculated for deet and IR 3535, 78 using modelling first used by Rutledge et al. in This is because KBR 3023 evaporates at a slower rate than deet. Were it not for the lower volatility of Picaridin, it would probably be less effective, because dose for dose it is less repellent than deet when freshly applied. 134 Other studies have shown a similar performance when compared to deet in field trials: against Anopheles spp. 76 and Verrallina lineata 87 in Australia, Aedes (Ochlerotatus) taeniorhynchus in U.S., 68 Aedes albopictus, Culex quinquefasciatus and Anopheles spp. in Malaysia, 135 as well as one field trial under biting pressures of 1,200 2,400 Aedes cantans and Aedes annulipes landings/person/h. 128 KBR 3023 has also shown similar efficacy to deet against Aedes aegypti, Anopheles gambiae, 136 and Amblyomma hebraeum 98 in laboratory tests. It is this combination of efficacy, safety, and cosmetic appeal that has led to the WHO designating KBR 3023 as its repellent of choice for malaria prevention. 137 In addition, the Centers for Disease Control and Prevention (CDC) recommended both deet and KBR 3023 for West Nile virus and malaria prevention. 138 It is also being investigated for incorporation into military repellents after outperforming the standard Australian Defence Force formulation of 33% deet. 87,132 AI and AI The piperidine compounds 1-[3-cyclohexen-1-ylcarbonyl] piperidine, called AI , and 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine, also known as AI , were first synthesized by the USDA in It should be noted, however, that neither of these compounds is available commercially. Research on AI showed it to have similar efficacy as deet against Anopheles albimanus, Anopheles freeborni, Anopheles gambiae, Anopheles stephensi, and Phlebotomus papatasi 88 ; Prosimulium mixtum, and Prosimulium fuscum 140 ; Anopheles stephensi and Culex quinquefasciatus 141 ; as well as Culex pipiens, both in the laboratory and the field. 88 A was dropped from the Army research program, despite its impressive efficacy, because it caused an uncomfortable liniment-like warming reaction on some peoples skin (Dan Strickman, pers. com.). However, recent interest has focused on AI , a compound consisting of a racemic mixture of four isomers. 142 This mix has proven highly effective against a variety of blood-feeding arthropods, including Anopheles albimanus, Anopheles freeborni, Anopheles gambiae, Anopheles stephensi, and Phlebotomus papatasi 88 ; Prosimulium mixtum and Prosimulium fuscum 140 ; and Aedes communis and Simulium venustum. 67 In fact, its longevity was shown to be superior to that of an equivalent concentration of deet in field trials with Anopheles farauti in Australia 143 and Papua New Guinea, 86 Anopheles dirus, 144 Anopheles funestus and Anopheles arabiensis, 80 Leptoconops americanus, 145 Amblyomma americanum 96 and a laboratory trial with Anopheles stephensi. 88 It has undergone extensive toxicology testing and has been deemed safe. 146,147 However, it should be noted that it has not yet undergone all of the necessary toxicological testing to support registration. SS220 The latest development in synthetic skin repellents is optically active (1S,2S)-2-methylpiperidinyl-3- cyclohexen-1-carboxamide, discovered by the USDA and dubbed SS220. It is derived from AI insomuch as it is the most repellent of the four isomers that comprise racemic AI , and is 2.5 times as effective as the racemic mixture against Aedes aegypti. 148 Laboratory tests showed SS220 to be equal to deet against Anopheles stephensi and Aedes aegypti, and better than KBR 3023 against Aedes aegypti. 134 Against the tick, Ixodes scapularis, SS220 outperformed deet and was as good as deet against Amblyomma americanum. 149 However, SS220 is less effective than deet against Anopheles albimanus. 150 To date, no field studies have been published, although a USDA report stated that SS220 equals the effectiveness of 33% deet. 151 It has been reviewed by the U.S. military as the new active ingredient to

9 History of Insect Repellents replace deet. Extensive toxicological tests have shown low irritation and toxicity In addition, SS220 has a low rate of evaporation that will improve longevity. Using a smaller amount of long-lasting repellent makes for a more cost-effective and safe product because potential dermal absorption will be reduced. User acceptability is also likely to be higher because it has a slightly fruity odor, does not have an oily consistency, and has little plasticizing effect. 157 The disadvantage of SS220 lies in the fact that it is a single stereoisomer, and will, therefore, be more costly to produce than a racemic mixture. Furthermore, SS220 has not yet been registered, and the huge costs associated with this process, although necessary, mean that many promising new compounds may never be realized, as developers need to consider the potential financial benefits of registering a compound versus the initial large monetary outlay. Repellent Delivery Methods Insect repellents are effective in the vapor phase, defined by Browne as a chemical that, acting in the Q2 vapor phase, prevents an insect from reaching a target to which it would otherwise be attracted. 158 As the repellent molecules are volatile, temperature, humidity and wind affect evaporation of the repellent and therefore its longevity Perspiration and abrasion will also reduce the longevity of the repellent. 162,163 Many effective repellents have a high vapor pressure and are therefore volatile. At high mosquito densities, a heavy dose of a low vapor pressure repellent may be necessary to repel mosquitoes initially, whereas repellents with high vapor pressures may offer protection at low concentration. Subsequently, the lower evaporation rate of a repellent with less volatility means that it will continue to repel for a longer time period. 164 For instance, citronella (Cymbopogon nardus) essential oil and pure deet have similar ED 90 values of and 95.5 nl/cm 2, respectively, for Anopheles gambiaedes. 74 However, pure citronella oil at a dose of 3.33!10 K3 ml/cm 2 provides protection for only 2 h. 165 Citronella contains actives such as citronellol that has a vapor pressure of kpa vs kpa at 208C for deet. 166,167 The high rate of loss of repellents was overcome by using extremely high concentrations of deet, especially among military personnel. Standard deet concentrations for military repellents were 75% (U.S.) and 95% (Australia). 132 However, repellents may also be lost through dermal absorption. Absorption of deet is generally high, at 0.8%/h in humans. 168 The high rate of dermal absorption raised safety concerns for adverse side effects associated with using high concentrations of deet. This prompted several collaborative research studies that eventually resulted in the development of slowrelease formulations based on creams, polymer mixtures, or microcapsules that are available on the market today. Increased repellent longevity may be achieved in one of three ways: (1) controlling release or lowering vapor pressure, (2) preventing or reducing repellent absorption, or (3) improving resistance to abrasion and sweat. Formulations can prolong the effect of repellents. Initially, additives such as olive oil 169 and mineral oil 170 were used. They may improve repellent longevity by inhibiting loss of repellent volatiles and loss through sweating and abrasion. 171 Additives such as perfume fixatives were also researched. Fixatives are large branching molecules that lower the vapor pressure of repellents. These included Tibetene and vanillin, both of which have a significant effect on repellent longevity, increasing it by 29 and 95%, respectively, when used with deet at a 1:1 ratio. 172,173 In the early 1970s, an intense research program involving military, federal, academic, institutional, and industrial investigators began with the aim of providing a non-toxic, cosmetically-acceptable, and effective repellent system that would repel insects for 12 h under tropical conditions. They aimed to develop a repellent that would provide 24-hour protection under conditions that induced sweating through a two-pronged approach: (1) searching for agents with higher intrinsic repellency and

10 12 Insect Repellents: Principles, Methods, and Uses (2) enhancing repellent protection time of deet using hydrophobic agents to maintain the repellent on sweating skin. 174 The research on development of a binding agent was carried out by the Letterman Army Institute of Research (LAIR). The formulation of deet with film-forming polymer resins was aided by enlisting the help of industrial and institutional laboratories. Evaluation of cosmetic properties, dry-skin protection time, and wash resistance was carried out at LAIR using radio-labelled deet formulated with polymerfilm formers to study evaporation, skin penetration, and wash resistance. 174 During the first year of skin testing, several formulations were developed that were far superior to deet in both dry protection time and wash resistance, but few were cosmetically acceptable. However, the basic premise that film formers are extremely effective in enhancing protection time was confirmed. Then, almost a year was spent attempting to upgrade the cosmetic properties of those formulations that had superior wash resistance. The majority of research used silicone and carboset acrylic polymers and showed dramatically enhanced protection times. One example was the use of silicone that improved the dry protection time of deet by a factor of two, although it did not impart appreciable wash resistance. Tests with carboset acrylic polymers enhanced the dry protection time of deet and significantly improved wash resistance. Over 150 reformulations were prepared, examined, and about half were studied for wash resistance and cosmetic appeal on volunteers. However, little success was realized: cosmetically-elegant formulations had inferior wash resistance, whereas systems having superior wash resistance were sticky or brittle on the skin. A further year was spent expended in attempting to reformulate carboset polymers to improve their cosmetic appeal without sacrificing their excellent wash resistance. Formulations of carboset/deet were combined in increments with silicone polymer (decreasing carboset content in each member of the series) trying to upgrade cosmetic acceptability without losing the excellent wash resistance. 174 This research at LAIR in the late 1970s and early 1980s established the physical parameters and theoretical framework that demonstrated the feasibility of polymer and microcapsule mechanisms to release deet at a predetermined rate. The formulations tested in those early studies utilized microcapsule and polymer systems designed to provide continuous long-term release of the active ingredient. In microcapsule formulations, the active ingredient is contained in tiny capsules produced by coacervation, interfacial polymerization, extrusion, and other processes. The release rate is determined by the size and number of the microcapsules, the composition and thickness of the microcapsule walls, the concentration and properties of the excipient, and other additives used. These formulations may also contain free active repellent in addition to that contained in the microcapsules. In polymer systems, the active ingredient is formulated with a polymer that will form a thin film over the skin. This film acts as a reservoir for the active ingredient and slows its absorption and evaporation. In microparticulate controlled-release systems, the active ingredient is absorbed on the surface of microparticles and released slowly over time. 70 Further research was conducted that looked at formulations based on hydrophilic vinyl polymer, polyvinylpyrrolidone (PVP), 133 before the 3M Corporation s proprietary polymer formulation was finally devised. The polymers and microcapsules in the formulations slow the absorption and evaporation of deet, thereby holding it on the surface of the skin, where it can continue to repel arthropods for an extended period of time. 171 Ultrathone * (3M) has been the military topical repellent of choice since 1990, when it first became available in the military supply system. The product contains 33% deet in a controlled-release polymer base, and is a nongreasy, white lotion with a mild, pleasant odor. 175 It was validated by the USDA 176 and chosen as a result of tests against a variety of mosquito species under three climatic regimes: (1) 248C and 98% relative humidity (RH), (2) 308C and 78% RH, (3) 378C and 31% RH. 70 In these tests, the polymer formulation performed as well as a microparticulate formulation of 42% deet (Biotek) or 75% deet in alcohol (former standard of the U.S. Army). Field trials in the Philippines with Anopheles flavirostris showed that 3M was significantly more effective than 71% deet in ethanol for between 6 and 12 h after application. 177 However, in Australian field tests against Anopheles farauti, 3M and Biotek * A registered trademark of 3M Corporation, Minneapolis, Minnesota, U.S.A.

11 History of Insect Repellents performed as well as the Army deet formulation. 176 In this study, the volunteers applied repellents themselves according to label instructions, therefore reflecting normal use conditions. There was a significant difference in the amount of product applied by the individuals, but due to differences in the deet concentration of the three formulations, the amount of deet applied was fairly consistent. 176 The 3M 33% deet polymer formulation was found to be just as effective in repelling mosquitoes in field tests by the Australian military, 87,178 and a 35% deet formulation in cellulose gel that lowered dermal absorption and evaporation of deet was placed into service in Australia in The British military now also uses 3M Ultrathon. 179 Importantly, the slow-release formulations have significantly lowered dermal absorption, compared to ethanol formulations with deet at comparable concentrations. 180 The addition of polymers also improves the cosmetic appeal of repellents by lowering the amount of deet available, thus reducing odor, stickiness, and plasticization, as well as improving abrasion and wash resistance. 181 Several polymer and microencapsulated formulations are available on the market, including Sawyer Controlled Release w, HourGuard w, Skedaddle w, and Ultrathon. 110* Gel-based slow-release deet formulations, such as Ultrathone, have low acceptability among troops. Recently, 10% of American soldiers serving in Kuwait, Haiti, and Bosnia used the U.S. Army repellent containing 33% deet alone, 29% used commercial formulations, 34% used both types, and 27% used neither. 182 A similar situation was witnessed among Australian troops. Only 26 out of 955 soldiers interviewed used the standard issue 35% deet formulation in gel base. 183 The main reason given for nonuse is the sticky feel of the repellent on the skin. 183 Soldiers do not use military-issued repellents for several reasons, including their previous familiarity with nonmilitary products before joining the military, availability of commercial options, and advertising of repellents in various commercial media. 182 Additionally, soldiers perceptions of what is acceptable or good has been demonstrated by Gambel et al. (1998), 182 who observed American soldiers declining free military issue (33% deet formulation) repellent in an olive-green tube for a commercial product that was identical to the military issue, except that it was packaged using a different name and supplied in a brightly colored tube. Area Repellents There has been a recent increase in interest in area repellents that repel all biting insects within a set distance of the source of repellent molecules. Mosquito coils that are area repellents continue to be the most popular form of personal protection in use today. 40 In addition, citronella candles are commonly used as insect repellents in backyards and can provide 42% protection. 184 Spatial repellents have been defined as an inhibiting compound, dispensed into the atmosphere of a three dimensional space which inhibits the ability of mosquitoes to locate and track a target such as a human or livestock. 185 As repellents act in the vapor phase, they may potentially have a long-range effect through toxicity or confusing signals that indicate the presence of a host, established by saturating a zone or space with the spatial repellent. 186 One important concern with area repellents is the fact that they may only be used under conditions where air flow is minimal for instance, in forests as the repellent volatiles may be diluted with significant air flow. 28 A new development in spatial repellent technology is the ThermaCELL w Mosquito Repellent system, consisting of a butane-fueled generator that heats a metal plate to volatilize cis or trans-allethrin from an impregnated pad. The repellent is effective over a distance of 7 m and provided O90% protection over 6 h from sand flies and mosquitoes in a field trial in Turkey. 187 The system vaporizes the active ingredient from paper mats that are heated. These are highly effective, even under drafty conditions, as shown by laboratory 188 and field trials. 189 The various devices that heat such impregnated mats are second only to mosquito coils in global consumption. 40 The ThermaCELL system is an excellent development in * Sawyer Controlled Release is a registered trademark of Spectrum Brands; HourGuard is a registered trademark of 3M Corporation; and Skedaddle is a registered trademark of Multicrop International Pty. Ltd. A registered trademark of Schawbel Corportation, Bedford, Massachusetts, U.S.A.

12 14 Insect Repellents: Principles, Methods, and Uses repellent research, as it allows several people to be protected at once. However, it costs approximately U.S. $1.00 per hour to run, making it too expensive for use in the developing world. Several experiments with y-port olfactometers have recently shown the repellent effect of plant-based components such as the essential oil of catnip. 190 Plant-based repellents usually have a short longevity when applied to the skin, as they have high vapor pressure. 166,191 However, it is this feature that makes them excellent spatial repellents. In a field test, mint (Mentha arvensis) oil volatilized using a kerosene lamp significantly protected volunteers from Mansonia titillans, 28 and field experiments have demonstrated the spatial repellent effect of volatilization of plant oils using heated plates against Anopheles gambiae. 29 The Evolution of Repellent Testing Kairomones Progress in the development of new repellents has been slow until the recent breakthroughs, perhaps due to improved understanding of the repellents modes of action on the target organisms. However, this is now changing, and many papers have now been published on the mode of action of host kairomones on host-seeking insects. 14, Delicate methods, such as electroantennogram readings of the response of sensory neurons in insect antennae to attractive and repellent compounds, have allowed greater understanding of insects sensory systems. 19, Y-port olfactometers have also proved very useful in discriminating the effect of kairomones, as well as insect repellents and inhibitors. They have shown the importance of the interplay between whole host odor and repellents. Olfactometer experiments with deet have shown that it is not a true repellent insomuch as it causes insects to make oriented movements away from its source, but it is an inhibitor that prevents insects from feeding on a host in its presence (in this case a host-derived odor blend). 186,214 Many groups are working on quantifying what elements of human skin and breath are actually attractive to host-seeking insects, and, in particular, highly-anthropophilic disease vectors. Cork and Park (1996) 215 chemically fractionated human sweat samples into acid and nonacid components. They measured the electrical response of sensillae in the antennae of mosquitoes, and found that short-chained aliphatic acids (C 2 C 8 ) elicited significantly greater responses than the longer-chained acids. These acids elicit a landing response 198 and they have a significant effect on mosquito host-seeking behavior. 203 There is a growing body of evidence indicating short-chained fatty acids are reliable cues; however, these require complex blends, including synergists such as ammonia and lactic acid. 206,216 Therefore, the potential for an olfactometer with a reliable synthetic lure for repellent testing is some way off. Choice Should olfactometers become used regularly in the future for repellent/inhibitor screening, they will only ever be suitable for preliminary screening because olfactometers allow insects to choose between one or more targets. This is a disadvantage, as it causes an inflation of repellent efficacy: it shifts the point of reference for the ED 50 to a lower level. 217 It was argued that free choice between repellent-treated and untreated areas more accurately reflects use conditions where mosquitoes will feed on untreated areas of a repellent user, or their untreated companions. 218 This, however, is not a useful scenario, as a single infected bite is sufficient to transmit vector-borne pathogens. Therefore, recent publications have stressed the importance of high (O95%) protection, where the mosquito has no choice but to feed on repellenttreated skin if they wish to feed at all. 78,219 In addition, several experiments have demonstrated that offering mosquitoes free choice in laboratory overestimates repellency. 74,220 A free-choice test calculated the ED 50 of deet as mg/cm 2, 221 whereas a similar test with no choice calculated it as 0.35 mg/cm In field tests, Barnard et al. 68 showed that the application of a repellent to one limb

13 History of Insect Repellents where the other was used as a control inflates repellency, while the use of repellent using a repellentwearing bait individual with an untreated collector also overestimates repellency. 222 A study in Vietnam was performed with Anopheles dirus using one pair (one wearing deet, and one solvent control) and one individual (wearing deet) sitting alone. 223 In this case, the repellent wearer that was sitting alone received 3.5 times more mosquito bites than the repellent wearer that was sitting close to an alternate blood source. This is because the mosquitoes will always feed on the easiest option with least repellent, be that an adjacent area of skin, an alternate limb, or another individual. When the protection afforded by the repellent wanes, mosquitoes will start to feed through the repellent. However, if there is an unprotected alternative, they will be diverted and feed upon it. This also applies in field tests if individuals are less than 10 m apart because this is the limit of short-range attraction. 224 In Vitro and Animal Tests Tests on repellents, from the 1920s until recently, were often performed on shaven animals including rabbits, dogs, 225 guinea pigs, 226 and chicks. 227 This method, despite questions regarding the ethical treatment of animals, may distort the results of repellent tests. Nicolaides et al. (1968) 228 compared the skin of humans and other domestic animals. They concluded that humans excrete mainly triglycerides and are, therefore, unique in having fatty acids as breakdown products on the skin surface. This means that short-chained aliphatic acids are reliable host cues for anthropophilic mosquitoes, and, therefore, testing repellents on animals will not give representative data of how the repellent will perform when applied to human skin. In addition, the most efficient malaria vectors are extremely anthropophilic, and will be less attracted to nonhuman hosts, 212 possibly due to genetically mediated innate preferences. 229 Thus, this method gave a distorted measure of repellency. Indeed, Rutledge et al. directly compared measurements of repellent efficacy obtained using rabbits 171 and mice 230 with that obtained using human arms. In both cases, repellents showed greater variability and greater persistence when applied to animals than humans. Other studies have utilized membrane blood feeders, commonly used for feeding mosquitoes in insectaries, to measure repellency. 217 Although the data obtained using this method roughly corresponds to data obtained with human-arm tests, 220 this method should be used only for rapidly screening large numbers of repellents to narrow down candidates for further testing. This is because membrane feeder tests differ from human-arm tests because mosquitoes do not respond as enthusiastically to a feeder as they do to a living host, and there is much interspecific variation in readiness to feed from membrane feeders. 231 Another testing method employs disks of paper impregnated with a test repellent, and the numbers of insect landings on impregnated and unimpregnated control disks are counted. This was shown to be an excellent method for testing irritancy of a chemical, but is not a measure of repellency. 232 In vitro methods are cheap, and yield many results rapidly with no risk to human subjects, but they do not accurately mimic the conditions of repellent usage. Thus, different methodologies cannot be compared, nor can their results be directly extrapolated to the end user. This is particularly important since the discovery that deet, the leading insect repellent, is an inhibitor and not a true repellent. 214 Test Standardization Other recent developments in repellents research have followed after the call of the WHO in to standardize repellent testing protocols. It would appear that tests are becoming far more stringent and standardized. It is always preferable to conduct tests on human volunteers for greatest accuracy, provided that laboratory-reared mosquitoes are used to eliminate the risk of pathogen transmission, and the selected volunteers show mild or no allergic reaction to mosquito bites. 234 It is conventional to use Aedes aegypti mosquitoes for repellent testing, but people generally show milder reactions to Anopheles bites. Aedes aegypti are commonly used, as they are easy to rear under laboratory conditions, and are avid biters. However, several other species also fulfill these criteria, including Anopheles stephensi, Anopheles