2.5. Repellent Test

The repellent test was based on the WHO protocol (2009) [18] with a few modifications. The test was performed in a metal cage (30 cm × 20 cm × 20 cm) covered with a net. Fifty females (5–7 days old) were raised together with males to ensure copulation. They had no previous blood meal and had been fed on a sucrose solution of 10%. These female mosquitoes starved for 12 h before the tests. Two mosquito cages were randomly assigned to each participant. One cage was used for testing the essential oil solution and the other for the positive control (20% DEET standard solution in ethanol). The essential oil was tested at 25%, 50%, 100% concentration. In addition, a test solution containing the essential oil (25%) and vanillin (5%) was performed. Both forearms were covered with a rubber sleeve except a rectangle area (3 cm × 10 cm), thus the skin within these rectangle areas were directly exposed to the mosquitoes. Before each test, the forearms were treated with negative control (only ethanol) in order to ensure host-seeking behavior. On the forearms, each rectangle area was treated with 0.1 mL of repellent (essential oil solution or DEET 20% solution). After that, the protection time was considered started and each treated forearm was then inserted into a different cage for 3 min. If there was no mosquito bite, the test was repeated after a 30-minute interval. Once a mosquito sucked blood, the repellent test was considered finished. New mosquitoes were used for each test. Four volunteers who worked at the Faculty of Pharmacy and had no history of allergic reactions to mosquito bites were recruited. Following WHO’s guideline [18], we selected equal numbers of female and male volunteers. To avoid the problem of the limited number of volunteers, each participant was tested twice on different days to confirm the results.