INFLUENCE OF SEX RATIO AND DENSITY OF MOSQUITOES ON THE EFFICACY OF IR3535® AND DEET 199 a non-DEET molecule, registered by the Environmental Protection Agency as a biopes- ticide repellent IR3535® [3-(N-acetyl-N-butyl) aminopropionic acid ethyl ester]. We used DEET as a positive control. The aim of the study was to examine those factors and further understand the dynamics in the infl uence of the study outcome by comparison of the repellent effi cacy of IR3535® with that of DEET. If indeed sex ratio and density have an infl uence on study outcome, this might provide a rationale guiding future revisions of label instructions that provide safe and effective consumer use. MATERIALS AND METHODS This stud y was conducted by BioAgri Laboratories, Bela Vista, Charqueada, Sao Paulo, Brazil. Testing was conducted in two periods. A group of fi ve volunteers participated in tests with Aedes mosquitoes in June 2011, and a second group of fi ve volunteers partici- pated in tests with Anopheles and Culex mosquitoes in October and November 2011. Testing in these two periods was conducted under similar environmental conditions as detailed in the section Exposures to Mosquitoes. Only very broad comparisons are made between species this reduces the risk that temporal division confounds important interpretations. Testing was conducted using an EPA protocol. Test materials were applied to volunteers by trained technicians at BioAgri Laboratories. REPELLENT S The test repellent formulation consisted of a hydroalcoholic pump-spray containing 20% IR3535® with 2% of a fi lm-forming agent, a PVP/VA copolymer—C10H15NO3, added to the product to improve effi cacy. The comparison article was a commercial 20% DEET in 80% ethyl alcohol formulation. Both test materials were prepared in the laboratory of Merck KGaA (Darmstadt, Germany) in Brazil. IR3535® (F igure 1) was developed by Merck KGaA in 1975, and has been marketed in Europe for more than 20 years and elsewhere in recent years. Its mechanism of repel- lency is similar to that of DEET. IR3535® acts by forming a vapor barrier surface on skin that inhibits feeding by certain blood-feeding arthropods (11). IR3535® was reg- istered by the U.S. EPA in 1999 as a biopesticide because it is functionally identical to naturally occurring beta-alanine. IR3535® is a substituted β-amino acid that contains 98% 3-(N-acetyl-N-butyl) aminopropionic acid ethyl ester as the active ingredient and 2% inert ingredients. Biopesticides receive special consideration by EPA because they are generally considered to be less toxic and are more specifi c than conventional pesti- cides (12,13). Registered IR3535® formulations are labeled as repellents against mos- quitoes, deer ticks, body lice, and biting fl ies globally depending on the registration in specifi c countries. The repellent ef fi cacy of IR3535® has been investigated in numerous fi eld and laboratory tests around the world against a wide variety of arthropods. Effi cacy is shown to vary for similar concentrations of active ingredients (14–17) perhaps in part because of differences in formulation (emulsion, spray, or lotion). IR3535® performs similarly to DEET when tested against mosquitoes and ticks using comparable emulsion systems and concentrations (18).

JOURNAL OF COSMETIC SCIENCE 200 HUMAN TEST SUBJECT S Volunteer recruitm ent was made by BioAgri Laboratories. During subjects’ selection for the study, the physician in charge certifi ed that the subjects have no pathologies that might interfere with the study results. The physician was also responsible for all informa- tion contained in the subject’s evaluation form, by checking all inclusion and noninclu- sion criteria for admission of each subject in the study. The study was conducted according to the recommendations of the National Council of Health, Resolution number 196/96, an Institutional Review Board (IRB) of Brazil. Each experiment wa s carried out with fi ve healthy test subjects of both genders, between 18 and 65 years of age. Each subject stated that he or she had either no or slight dermal irritation when bitten by mosquitoes. Subjects read information sheets and signed in- formed consent forms before participating in the study. They agreed not to use scented cosmetic products or ingest alcohol or caffeine beginning 12 h before the test. Subject exclusion criteria included attractiveness to mosquitoes. This criterion was assessed in a preliminary exposure in which each candidate exposed a bare, untreated forearm in an individual cage containing the mosquitoes. Mosquito species tested were Aedes, Culex, and Anopheles, using different sex ratios: 100 females:100 males 200 females:200 males 200 females:60 males and 200 females:0 male (female only), for a period of 30 s. Volun- teers who showed natural repellency, defi ned as no landings, were excluded from the test (Figure 1). MOSQUITO SPECIES I N THE STUDY Three mosquito spe cies were evaluated: Aedes aegypti, Culex quinquefaciatus, and Anopheles aquasalis. Ae. aegypti is important in the transmission of pathogens that cause dengue fever, yellow fever, and chikungunya. Cx. quinquefasciatus vectors West Nile fever and equine Fi g ure 1. Repellent activity of 20% IR3535(R) against Aedes aegypti in a cage test with 5 volunteers, shown as mean complete protection time.