INFLUENCE OF SEX RATIO AND DENSITY OF MOSQUITOES ON THE EFFICACY OF IR3535® AND DEET 201 encephalitis pathogens. An. aquasalis is an important vector of the malaria pathogen Plas- modium vivax in coastal regions of the neotropics. All mosquitoes were reared in the labo- ratory at 23–27°C and 50–70% relative humidity and in a 12:12 light-to-dark cycle. On eclosion, the new adults were segregated by gender and maintained on a 10% sucrose solution in water. Mosquitoes used in the study were obtained from a population main- tained in the laboratory. APPLICATION OF TEST MATERIALS Forearm dimensions of qualifi ed subjects were measured and entered into the following formula to calculate the quantity of test material needed by each to receive a dosage of 1 g per 600 cm2 of forearm skin: x M1 M2 M3 M4 Amount 4 600 L g Here, L = forearm length [cm], M1 = wrist circumference [cm], M4 = elbow circumfer- ence [cm], and M2 and M3 = two equally distant forearm circumference measures from the wrist to elbow [cm2]. Subjects prepared fo r repellent application by fi rst washing their forearms with soap, fol- lowed by a thorough rinse with water and drying with paper towels. Subjects were then supplied with the calculated amount of the test product. While protecting the hands with rubber gloves, a technician evenly spread the supplied test product on one of the forearms of each volunteer. All test subjects were directed not to touch or rub the treated arm between evaluation periods. EXPOSURES TO MOSQUIT OES The mosquitoes used for testing were 5- to 10-d-old, disease-free, adult males and fe- males. Both sexes were virgin, and the females had no prior opportunity to blood-feed. On each test day, they were released in groups by species into individual metal-screened cages (61 × 61 × 61 cm3). Test conditions were adjusted to account for the biting behav- ior of the species tested. For Ae. aegypti, the room was kept illuminated with temperature at 25° ± 2°C and humidity at 50–70%. For Cx. quinquefaciatus, the room was kept dark with infrared illumination from the top to the bottom of the cage, or laterally during the test. This species bites at night, and temperature was set for 25° ± 2°C with humidity between 50% and 70%. For An. aquasalis, the illumination was penumbra, partial light, with relative humidity not lower than 70% and temperature between 24° and 28°C. Across four treatments, the total number of mosquitoes per cage was varied between 200 and 400, and the female: male ratio was varied between 1:1 and 1:0 (Figure 1). IR3535® was tested against all three mosquito species at all densities and sex ratios. The DEET positive control was tested solely using 200 females (no males) per cage. Females-only is the standard confi guration used for cage-based testing of mosquito repellent effi cacy. Tests were conducted at 23–27°C and 7 0% RH. Beginning approximately 30 min after test materials were applied, subjects inserted their treated forearms individually through

JOURNAL OF COSMETIC SCIENCE 202 a voile sleeve into the test cage and exposed them to the mosquitoes for 5 min, after which the arms were withdrawn from the cage. Reexposure occurred every 30 min. The test ended for an individual subject when there was a confi rmed bite. A confi rmed bite was defi ned as the occurrence of more than one bite within the same exposure period (in this case, a period of 30 min) or when one bite was followed by another in a consecutive expo- sure, which confi rmed the result of the previous period. The number of landings was also recorded until the end of the test. DATA SCORING, TABULATION, AND ANALYSIS The duration of repellency for each sub ject in each exposure was calculated as the time between test product application and confi rmed bite. This duration is the complete pro- tection time (CPT). Mean CPT (±1 standard deviation) was determined for each test subject. RESULTS Infl uence of density and sex ra tio on CP T by IR3535®. AE. AEGYPTI The sex ratio and density ma nipulations s trongly infl uenced CPT in Ae. aegypti (p 0.003 Figure 2). CPT averaged approximately 3 h or slightly longer when tested against 200 females alone or 200 females:200 males and was almost halved at the 3.3:1 ratio Figur e 2. Repellency of 20% IR3535® against Culex quinquefaciatus as cage test. The values of fi ve volunteers are shown as mean CPT ± standard deviation.