For each test, 10 disease-free, laboratory-reared Aedes aegypti
female mosquitoes that were between 7 and 24 days old were placed
into separate laboratory cages measuring 30 cm by 22 cm by 22 cm.
A batch of 10 mosquitoes that had not been exposed to the repellent
being tested was used for each arm insertion. Mosquitoes were
provided with a constant supply of 5 percent sucrose solution.
Cages were placed in a walk-in incubator measuring 2.2 m by 2.2 m
by 2.2 m, in which the temperature was maintained at 24 to 32°C,
the relative humidity at 60 to 70 percent, and the light–dark cycle
at 12 hours of light followed by 12 hours of darkness.
Fifteen volunteers (5 men and 10 women) were recruited from
the staff of the Medical Entomology Laboratory at the University
of Florida. The study was reviewed and approved by the institutional
review board of the University of Florida, and subjects gave
written informed consent before participating.
As repellents were purchased, they were labeled sequentially from
1 to 16. A random-number generator was then used to determine
the order in which the products would be tested on each subject.
A total of 720 individual tests were conducted, with each repellent
being tested three times on each subject. Most subjects only
completed one test per day. The average time to completion of
all three tests was 10.2 days. In the case of repellents that were
identified as very short-acting in the initial test, subjects were permitted
to conduct all three tests of the repellent in a single day,
washing the skin with an unscented soap before each application
of the repellent. Subjects did not test more than one repellent
product on a single day. No information on the likely duration of
action of each repellent was provided to subjects before they began
their tests.
Before each test, the readiness of the mosquitoes to bite was
confirmed by having subjects insert their untreated forearm into
the test cage. Once subjects observed five mosquito landings on
the untreated arm, they removed their arm from the cage and applied
the repellent being tested from the elbow to the fingertips,
following the instructions on the product’s label. After the application
of the repellent, subjects were instructed not to rub, touch,
or wet the treated arm. Repellent-impregnated wristbands were
worn on the wrist of the arm being inserted into the cage. Subjects
were provided with a standardized log sheet to ensure accurate
documentation of the duration of exposure and the time of
the first bite. The elapsed time to the first bite was then calculated
and recorded as the “complete-protection time” for that subject
in that particular test.
Subjects were asked to follow the testing protocol shown in
Figure 1. Subjects conducted their first test of each repellent by
inserting the treated arm into a test cage for one full minute every
five minutes. If they were not bitten within 20 minutes, then the