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Standard Methods for Testing Mosquito Repellents

Article · January 2007

DOI: 10.1201/9781420006650.ch5 · Source: OAI

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5H.
Standard Methods for Testing Mosquito Repellents

Donald R. Barnard, Ulrich R. Bernier, Rui-de Xue, and Mustapha Debboun

CONTENTS
Introduction...................................................................................................................................103
Laboratory Repellent Bioassay Methods......................................................................................104
World Health Organization Method ................................................................. . ........................ 104
American Society for Testing and Materials Method E951-94 (Revised 2000) .....................105
ScreenedCage Method ............................................................................................................ ..105
K&D Module Method ...............................................................................................................106
Field Repellent Bioassay Methods ...............................................................................................107
World Health Organization Method..........................................................................................107
American Society for Testing and Materials Method E939-94 (Revised 2000) .....................107
U.S. Environmental Protection Agency Test Guidelines .............................................................. 107
Sources of Variation in Repellent Bioassays ...............................................................................108
AbioticFactors ..........................................................................................................................108
BioticFactors ....................................... . ..................................................................................... 108
Conclusions......................................................................................................................................109
References.....................................................................................................................................109

Introduction
Testing of mosquito repellents, whether in the laboratory or the field, is performed using a process called
biological assay (bioassay for short).' Bioassays can be used to answer three questions about repellents:

1. Is the candidate material repellent?

2. What quantity of material is required for repellency?
3. How long does repellency last?

There are three repellent bioassay procedures documented as standard methods in the literature and
one set of repellent testing guidelines available on the internet:

I. World Health Organization (WHO), WHO/Control of Tropical Disease/WHO Pesticide

Evaluation Scheme/Informal Consultation (WHO/CTD/WHOPES/IC 96.1). Report of
WHOPES Informal Consultation on the Evaluation and Testing of Insecticides.2

• • . • . I • I ri 1!. I. It. I 103


104 Insect Repel/em's: Principles, Methods, and Uses

2. American Society for Testing and Materials (ASTM) E951-94 (revised 2000). Laboratory
Testing of Non-Commercial Repellant Formulations on the Skin.3
3. American Society for Testing and Materials (ASTM) E939-94 (revised 2000). Field Testing
Topical Applications of Compounds as Repellents for Medically Important and Pest
Arthropods. I. Mosquitoes.4
4. U.S. Environmental Protection Agency (EPA) Office of Prevention, Pesticides, and Toxic
Substances (OPPTS) 810.3700. Product Performance Test Guidelines, Insect Repellents for
Human Skin and Outdoor Premises. EPA #712-C-99-369, December 1999 (available in
public draft on the internet: see below for URL).

A fifth repellent bioassay system, the screened cage method, is frequently cited in the literature, 58 and
a sixth system, modified from ASTM E951-94 and adaptable to both in vivo and in vitro testing, has
recently been published.'

Laboratory Repellent Bioassay Methods

World Health Organization Method

Laboratory repellent bioassays based on the WHO protocol require a mosquito-filled. screened cage and
use deet as a positive control. Human test subjects are preferred over laboratory animals or artificial
membranes. Aedes aegvpri, the normal test species, is used in variable numbers, but other mosquito
species can be substituted depending on the needs of the experiment. An area of skin ranging from that
covering the entire forearm to as little as 25 cm 2 is treated with repellent and exposed to caged
mosquitoes. Untreated skin is covered with a glove or other protective material. For compounds of
unknown toxicology, the repellent may be applied to a cotton stockinette sleeve, and the treated sleeve
may be pulled over a second untreated stockinette on the arm to prevent skin contact with the repellent.
At least five variations of the WHO method have been developed to meet the testing needs of different
institutions. 2 These meathods emphasize either the determination of protection time after treatment with
a single repellent dose or the percent protection in relation to repellent dose. The protocols are as follow:

1. A 25 cm area on a subject's forearm is treated with an ethanolic solution of repellent

(treatment), and the same-sized area on the adjacent forearm is treated with alcohol (negative
control). Both arms are simultaneously introduced into one cage, and the numbers of
mosquitoes biting each arm in 5 min is recorded. Percent protection is calculated by
comparing biting rates on the treatment and control arms.
2. A subject's feet and legs are treated with repellent, exposed to 25 female mosquitoes in a
mosquito-proof enclosure (I m I m X 3 m high), and the number of bites in 10 mm
is recorded.
3. A subject's forearm is treated with I mL of a 25 17c ethanolic repellent solution and introduced
into a mosquito-filled cage for 3 min once every 30 mm. Protection time is that elapsed
between repellent application and the first mosquito bites followed by a confirmatory bite in
the same, or next, exposure period.
4. One gram of repellent is dissolved in sufficient acetone to saturate 280 cm of cotton
stockinette. The stockinette is drawn over the arm of a subject and exposed to 1,500 caged
female mosquitoes for I mm. at daily or weekly intervals, until 5 bites are obtained.
5. A subject's untreated arm is exposed to 50 caged female niosquitoe. follosed b y repeated
exposures of the same arm \ ith iiicieasiuI hi -2h doses of repellent. In each exposure. the
 105
Standard Methods for Testing Mosquito Repellents

arm is withdrawn before the mosquitoes can imbibe blood. Probit analysis is used to calculate
the ED 50 . When the dose giving 100% repellency is identified, the arm is re-exposed at 60 mm
intervals until repellency declines to 50%.

American Society for Testing and Materials Method E951-94 (Revised 2000)
This method comprises the use of a rectangular (18 cm length X 5 cm width X 4 cm height) clear plastic
test cage with five 29-mm-diameter openings in the bottom. A template is used to place four repellent
dosages and a control on the skin of a human volunteer in a pattern that matches the openings on the test
cage bottom. The cage is strapped to the arm or leg of a volunteer, bottom-side to the skin, with 10-15
nulliparous, 5-15-day-old female mosquitoes placed into the cage through a 13-mm opening at one end.
A test commences when the plastic slide (0.3-mm thick) that blocks the openings in the test cage bottom
is withdrawn, allowing mosquitoes access to the repellent treated skin. The number of mosquitoes that
land on and probe the skin in 2.5 min is recorded. The dose-response data obtained with ASTM E951-94
has been used to calculate median (ED 50 ) and 95% effective doses (ED 95 ) 1213 and to describe functional
responses, in time, of mosquitoes to topical repellents.'2

Screened Cage Method

The screened cage bioassay method employs a 40 cm aluminum-frame cage with a metal bottom,
screened top and back, clear acrylic sides (for viewing), and a front stockinette sleeve for access. Two
hundred human host-seeking' 4 nulliparous, 7-8-day-old female mosquitoes are placed in the cage 1 h
before the test. Treatment consists of a 25% ethanolic solution of repellent active ingredient applied to
the forearm of a volunteer (between the wrist and elbow) at the rate of 1 mL/650 cm of skin surface area.
The treated forearm is inserted into the cage (a glove is used to protect the hand from mosquito bites.
Figure 5.1) and the number of mosquitoes that land and probe the skin in 3 min is observed and recorded.
The observations are repeated every 30 or 60 min. Two bites in one 3 min test or one bite in one 3 mm
test, followed by one or more bites in a second test 30 min later ends the test for the repellent. A second
cage of mosquitoes is used as a positive or negative control. Depending upon the requirements of the
experiment, protection time is calculated as either the time elapsed between repellent application and the

FIGURE 5.1 The screen cage method of testing trial repellent formulations.
 106 Insect Repellents: Principles, Methods, and Uses

first confirmed mosquito bite, or the time between repellent application and the observation period
immediately preceding the first confirmed bite. Data obtained with this bioassay method can be used to
calculate complete protection time (CPT).

K&D Module Method

An extension of ASTM E951-94 is the "K&D module". 9 This apparatus is reported to minimize the
likelihood of treatment interactions. It increases the number of possible treatments per replicate and
permits large numbers of replicated observations for each human test subject. The module can be used to
test the responses of more than one mosquito species at a time to one dose of repellent or to evaluate
repellent responses in the same species using specimens from geographically distinct locations.
In vivo bioassays using the K&D module (Figure 5.2) are conducted in a walk-in incubator (27°C and
80% RH) under fluorescent light. A template is used to delineate 3 cmX4 cm areas on the skin that
correspond to each of the six cell openings on the bottom of the module. A treatment is administered by
pipette onto a 4 cm, X 5 cm rectangular area of skin centered over one of the individual template marks on
a human volunteer and consists of 55 iL of ethanol containing 8.73 nmol of candidate repellent per
microlitre of ethanol. This process results in a 24 nmol dose of the treatment on 1 cm 2 of skin. Skin
treated with ethanol serves as the control. The module, with five mosquitoes in each cell, is then
positioned over the treated skin area, each cell door is opened, and the number of mosquitoes that bite the
skin or become blood-engorged in 2 min is recorded.
The in vitro system '°" 1 (Figure 5.3) consists of six reservoirs (3 cmX4 cm) warmed to 38°C by a
water bath. Each reservoir is filled with 6 mL of outdated human blood and covered with a Baudruche or
collagen membrane. Trial repellent compounds dissolved in 110 1.tL of ethanol are applied in random
order to six 4 cm 5 cm pieces of organdy cloth. Each cloth is allowed to dry and then placed over one of
the membrane-covered, blood-filled cells. The module, with five mosquitoes in each cell, is positioned
over the treated cloth, and the doors are opened. The number of mosquitoes with their probosces inserted
through the cloth into the Baudruche or collagen membrane into the blood after 2 min is recorded.

ANA

FIGURE 5.2 The in vivo K&D module apparatus for bioassay of repellent active ingredients and formulations.
p Standard Methods for Testing Mosquito Repellents
107

-a

FIGURE 5.3 The in vitro K&D module apparatus for bioassay of repellent active ingredients and formulations.

Field Repellent Bioassay Methods

World Health Organization Method
When using the WHO field method, 2 repellent tests are made in the vicinity of human domiciles.
Mosquito biting rate and the assessment of repellency is based on the capture of mosquitoes attacking
human volunteers; thus, tests are timed to exploit the biting cycle of the target mosquito species. Test
subjects are spaced 10 m apart and rotated in a randomized manner throughout the experiment to
minimize positional errors. Appropriate criteria for repellency include 80% reduction in biting rate for
6-8 h without adverse user side effects.

American Society for Testing and Materials Method [939-94 (Revised 2000)
In this method, 4 1.5 mL of repellent solution is applied to the forearm (between the wrist and elbow) or
lower leg (between the knee and ankle) and the treated limb is exposed continuously to biting mosquitoes
as the subject moves through mosquito-infested habitat. Biting mosquitoes are collected from treated and
untreated skin (usually an exposed forearm) at regular intervals to determine mosquito biting rates and
for species identification. This procedure is used to determine CPT, but percent repellency can also be
calculated when a negative control is used.

U.S. Environmental Protection Agency Test Guidelines

The OPPTS guidelines have been developed for laboratory and field evaluation of pesticides and
toxic substances and for acquiring test data submitted to the EPA for review under the Toxic
 108 Insect Repellents: Principles, Methods, and Uses

Substances Control Act (TSCA) (15 U.S.C. 2601) and the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136 et seq.). The product performance test guidelines contained
in OPPTS 810.3700 describe specific methods for evaluating insect repellents and reflect the EPA's
minimum recommendations for developing reliable repellent product performance data. The draft
guidelines are available electronically in portable document format (pdf) at http://www.epa.gov/
opptsfrs/publicatjons/O pp T5Harmonjzed/8 I O_Product_Performance Test Guidelines/Drafts/S 10-
3700.pdf

Sources of Variation in Repellent Bioassays

Abiotic Factors
Many factors influence the outcome and interpretation of repellent bioassays. Skin-mediated effects
comprise absorption and penetration of repellent on skin, but evaporation, abrasion (contact with
clothing), washing or rinsing of treated surfaces, and perspiration also result in repellent loss. 1515 These
physical factors are difficult to control in a bioassay, but their contribution to experimental error can be
minimized by random selection of test subjects, the use of appropriate sample sizes in bioassays, and by
recognizing and avoiding pseudo replication. Loss of repellent by abrasion or by washing or rinsing from
treated skin can be minimized by rigorous oversight of the test proceedings and by diligence on the part
of the test subject.
Light, temperature, humidity, and air quality at the testing venue are important environmental
influences in repellents bioassays.i7.i920 These factors can be manipulated to desired levels in the
laboratory, but in nature their variation profoundly affects mosquito responses to repellent stimuli.
Therefore, field bioassays should be standardized with respect to season, geographic location, and the
time within the diel period in which observations are made. When this is not possible, tests should be
designed so that estimates of important physical and climatic parameters are included as treatment
variables in the statistical analysis.
Additional environmental sources of variation in bioassays are repellent dose and exposure time 15 and
test cage configuration.7821 In the latter case, research suggests relationships between protection time,
mosquito test population size, and the mosquito biting rate. However, investigations using different test
cage configurations and mosquito population sizes 19,21-24 have not led to a consensus regarding the
optimal mosquito biting rate and density for repellency tests. One reason is that test cage shape and size
and mosquito density effects vary between mosquito species. For Aedes aegvpti, for example, repellent
protection time is inversely related to cage size but is not affected by mosquito density; whereas, for
Anopheles quadrinaculatus, protection time is short in large (125-L).cages with high mosquito densities
(49 cm 3 per mosquito) and long in medium (65-L) cages with low mosquito densities (640 cm 3 per
mosquito).

Biotic Factors
Biological factors in repellent bioassays consist of larval nutrition, carbohydrate availability to adult
mosquitoes, age and parity in female mosquitoes, partial blood engorgement, and innate differences
among repellent-treated test subjects. 8.22,25,26 An important behavioral factor that affects bioassay results
is the timing and intensity of mosquito biting activity. 5 '27 Ignorance of temporal feeding patterns can
compromise estimates of protection time for repellents that have extended activity, as can poor
knowledge of biting rates. In screened cage tests, biting patterns can vary with the size of the cage,
and this factor can affect the determination of repellency.8
 109
Standard Methods for Testing Mosquito Repellents

Conclusions
A comprehensive understanding of the parameters that affect repellent bioassays can minimize false
positive responses in the early stages of repellent screening. Rigorous bioassay standards in the later
stages of testing facilitate identification of the most promising new repellents and provide a sound basis
for selecting new repellents for toxicology testing and evaluation in field tests. The selection of a
repellents bioassay procedure should always be based on the biological relevance of the method and its
capacity to yield precise experimental data. When these two outcomes are achieved, one can correlate the
results from different bioassay techniques to obtain an accurate estimate of the repellency of
any compound.

References
1. J. L. Robertson and H. K. Preisler, Pesticide Bioassays with Arthropods, Baca Raton: CRC Press,
1992.
2. World Health Organization [WHO], Report of the WHO informal consultation on the evaluation and
testing of insecticides, World Health Organization, Control of Tropical Diseases, Pesticide Evaluation
Scheme, Informal Consultation 96.1, Geneva, 1996.
3. American Society for Testing and Materials IASTMI, Laboratory testing of non-commercial repellant
formulations on the skin, ASTM-E951-94, 2000.
4. American Society for Testing and Materials [ASTM], Field testing topical applications of compounds
as repellents for medically important and pest arthropods, 1. Mosquitoes, ASTM-E393-94, 2000.
5. H. K. Gouck and C. N. Smith, The effect of age and time of day on the avidity of Aedes aegypti, Fla.
Entomol., 45, 93, 1962.
6. C. N. Smith et al., Factors affecting the protection period of mosquito repellents, USDA Tech. Bull.,
1258, 36, 1963.
7. C. E. Schreck, Techniques for the evaluation of insect repellents: A critical review, Annu. Rev.
Entomol., 22, 101, 1977.
8. D. R. Barnard, Mediation of deet repellency in mosquitoes (Diptera: Culicidae) by species, age, and
parity, J. Med. Entomol., 35, 340, 1998.
9. J. A. Kiun and M. Debboun, A new module for quantitative evaluation of repellent efficacy using
human subjects, J. Med. Entomol., 37, 177, 2000.
10. P. J. Weldon et al., Benzoquinones from millipedes deter mosquitoes and elicit self-anointing in
capuchin monkeys (Cebus spp.), Naturwissenshaften, 90, 301, 2003.
11. A. J. Klun et al., A new in vitro bioassay system for discovery of novel human-use mosquito repellents,
J. Am. Mosq. Control Assoc., 21, 64, 2005.
12. M. D. Buescher et al., The dose-persistence relationship of deet against Aedes aegypti, Mosq. News,
43, 364, 1983.
13. L. C. Rutledge et al., Comparative sensitivity of representative mosquitoes (Diptera: Culicidae) to
repellents. J. Med. Entoinol., 20, 506, 1983.
14. K. Posey and C. E. Schreck, An airflow apparatus for selecting female mosquitoes for use in repellent
and attraction studies, Mosq. News, 41, 566, 1981.
36, 141,
15. M. L. Gabel et al.. Evaporation rates and protection times of mosquito repellents, Mosq. News,
1976.
16. L. C. Rutledge et al., Mathematical models of the effectiveness and persistence of mosquito repellents,

I
J. Am. Mosq. Control Assoc., 1, 56, 1985.
17. R. Gupta and L. C. Rutledge, Laboratory evaluation of controlled release repellent formulations on
human volunteers under three climatic regimens, J. Am. Mosq. Control Assoc., 5, 52, 1989.
18. L. M. Rueda et al., Effect of skin abrasions on the efficacy of the repellent deet against Aedes aegvpti,
J. Am. Mosq. Control Assoc., 14, 178. 1998.
1 110 'Jnsect Repellents: Principles; Methods, and Uses

.19. S. P. Ftancds et al., Laboratory and field evaluation of deet ClC-4 and 'A13-37220 against Anopheles
dirus (Diptera: Culicidae) in Thailand, J. Med. Entoniol., 33, 511, 1996.
20. W. G. Rcinfenrath and T. S. Spencer, Evaporation and penetration from the skin, in Percutaneou
Absorption: Mechanisms—Methods—Drug Delivery, R. L. Bronaugh and H. I. Maibach (Eds.),
2nd ed:, New York: Marcel Dekker, 1989, pp. 3 13-334. 1'
21.' L. L. Lomax' and P.1 Grinett, Current laboratory' jrocedurés for the development of improved insect
repellents at Rátgers-The State University, Proc. 'NJ Mosq. Exterrh.'Assoc., 58, 41, 1971.
22. M. Bar-Zeev and D. Ben-Tamar, Evaluation of mosquito rcpellánts, Mosq. News, 31, 56, 1971.
23. A. A. Khan et al., Insect repellents: Effect of mosquito and repellent related factors on protection time.
J. Econ. Entomnol.,. 68, 43, 1975..
24. S. P. Frances et al., Response of Anopheles diru.s and Aedes albopictus to repellents in the laboratory,
J. Am. Mosq. Control Assoc., 9, 474, 1993.
25. P. V. Wood, The effect of ambient humidity on the repellency of ethylhexanediol ('6-12') to Aedes
aegypti Can. Entomnol., 100, 1331, 1968.
26. R. D. Xue and D. R. Barnard, Effects of partial blood engorgement and pretest carbohydrate
availability on the repellency of deet to Aede.c albopictus, J. Vector Ecol., 24, Ill, 1999.
27. R. D: Xue and D. R. Barnard, Human host avidity in Aede.c aihopicrus: Influence of mosquito body
size, age, parity, and time of day, J. Am. Mo.sq. Control Assoc., 12, 58, 1996.

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