2001 ANNUAL SCIENTIFIC SEMINAR 417 NEW METHOD FOR TESTING MOSQUITO REPELLENCY: HIGHLIGHTING A NEW BENEFIT FOR AN EXISTING INGREDIENT Heather Eyre, Steve Watkins and Richard Birch Quest International, Ashford, Kent, TN24 OLT, United Kingdom Back,qround Mosquitoes are found all over the world except Antarctica. There are around 170 different species of mosquito found in Nodh America alone (1) and, even within the United States, cedain species are known to be responsible for spreading disease (2). Even a harmless bile often results in a red, itching welt on the skin, caused by an allergic reaction to the mosquito saliva. (3) An Effective Deterrent Various attempts have been made to control mosquito populations, but this work has had limited success and so there is still a need to use repellent products to avoid being bitten. Ideally, a product sold as a repellent should achieve high [ 90%] repellency on initial application and maintain that efficacy for a number of hours. Unfodunately, there is no regulation stipulating how long a product should last and not all products give the same level of protection for the same length of time. In pad, this is due to the large number of variables involved in assessing the repellency. The formulator has a limited palette of effective actives from which to choose. Diethyl Methyl Benzamide (DEET), developed by the US Army in 1946 (4), is still considered the most effective repellent active available today. There is evidence to suggest that its main mode of action may be through inhibiting lactic acid receptors on the mosquito antennae (5). In normal use there is a consensus that 50% DEET will provide acceptable levels of protection for 3-4 hours. Using 100% DEET may extend this range by a further hour. However, there are a number of drawbacks to its usage: it is a good plasticiser, so can damage equipment. It is aesthetically unpleasant which will discourage frequent product usage, and most seriously, despite rigorous and repeated safety tests showing that it has no adverse effects when used properly, there are still concerns over the potential toxicity when used by children and pregnant women (1). In addition, allergic skin reactions are occasionally reported (6). DEET is irritant to the eyes and mucous membranes and care should be taken when applying it Io the face. Other actives include encapsulated DEET, ethyl butyl acetyl amino proprionate (Merck IR 3535), Bayrepel and dimethyl phthalate. IR 3535 has recently been introduced to the US as a 'biopesticide' alternative to DEET, with a good safety record from its a long history of use in Europe. It is claimed to offer the skin effective protection from insects without toxic allergic or sensitizing propedies, without skin penetration, but its use has so far been limited in the US due to an exclusivity agreement with one manufacturer, and the limited data is somewhat conflicting. • There are also a number of natural products available, which usually contain a blend of volatile oils as active ingredients. Published literature is only available to any significant extent for citronella, which, while effective, has an extremely short length of action, often under one hour (7). In summary, few alternatives exist and, whichever active is selected, the need to use high concentrations in order to achieve the desired activity is a concern. A possible way around this is to use lower levels of the main active by blending repellents or by identifying existing cosmetic ingredients that can contribute to the overall repellency. Bitin.q Behaviour There are a number of factors that attract the mosquito to its human prey (7): ß Chemoreceptors on the insect's antennae can detect minute quantities of substances of chemical attractants. The most important of these are believed to be lactic acid from skin, and carbon dioxide in exhaled breath. ß Mosquitoes rely on body heat and moisture to determine where to bite once they are within close range ß Anopheles mosquitoes bite at night, with the most intense outdoor activity being in the early evening ß Aedes mosquitoes are daytime feeders and are most active in the afternoon ß There appears to be a gender difference in attracting mosquitoes, with females receiving significantly more bites in trials (8). In addition, children tend to be bitten less than adults. Larger people tend to be bitten more than smaller individuals Repellent action The mechanism of action of repellents applied to the skin is not well understood, but undoubtedly they interfere with the chemical stimuli that attract mosquitoes. Repellents can be thought of as having a vaporising effect: molecules of repellent evaporate from the skin and interfere with the "homing mechanism" that mosquitoes use (6). There is an enormous range of species of mosquito with varying sensitivity to repellents. This variation is even present within species. For instance, DEET is highly effective against Anopheles stephensi but much less active against Anopheles albimnus. (9). Mosquitoes seem to prefer to bite a person with no repellent on their skin than one wearing a repellent, even if the repellent is not very effective and/or is present in quite low concentrations. (6)

418 JOURNAL OF COSMETIC SCIENCE Screeninq Ingredients that might contribute to Repellency Numerous methods for testing the effectiveness of mosquito repellents have been described. One of the most popular and convenient is the "cage test". A volunteer places an arm into the mosquito cage, and the number of bites received in a particular time is noted. In contrast, a "free flight" test necessitates the individual standing in a large cage containing mosquitoes. This allows the testing of different body parts, for direct comparisons. Cage test methods are mainly used to calculate the minimum effective dose, i.e., potency, of a repellent. Ultimately, the best test is with large field trials but this is expensive and is not an option when considering screening many different compounds. It had long been reported that certain aromatic oils had mosquito repellent activity (10, 11). However, the available data was not always reliable and proved difficult to compare. We initiated a screening program in conjunction with The University of London, Wye College, to identify a suitable screening technique that could compare many different compounds. This was then used to evaluate several hundred Perfumery ingredients, including a number of Cosmetic ingredients. The patented protocol (12, 13) used test cages containing a warmed moist skin 'biting target'. The ingredient was applied to the target (dosage 0.2mg/cm2), then hungry female Aedes aegypti mosquitos were released into the chamber, and their biting behaviour monitored by video. The mean numbers of bites immediately after introduction, and one hour later, were calculated. The method allowed a positive control (DEET) to be run alongside 3 test materials, giving direct comparisons between ingredients regardless of time of test. Materials were ranked according to their performance initially and at one hour, "good" materials achieving at least 50% repellency at both time intervals. The results showed that DEET typically gave 93% initially, and 75% at 1 hour, and IR 35353 gave 86% and 25% respectively. One of the interesting results showed that a commercially available coolant and skin moisturiser, Menthyl Pyrrolidone Carboxylate (MPC), performed very well, giving an equivalent of 80% repellency initially, and 59% after one hour. The test was repeated using 2% active in skin cream, again compared to DEET, to prove that this benefit would be manifested in a more 'real life' situation. We have no explanation for this ability, whether it is due to the odour, taste or chemical properties of MPC. However, MPC is known to liberate menthol on the skin due to hydrolysis, so we conducted a further test against menthol. MPC was significant•ly better at both time points, confirming that menthol cannot be the sole contributor to this effect. MPC is not registered or marketed as a repellent, but promoted as a cosmetic ingredient for its cooling, moisturising and soothing benefits. This data demonstrates that MPC could also contribute to the overall repellency of a finished formulation, perhaps allowing for the reduction of the main active. This would be of particular interest for suncare formulations, where the performance of DEET is known to be inhibited by the presence of sunscreens (14). References 1 Annals of Internal Medicine online: http:fiwww.acponline.orgljournalslannalslO1jun981mosquito.htm 2 United States Environmental Protection Agency website: http:fiwww.epa.govlpesticideslfactsheetslskeeters.htm 3 United States Environmental Protection Agency website: http:llwww.epa.govlpesticideslcitizenslmosquito.htm 4 United States Environmental Protection Agency website: http:fiwww.epa.govlpesticideslcitizensldeet.htm 5 Dogan EB, Ayres JW, Rossignol PA. Behavioural mode of action of DEET: inhibition of lactic acid attraction. Med Vet Entomo1:13:97-100 (1999). (in 6) 6 The Pharmaceutical Journal online: http:llwww.pharmj.comlEditorial1200008261educationltravel_bites.html 7 Fradin, MS. Mosquitoes and mosquito repellents: a clinician's guide. Ann Internal Med 128:931-40 (1998). (in 6) 8 Golenda CF, Solberg VB, Burge R, Gambel JM, Wirz RA. Gender-related efficacy difference to an extended duration formulation of topical NN-diethyl-m-toluamide (DEET). Am J Trop Med Hyg 60:654-7 (1999). (in 6) 9 Robert LL, Hallam JA, Seeley DC, Roberts LW, Wirtz RA. Comparative sensitivity of four Anopheles (Diptera:Culicidae) to five repellents. J Med Entomol 28:417-20 (1991). (in 6) 10 Brown M, Hebert AA. Insect repellents: an overview. J Am Acad Dermatol. 36(2 Pt 1 ):243-9 (1997). 11 Das NG, Nath DR, Baruah I, Talukdar PK, Das SC. Field evaluation of herbal mosquito repellents. J Commun Dis. 31 (4):241-5 (1999). 12 WO 96/08147, Quest International. 13 Sharpington P J, Healy TP, Copland M J: A wind tunnel bioassay system for screening mosquito repellents. J Am Mosq Control Assoc. 16(3):234-40 (2000). 14 Montemarano AD, Gupta RK, Burge JR, Klein K. Insect repellents and the efficacy of sunscreens. Lancet. 349:1670-1 (1997). (in 1).